Full length articleEnhanced targeting of invasive glioblastoma cells by peptide-functionalized gold nanorods in hydrogel-based 3D cultures
Graphical abstract
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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