Glucose decorated gold nanoclusters: A membrane potential independent fluorescence probe for rapid identification of cancer cells expressing Glut receptors
Graphical abstract
Introduction
Due to ultra-small size and high intrinsic fluorescence property, gold nanoclusters (AuNCs) have gained applications in several avenues of biomedicines, such as fluorescent chemosensors, chemical catalysts, cancer cell imaging and radiotherapy sensitizer [1], [2], [3], [4], [5]. Although several methods have been reported for the facile synthesis of AuNCs and found preferential accumulation in cancer tissues, however, a detailed mechanism is still infancy. Zhang et al. [5] have reported the synthesis of glutathione (GSH) coated AuNCs showing preferential accumulation in tumor cells via improved EPR (Enhanced Permeability and Retention) effect. EPR effect is a passive targeting strategy, which depends on several physiological factors of organisms. Therefore, EPR effect may not give the desired therapeutic efficacy consistently. Additionally, arginine-glycine-aspartic acid (RGD) peptide and folic-acid-conjugated AuNCs have also been reported to show the active targeting and imaging of tumor cells [1], [6]. These peptides are uniquely expressed on certain types of cancer cells, therefore the imaging strategies would be limited to few cancer types. Further, these studies lack in providing information about cell membrane potential based internalization of AuNCs. It has been well established that nanoparticles enable their internalization followed by interaction with cells orchestrated by cell membrane potential and surface receptors. Further, cellular internalization of nanoparticles also depends on nanoparticle shape, size, composition and capping molecules [7].
Cancer cells are characterized to have accelerated metabolism and high glucose uptake. Due to this, cancer tissues are imaged by PET (Positron Emission Tomography), which depends on the high uptake of radiolabeled [18F]-2-fluoro-2-deoxy-d-glucose (FDG), which is a glucose analog [8]. Glucose transporter (Glut) proteins, present across the plasma membrane of mammalian cells, leads to the transport of glucose in the cytoplasm. Out of several Glut proteins, Glut-1 has been assigned to be involved in increased transport of glucose in cancer cells. High expression of Glut-1 proteins in cultured mammalian cells has been shown to rapidly transport the glucose in the cytoplasm and also correlated with poor survival of patients in human studies [9].
All cells display an electrical potential across the plasma membrane controlled by an ion gradient coupled with selective permeabilities [10]. Under normal condition the ion gradient varies between −10 and −100 mV potential across the cell membrane exhibiting a net negative charge on the cytoplasmic side. The cytoplasmic and extracellular potassium concentrations regulate the cellular membrane potential. The plasma membrane is relatively permeable to K+ than negatively charged ions and proteins present in the cytoplasm. Therefore, under standard condition, a high concentration of K+ exists in the cytoplasm than in the extracellular medium resulting in a net efflux of K+ out from the cell. There exist K+ channels, through which these ions are passively cross the plasma membrane. This efflux of K+ and retention of negatively charged molecules lead to the generation of net negative charge in the interior of the cell membrane. At this state of “resting potential”, cells are referred as “polarized”. A state of less negative voltage in the interior of the cell membrane is called “depolarized” and conversely, a high negative voltage is “hyperpolarized” state. Since the polarization state of cells greatly influences the nanoparticle binding and internalization, therefore, any therapeutic nanosystem developed must be explored for membrane potential dependent cellular binding and internalization. As reported earlier, hyperpolarization of cells leads to increased internalization, whereas, depolarization results reverse the trend. Therefore, these factors may provide a control mechanism to selectively deliver the therapeutic nanosystems to the diseased cells. However, several diseases exhibit altered membrane potential, for example in cancer cells and defective neutrophils, the membrane potential is significantly lower than corresponding normal cells, therefore, membrane potential based uptake may not be useful [11], [12], [13], [14], [15].
Another method by which cells internalize the macromolecules, including nanoparticles, is through the action of integral membrane proteins and surface receptors. Endocytosis is the process in which membrane-bound vesicles are derived in from the invagination followed by pinching-off as the pieces of the plasma membrane. Phagocytosis and pinocytosis (cell drinking) are the two broad categories of endocytosis. Pinocytosis can be further categorized into four distinct processes, namely, clathrin-mediated, caveolae-mediated, clathrin and caveolae-independent and macropinocytosis [16]. It is well known that once internalized, the molecules are sorted and trafficked to specialized cell organelles, depending on several factors such as surface charge. Several groups have shown that endocytosis is a prevalent process for a variety of nanomaterials internalization into mammalian cells [17], [18], [19], [20].
Our goal was to investigate the cellular internalization of BSA-AuNCs and Glu-AuNCs in response to depolarized and hyperpolarized cancer (A431) and normal (HaCaT) cells. Cell membrane potential was regulated by using exogenous ion channels or varying the extracellular ion concentrations. Further, Glut-1 receptor-mediated internalization of BSA-AuNCs and Glu-AuNCs was also explored and compared. A431 cells exposed to genistein, Glut-1 receptor inhibitor, were also investigated for the uptake of Glu-AuNCs. Two techniques were used, flow cytometry and fluorescence microscopy, to measure the internalization of AuNCs. The experiments were carried out in one cancerous (A431) and normal (HaCaT) cell culture model for skin to investigate the preferential internalization of Glu-AuNCs in Glut-1 receptor expressing A431 cells. The results demonstrate that not only cellular membrane potential but the receptor-mediated internalization of targeted nano drug delivery or imaging system must also be considered for the design of suitable nanotherapeutics for biomedical applications. Further, this study revealed that Glu-AuNCs could be an excellent probe for targeted drug delivery, imaging of cancer cells and monitoring of therapeutic efficacy in real-time.
Section snippets
Materials
Gold (III) chloride trihydrate (HAuCl4·3H2O), fetal bovine serum (FBS), choline chloride (ChoCl), DiBAC dye, gramicidin A and genistein used in this study were purchased from Sigma–Aldrich (USA). Bovine serum albumin (BSA), sodium hydroxide (NaOH), glucose (C6H12O6), potassium bromide (KBr), formaldehyde, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), HEPES, potassium chloride (KCl), sodium dihydrogen phosphate (NaH2PO4), calcium chloride (CaCl2),
Particle characterization
The BSA-AuNCs were synthesized by the method reported by Khullar et al. [21]. However, the same method did not produce Glu-AuNCs, rather resulted in glucose coated gold nanoparticles, which do not exhibit fluorescence (data not shown). Therefore, Maillard reaction [22] was performed between BSA and glucose to produce BSA-glucose conjugate, which was used for Glu-AuNCs synthesis. As expected, the UV–vis spectra of BSA-AuNCs and Glu-AuNCs (Fig. 1A) did not show a clear surface plasmon resonance
Conclusion
In summary, cellular binding and uptake are the two most essential initial steps for any biomedical applications intended from nanomaterials. We have shown that Glucose coated fluorescence AuNCs can be successfully synthesized at room temperature. In contrast to the reported bare gold nanostructures, BSA-AuNCs and Glu-AuNCs are found stable in cell culture media conditions, without any apparent changes in optical properties. Using flow cytometry and fluorescence microscopy, we found that the
Conflict of interest
The author declares no conflict of interest.
Acknowledgements
The financial assistance provided for the establishment of Centre for Nanotechnology Research and Applications (CENTRA) by the Gujarat Institute of Chemical Technology (GICT) is gratefully acknowledged (project code: ILS/GICT/2013/003). Funding from the Department of Chemistry, Indian Institute of Technology Madras (grant number TTR1314010IITMTPRA) is also gratefully acknowledged. Dr Sanjay Singh thanks the Science and Engineering Research Board (SERB) for providing the Young Scientist Start-up
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2021, Sensors and Actuators, B: ChemicalCitation Excerpt :India). BSA-AuNCs were synthesized using method described by Singh in one of the reports [8]. In brief, 5 mL 50 mg/mL BSA solution was mixed with 5 mL 0.1 M chloroauric acid and 0.5 mL 1 M NaOH.