Elsevier

Acta Biomaterialia

Volume 58, August 2017, Pages 12-25
Acta Biomaterialia

Full length article
Enhanced targeting of invasive glioblastoma cells by peptide-functionalized gold nanorods in hydrogel-based 3D cultures

https://doi.org/10.1016/j.actbio.2017.05.054Get rights and content

Abstract

Cancer stem cells (CSCs) are responsible for drug resistance, tumor recurrence, and metastasis in several cancer types, making their eradication a primary objective in cancer therapy. Glioblastoma Multiforme (GBM) tumors are usually composed of a highly infiltrating CSC subpopulation, which has Nestin as a putative marker. Since the majority of these infiltrating cells are able to elude conventional therapies, we have developed gold nanorods (AuNRs) functionalized with an engineered peptide capable of specific recognition and selective eradication of Nestin positive infiltrating GBM-CSCs. These AuNRs generate heat when irradiated by a near-infrared laser, and cause localized cell damage. Nanoparticle internalization assays performed with GBM-CSCs or Nestin negative cells cultured as two-dimensional (2D) monolayers or embedded in three-dimensional (3D) biodegradable-hydrogels of tunable mechanical properties, revealed that the AuNRs were mainly internalized by GBM-CSCs, and not by Nestin negative cells. The AuNRs were taken up via energy-dependent and caveolae-mediated endocytic mechanisms, and were localized inside endosomes. Photothermal treatments resulted in the selective elimination of GBM-CSCs through cell apoptosis, while Nestin negative cells remained viable. Results also indicated that GBM-CSCs embedded in hydrogels were more resistant to AuNR photothermal treatments than when cultured as 2D monolayers. In summary, the combination of our engineered AuNRs with our tunable hydrogel system has shown the potential to provide an in vitro platform for the evaluation and screening of AuNR-based cancer therapeutics, leading to a substantial advancement in the application of AuNRs for targeted GBM-CSC therapy.

Statement of Significance

There is an urgent need for reliable and efficient therapies for the treatment of Glioblastoma Multiforme (GBM), which is currently an untreatable brain tumor form with a very poor patient survival rate. GBM tumors are mostly comprised of cancer stem cells (CSCs), which are responsible for tumor reoccurrence and therapy resistance. We have developed gold nanorods functionalized with an engineered peptide capable of selective recognition and eradication of GBM-CSCs via heat generation by nanorods upon NIR irradiation. An in vitro evaluation of nanorod therapeutic activities was performed in 3D synthetic-biodegradable hydrogel models with distinct biomechanical cues, and compared to 2D cultures. Results indicated that cells cultured in 3D were more resistant to photothermolysis than in 2D systems.

Introduction

Glioblastoma Multiforme (GBM) is a deadly and incurable form of primary brain cancer [1]. The current lack of efficient therapies for this disease is attributed to the infiltration of single GBM cancer stem cells (CSCs) throughout the brain, leading to its high recurrence [2]. CSCs or tumor initiating cells have a high tumor-forming capacity, and show strong radio/chemotherapy resistance [3]. They express genes associated with neural stem cells, and it has been reported that CSCs in GBM tumors express Nestin and Prominin-1 markers. Nestin, in particular, has been associated with invasive malignant cells [4], [5], [6], [7], [8], [9]. Recently, a Nestin-binding peptide with the ability to recognize Nestin proteins specifically expressed on the surface of glioma stem cells, was discovered by in vitro phage display technology [10], [11]. Even though the function of Nestin in GBM cells is not well understood, recent studies indicate that the Nestin-positive (Nes+) subpopulation of cells might be an important target for optimizing GBM treatment [9], [10], [11]. Interestingly, examples of nanoparticles that target GBM-CSCs directly are scarce, and none of them use Nestin for GBM-CSC targeting [12], [13], [14], [15], [16].

There has been increased interest in combining molecular targeting with photothermal therapy in the field of cancer therapy, and among the several synthesized nanomaterials for biomaterial applications, gold nanorods (AuNRs) have been shown to be excellent hyperthermal agents [17], [18], [19], [20], [21]. AuNRs act as highly localized energy transducers by absorbing photons to generate heat, which causes localized cell damage [22], [23], [24]. Moreover, the surface of AuNRs can be easily functionalized via conjugation, and their size can also be controlled during synthesis enabling tunable absorption wavelengths in the near-infrared (NIR) region [22], [23], [24]. This is important for in vivo applications, given the high penetration depth transparency of human tissue in the NIR spectral range [22].

Presently, preliminary screening of nanoparticle-based cancer therapeutics is typically conducted on two-dimensional (2D) monolayer cell culture systems, and although they are uncomplicated and easy to execute, 2D cultures usually select drug candidates that do not translate comparably to in vivo models or patients [25], [26]. Moreover, limitations in nanoparticle formulations are usually undiscovered until later stages of product development. Thus, to overcome the shortcomings linked with 2D monolayer cultures, researchers have developed various three-dimensional (3D) culture platforms seeking to recreate the highly controlled mechanical and molecular microenvironment characteristics of tumors in vivo [26], [27], [28], [29], [30], [31]. What makes 3D cultures better tumor models for in vitro studies is that they allow cancer cells to arrange into 3D structures, with increased cell-cell and cell-extracellular matrix (ECM) signaling, thus recreating more natural microenvironments than 2D culture models. GBM inspired tissue-engineered constructs that incorporate brain endothelial cells, have been recently developed to study GBM-tumor angiogenesis [32]. Other approaches utilizing hyaluronic acid-based scaffolds with properties that promote biologically relevant GBM invasion in vitro have been extensively investigated [33], [34], [35], [36], [37], [38].

In this study, we have engineered and produced a peptide containing a sequence that recognizes Nestin expressed on the surface of GBM-CSCs for AuNR surface functionalization. Biodegradable 3D hydrogels with tunable mechanical properties, composed of star-shaped polyethylene glycol (starPEG) covalently connected to matrix metalloproteinase-susceptible peptide and maleimide-functionalized heparin (starPEG-MMP-heparin) [39], were used as our 3D culture system. The peptide functionalized AuNRs were then evaluated regarding cell selectivity, cell uptake pathway, intracellular localization, and photothermal activity in 2D cultures and in 3D GBM mono- and co-culture systems containing Nestin-positive (Nes+) or Nestin-negative (Nes-) GBM cells. Finally, 3D cultures were compared with 2D to reveal different AuNR therapy resistance by GBM cells.

Section snippets

Materials

Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O) was purchased from Acros Organics (Geel, Belgium). Sodium borohydride (NaBH4) was acquired from Fluka (Munich, Germany). Ascorbic acid, cetyltrimethylammonium bromide (CTAB), sodium azide (NaN3), chlorpromazine, methyl-β-cyclodextrine, nocodazole, silver nitrate (AgNO3), poly(ethylene glycol)methyl ether thiol (PEG-SH, Mw 6000 Da), Triton X-100, glutaraldehyde, paraformaldehyde, osmium oxide (OsO4), potassium ferrocyanide,

Peptide design and synthesis

We have engineered a peptide containing specific amino acid sequences for the recognition of Nestin, and for the optimal functionalization of AuNRs, which was named as Nes-peptide. The Nes-peptide was designed using literature references, and protein databases [10], [11], [43], [44]. As specified in Fig. 1A, the Nes-peptide contains a 7 amino acid sequence, AQYLNPS, that specifically recognizes Nestin expressed on the surface of GBM-CSCs [10]. The spacer, CEKEKEKE, is composed of a cysteine and

Conclusions

The starPEG-MMP-heparin hydrogels reported herein simulated native tumor tissue and its associated microenvironments in a more physiologically relevant manner than 2D cultures. Depending on the hydrogel mechanical cues, GBM cells organized into distinct tumor-like structures, and expressed aggressive invasive GBM tumor traits. The hydrogel system was used as a platform for accessing the therapeutic efficiency of AuNRs on the selective targeting and destruction of single invasive GBM-CSCs. AuNR

Acknowledgements

R.D.R. and D.R.T.Z. acknowledge funding from the DFG Research Unit FOR1713. This work was performed in the context of the European COST Action MP1302 Nanospectroscopy (R.D.R.). L.J.B. was supported by the Endeavor Awards as part of the Prime Minister’s Australia Awards. T.L.S. and F.N.G. were supported by DFG through the cfaed, ESF contract 100111059 (MindNano), as well as a seed grant 043_2615A6 by the CRTD to T.L.S. This work was supported by the German Federal Ministry of Education and

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