Multiplexing surface anchored functionalized iron carbide nanoparticle: A low molecular weight proteome responsive nano-tracer

https://doi.org/10.1016/j.colsurfb.2021.111746Get rights and content

Highlights

  • Synthesis and surface-functionalized of Fe2C@PEG, Fe2C@NH2 and Fe2C@COOH.

  • TEM, FTIR and LC‐MS/MS Spectrophotometry were used to characterized NPs and proteins.

  • Total 119 proteins, 57 were LMW proteins of 82 % up-regulated and 17 down-regulated.

  • Surface coating help molecular, cellular & biological role for cell signaling process.

  • A STRING algorithm was used to study functional interaction network of proteins.

Abstract

Harvesting the low molecular weight (LMW) proteins from the cellular exudates is a big challenge for early disease detection. Here, we introduce a unique probe composed of surface-functionalized Fe2C NPs with different functional groups to harvest, identify and profile differentially expressed biomarker proteins. Three different functionalization of Fe2C NPs with Fe2C@NH2, Fe2C@COOH and Fe2C@PEG enabled to harvest 119 differentially expressed proteins from HeLa cell exudates. Among these proteins, 57 were LMW which 82.46 % were up-regulated and 17.54 % were down-regulated. The Fe2C@NH2 were able to separate 60S ribosomal proteins L7a, and L11, and leucine-rich repeat-containing protein 59. These proteins play a vital role in the maturation of large subunit ribosomal ribonucleic acid, mRNA splicing via spliceosome and cancer cell inhibitor, respectively. While, Fe2C@COOH identifies the 60S ribosomal protein types L7, 40S ribosomal protein S11, and 60S ribosomal protein L24. These proteins were important for large ribosomal subunit biogenesis, translational initiation, and assembly of large subunit precursor of pre-ribosome. Finally, the Fe2C@PEG extracted 40S ribosomal protein S2, splicing factor, arginine/serine-rich and 40S ribosomal protein S4, X isoform which were responsible for nonsense-mediated decay, oligodendrocyte differentiation and multicellular organism development. Thus, these results help us in defining oncogenic biomarkers for early disease detection.

Introduction

Alzheimer’s disease (AD) [1], type 2 diabetes (T2D) [2] and cancer are three of the major challenges facing mankind. All three pathologies involve spontaneous misfolding or degradation of proteins such as the growth of abnormally folded amyloid beta and tau proteins, turnover of protein in type 2 diabetic and mutations in proteins which leads to different types of cancers in the biological system [3]. Reported translational ribosomal protein are related to the development of cancer, as RPL15 (eL15) and RPL19 (eL19) were found overexpressed in gastric cancer RPL7A (eL8), RPL19 (eL19), RPL37 (eL37) in prostate cancer [4]. RPL27 (eL27), RPL37A (eL43) and RPL41 (eL41) found to be downregulated in nasopharyngeal carcinomas by the gene sequencing [5,6]. To save lives, efficient early-stage diagnosis is crucial which can be timely utilized to slow down or cure these diseases in initial stages. A wide range of microscopic and spectroscopic methods are employed in order to monitor the abnormalities associated to these three disorders [7,8] however, early detection for either of these diseases is currently not well established.

The rapid advancement in nanoparticle (NP) technologies, especially their surface functionalization for applications in bio-imaging [9], drug-delivery [10,11] and disease diagnosis and treatment, encourage the development of suitable nanoprobes for early disease detection [12]. Among metal NPs, iron carbide (Fe2C) NPs offer a possible conciliation between high saturation magnetization and high stability in biological media. Recently, many researchers have evidenced wide potential of iron bases species for wide applications including magnetic hyperthermia [13], magnetic resonance imaging, and photoacoustic tomography-guided photo thermal therapy [14], changing metal ions signaling protein. The cost-effective, biocompatible ferromagnetic Iron carbide nanoparticle encourages the development of biological active nanoprobes for the wide range of bio-medical prognostic and diagnostics applications [15]. This one-stepped synthesis of iron carbide NP provides with quick, easy, cost effective and significant result grabbing tool for adhering different functional groups, further detection and then harvesting of significant biomarker. Iron Carbide in our study has been proven an effective bio-material that have significant binding, harvesting and detecting tendency that can be easily introduced in the biological system without inducing toxicity within a limit and provide us reliable and positive results. Moving towards other particles such as gold, silica or any other nanoparticle, which need expenses and calibration for asses, but here we invested in obtaining the same or nearly same results with controlling expenses and calibrations. Introducing the Iron carbide as a bio-material for same purpose but new modification is the ultimate achieved goal. This opens new horizons of iron dependent detection in the field of bio-materials. Therefore, utilization of such NPs to investigate the proteomic alternation in the cells as a biomarker for the detection of any disease as its triggers in the body is the only possible method. However, the formation of protein corona around metallic NPs restricts their applications in diagnostics, since it changes the size and surface properties of the NPs [16]. For example, coating of NPs with protein corona compositions alters the in-vitro physiological response of HeLa cell [17,18]. Thus, it is critical to load a protective layer on the NP surface to achieve the required functionality. The surface of active metal NPs can be coated with a variety of polymers [19], carbohydrates, lipids, etc. in order to achieve desired physicochemical properties for biological environment [20]. In previous studies, biomolecules such as antibodies were used in various approaches like western blotting [21] and affinity chromatography [22] to distinguish proteins but these methods were unable to separate and detect the low molecular weight (LMW) proteins responsible for abnormal functions within the biological system. Therefore, detecting misfolded protein is extremely challenging and needs further exploration in probe designs which can withstand the environment in living cells as well as can provide the appropriate signaling for identification and classification of LMW proteins for early disease detection.

In this work, we have developed unique strategy for the separation of LMW proteins from the mixtures of cellular exudates in response to selectively modified Fe2C NPs. To capture LMW proteins, special functional groups are attached to NPs surface such as 3-aminopropyltriethoxysilane (Fe2C@NH2), N(trimethoxysilylpropyl) ethylenediamine-triacetic acid-trisodium salt (Fe2C@COOH) and polyethylene glycol (Fe2C@PEG). The functionalized Fe2C NPs harvested 119 differentially expressed proteins from HeLa cell exudates, among these, 57 are LMW proteins from which 47 (82.46 %) are up-regulated and 10 (17.54 %) proteins are down-regulated. The proteomic analysis of harvested proteins confirmed that these proteins are involved in key functionalities of cells and any change will trigger the respective disease. Thus, can be used as proteomic markers for early disease detection, which is possible through our innovative probe, a surface-modified Fe2C with different functional molecules.

Section snippets

Fe2C NPs were synthesized

Fabrication of Fe2C NPs was performed by modifying pre-existing method [23]. Octadecyl amine ODA (37.5 mmol) was blended in a four-neck flask and degassed for 1 h at 120 °C under a gentle N2flow, magnetically. 5 mmol Synthesized Fe NPs in 10 mL of hexane were added dropwise and heated at a temperature of 260–345 °C for 15–45 min. This amalgam was heated at 130 °C for next 30 min to remove hexane thoroughly. The black-brown colored solution was cooled down to room temperature. Further 27 mL

Structural properties

The surface coated Fe2C NPs were synthesized by a two-step methodology as shown in Fig. 1. In the first step, Fe2C NPs with homogenous particle size distribution were synthesized using a chemical route as reported elsewhere [26].

The NPs were then coated with three different type of surface layers to protect the NPs from corona protein as shown in step 2 of Fig. 1. The coated NPs were then used for harvesting proteins followed by expression studies as explained in subsequent sections.

The surface

Conclusions

In the present study, we have introduced an exclusive approach using surface functionalized Fe2C NPs for effective harvesting of LMW differentially expressed key proteins from complex HeLa cell exudates. High-performance liquid chromatography couple with electrospray ionization with ion trap mass analyzer (HPLC/ESI‐Orbitrap) used which efficiently identified 119 differentially expressed proteins among which 57 were LMW with 47 (82.46 %) up-regulated and 10 (17.54 %) down-regulated proteins.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported.

Acknowledgements

This work was supported in part by the Higher Education Commission (HEC) funded National Research Programme for Universities (NRPU) (9458). We also Acknowledge the Islamia University Bahawalpur, Pakistan. The authors would like to acknowledge the Vice-Chancellor fellowship scheme at RMIT University, the RMIT Micro Nano Research Facility (MNRF) in the Victorian node of the Australian National Fabrication Facility (ANFF), the RMIT Microscopy and Microanalysis Facility (RMMF) to support this work.

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