The Interplay Between Whey Protein Fibrils With Carbon Nanotubes Or Carbon Nano-Onions Part 1

Abstract: Whey protein isolate (WPI) fibrils were prepared using an acid hydrolysis induction process. 

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Carbon nanotubes (CNTs) and carbon nano-onions (CNOs) were made via the catalytic chemical vapor deposition (CVD) of methane. WPI fibril–CNTs and WPI fibril–-CNOs were prepared via hydrothermal synthesis at 80 ◦C. 

The composites were characterized by SEM, TEM, FTIR, XRD, Raman, and TG analyses. The interplay between WPI fibrils CNTs and CNOs was studied. The WPI fibrils with CNTs and CNOs formed uniform gels and films. CNTs and CNOs were highly dispersed in the gels. Hydrogels of WPI fibrils with CNTs (or CNOs) could be new materials with applications in medicine or other fields. 

The CNTs and CNOs shortened the WPI fibrils, which might have important research value for curing fibrosis diseases such as Parkinson's and Alzheimer's diseases. The FTIR revealed that CNTs and CNOs both had interactions with WPI fibrils. 

The XRD analysis suggested that most of the CNTs were wrapped in WPI fibrils, while CNOs were partially wrapped. This helped to increase the biocompatibility and reduce the cytotoxicity of CNTs and CNOs. HR-TEM and Raman spectroscopy studies showed that the graphitization level of CNTs was higher than for CNOs. 

After hybridization with WPI fibrils, more defects were created in CNTs, but some original defects were dismissed in CNOs. The TG results indicated that a new phase of WPI fibril–CNTs or CNOs was formed.

Keywords: whey protein fibrils; carbon nanotubes; carbon nano-onions; composites; interaction.

1. Introduction

Whey protein is common and easily obtained from bovine milk. It was of practical significance to prepare whey protein isolate (WPI) fibrils. Nowadays, self-assembled amyloid fibrils based on whey components are an important research field [1–3]. 

Generally, amyloid fibrils are derived from the association with amyloidosis. For example, islet amyloid peptide is associated with diabetes, and β-amyloid protein is associated with Alzheimer's disease [4]. 

Protein fibrils can also be synthesized in vitro. Additionally, βlactoglobulin (β-lg) can self-assemble fibrillar proteins [5,6]. The β-lg is a globular protein with a molecular weight of 18,400 g·mol−1 and a radius of about 2 nm [7]. 

It can induce fibril formation under prolonged heating (6–24 h) at 80 ◦C and has a pH of 2 and low ionic strength [8]. The average length of the fibrils is 1–8 µm, with a diameter of about 4 nm [9]. 

The proteinaceous material in these fibrils is held together by intermolecular β-sheets [10]. During fibril formation, the amount of β-sheets is increased. Carbon nanotubes (CNTs) are hollow tubes made of multi-layer graphite sheets rotating and curling around the same axis at a certain angle [11]. 

Their diameters range from 0.4 (SWCNTs) to 100 nm (MWCNTs); their length can reach several microns; and they have superior mechanical properties, chemical stability, and a large specific surface area [12]. Carbon nanotubes are often used as filling materials to prepare nanocomposites to improve the mechanical behaviors of matrix materials. 

The biological applications of carbon nanotubes have also been widely studied, such as in biosensors, drug and vaccine delivery, tissue engineering [13], and new biomaterials [14]. However, pristine CNTs have poor solubility and potential cytotoxicity [15]. Attached biomacromolecules such as protein, DNA, and RNA can promote the dispersion of CNTs [16]. 

The physical interactions with biomacromolecules might change their biological activity in vivo [17]. After functionalization and modification, the CNTs can load different types of drugs for targeted purposes [18]. Biocompatible-CNT-based systems can load multiple therapeutic, targeting, and probing agents for cancer therapy. 

It has been proven that functionalized CNTs can cross the plasma membrane through different mechanisms, notably through endocytosis [19–21]. Carbon nano-onions (CNOs) comprise multiple concentric shells of fullerenes. 

Their cage-within-cage structures generate some unique physiochemical properties. Unlike any other carbon allotropes [22,23], CNOs are equally important as CNTs and fullerenes, which are ideal for drug delivery applications due to their ability to remain in systemic circulation for hours, increasing their chances of accessing the target site [24–28]. 

In tissue engineering, modified CNO scaffolds display tissue regeneration capability [28]. Far-red fluorescent CNOs have been developed for cellular imaging purposes [29]. 

Despite this immense potential, it appears that the role of this novel nano-system in the biomedical field has been overlooked for many years. The research on protein fibrils–carbon nanomaterial systems will be of great significance in treating human diseases, reducing the cytotoxicity of carbon nanomaterials, and developing new technologies. 

The formation of amyloid fibrils in vivo could lead to a variety of diseases, such as Alzheimer's and Parkinson's neurodegenerative diseases. Researchers are looking for substances that can inhibit amyloid fibrosis or destroy the amyloid fibrils [30,31]. Table 1 summarizes some of the studies on the interplay of carbon nanomaterials with amyloid fibrils [32]. 

Some studies have shown that carbon nanomaterials can interact with various biological proteins [33]. CNTs are covered by adsorbed biological macromolecules in the biological solution because of their high specific surface area and hydrophobic surface [34]. 

The adsorbed proteins gather on the surface of carbon nanomaterials to form a "protein crown" [34]. The interaction between CNTs and proteins also plays an important role in the formation of β-sheets. 

Ghule et al. found that multi-walled carbon nanotubes (MWCNTs) provided interaction surfaces for protein adsorption or encapsulation. This could inhibit the ability of the nonpolar surface of proteins to bind protein fibrils, therefore preventing the protein from further fibrosis [35]. 

Jana and Sengupta [36] and Wei et al. [37] studied the self-assembly of Aβ-peptide in the presence of single-walled carbon nanotubes (SWCNTs) by using molecular dynamics (MD) simulation. The Aβ-peptide is a short amphiphilic peptide, and its aggregation is closely related to the pathogenesis of Alzheimer's disease [38]. The strong hydrophobic effect of CNTs can help locate peptides on the surface of SWCNTs. 

This prevents diffusion and inhibits the fibrosis of peptides. Proteins such as insulin, lysozyme, β-lactoglobulin, and cytochrome c can pattern on graphite [39,40]. This nanopatterned graphite is capable of template-guiding the alignment of amyloid fibrils [39]. The interaction between fullerenes and protein materials has also been studied. 

Through ThT fluorescence measurements, Kim and Lee found that fullerene could inhibit the fibrosis of protein. Fullerene could specifically bind to the central hydrophobic motif KLVFF, thus hindering the aggregation of Aβ-peptide [41]. 

It was found that hydrated fullerenes could not only destroy mature amyloid fibrils but also prevent the formation of new fibrils [42]. Podolski et al. found that hydrated fullerenes could effectively block the aggregation of Aβ25–35 [43]. 

There are few studies on the interplay between CNOs and amyloid fibrils. CNOs are a new allotrope with low toxicity and good biocompatibility. The study of the interaction between CNOs and amyloid fibrils is desirable.

On the other hand, some carbon nanomaterials have been combined with biological macromolecules to prepare hybrid nanocomposites for tissue engineering or drug delivery because of their mechanical and electrical advantages [55–57]. 

The amyloid fibrils also have certain mechanical behaviors and amino acid surfaces, which are used to prepare nanowires [58], hydrogels [59], fibrous cell scaffolds [60,61], and solid functional organic films [62]. The proteins are attached to the surfaces of CNTs in the form of monomers or oligomers [63,64], to improve their water solubility and reduce their cytotoxicity. 

CNTs change the structural properties of protein fibrils through hybridization and recombination to target the delivery of therapeutic drugs in vivo and destroy cancer cells [64,65]. Hendler et al. used the "co-assembly" method to form hybrid amyloid-fullerene composite fibrils [66], which are used for the preparation of color separation nanomarkers, diagnostic materials, and optoelectronic devices. 

The special properties of protein fibrils and carbon nanomaterials (such as the mechanical and electromagnetic properties of carbon nanomaterials and the biological properties of protein materials) can benefit each other, and their combination will greatly broaden the application ranges of these two kinds of nanomaterials. 

However, there is still a long way to go to fully understand the interaction between protein fibrils and carbon nanomaterials. In this research, we studied the interaction of WPI fibrils with CNTs (or CNOs) and characterized the composites of WPI fibril–CNTs (or CNOs) by SEM, TEM, XRD, Raman, FTIR, and TG. WPI fibrils were prepared by using an acid hydrolysis induction process. WPI fibril–CNTs (or CNO) composites were made using hydrothermal synthesis.

2. Materials and Methods

2.1. WPI Fibril Formation

WPI-1 was purchased from Davisco Foods International Inc. (97.8% without lecithin, NM, USA) and WPI-2 was purchased from Hilmar Ingredients (90.39% with lecithin, Hilmar, CA, USA). 

A stock solution (about 6 wt.%) was made by dissolving WPI in Millipore water. The pH of the solution was then adjusted to 4.75 by adding 1 M HCl, followed by centrifugation (10,000 rpm, 60 min, 4 ◦C) and filtration of the supernatant (FP 030/0.45 µm, Schleicher and Schuell). After filtration, the pH of the filtrated solution was set to 2 by using 6 M HCl. 

The protein concentration of the stock solution was determined using a UV spectrophotometer (UV-1800PC, MAPADA, Shanghai, China) and a calibration curve of known WPI concentrations at a wavelength of 278 nm. 

The stock solution was diluted to a protein concentration of 2 wt.% with an HCl solution of pH 2. The WPI solution was then heated and stirred (about 290 rpm) for 20 h at 80 ◦C to form fibrils.

2.2. CNTs and CNOs Preparation

2.2.1. Preparation of CNTs

Preparation of the La2NiO4 catalyst: La(NO3)3·6H2O and Ni(NO3)2·6H2O (molar ratio of La/Ni = 2:1) were dissolved in deionized water, then citric acid was added. The solution was heated at 80 ◦C for 1 h with stirring, and finally, it turned into a colloidal substance. 

The colloidal substance was calcined in a muffle furnace (10 ◦C/min in air; 300 ◦C for 1 h, then 800 ◦C for 5 h). Catalytic chemical vapor deposition (CVD) of methane to make CNTs: The fixed-bed gas-solid catalytic reactor was adopted for methane CVD to make CNTs. 

The La2NiO4 catalyst (0.5 g) was placed inside quartz boats in a tubular quartz reactor. Firstly, nitrogen (30 mL/min) was used to flush the reactor for 30 min, and then hydrogen (10 mL/min) was used to reduce La2NiO4 at 600 ◦C for 1 h. 

Afterward, the gas was switched to methane (60 mL/min) for catalytic CVD at 800 ◦C for 8 h to synthesize CNTs. Purification of CNTs: The CNTs mixed with catalysts were purified in 0.1 M nitric acid at 80 ◦C with stirring for 5 h. 

It was filtered and washed with deionized water five times. Finally, the sample was dried at 120 ◦C for 6 h.

2.2.2. Preparation of CNOs

Pretreatment of stainless steel mesh carrier: SS316 stainless steel meshes measuring 20 mm × 20 mm were ultrasonically cleaned for 30 min in 0.1 M HCl solution. Then, the meshes were placed in a tubular quartz reactor. 

Nitrogen gas-carrying water vapor (90 ◦C water vapor) was introduced into the quartz tube. The quartz tube was heated to 300 ◦C for 1 h. The surface of the stainless steel was used as a catalyst carrier after such treatment. 

Loading of catalyst: The above pretreated stainless steel mesh was immersed in nickel oxalate solution. Citric acid was added with stirring for 1 h. The solution was heated at 80 ◦C and finally turned into a colloid. The colloid and stainless steel meshes were put into a crucible and calcined in a muffle furnace (Zhonghuan, Tianjin, China) at 900 ◦C (10 ◦C/min, in the air) for 3 h. 

Finally, the stainless steel mesh loaded with catalyst was obtained. Catalytic CVD of methane to make CNOs [67]: A fixed-bed gas-solid reactor (Zhonghuan, Tianjin, China) was also used. The stainless steel mesh catalyst was placed in a quartz tube. 

Nitrogen (30 mL/min) was used to purge the reactor at room temperature for 1 h, then the temperature was raised to the reaction temperature of 900 ◦C and the nitrogen was switched to methane (30 mL/min) for 8 h for catalytic cracking. 

At last, the methane was switched back to nitrogen gas, and the reactor was cooled down to room temperature. Finally, the stainless steel mesh catalyst and CNOs were taken out. Purification of CNOs: A CNO sample was first sieved to remove the free catalyst particles. 

It was then mixed with concentrated HNO3 and refluxed at 90 ◦C for 40 h. After dilution and cooling, it was centrifuged at 4000 rpm for 10 min and the acid solution was removed. 

The remaining CNOs were rinsed thoroughly using distilled water several times until reaching neutral pH. Finally, the purified CNOs were dried.

2.3. Preparation of WPI Fibril–CNTs (or CNOs)

WPI fibril–CNTs (or CNOs) were synthesized using the hydrothermal method. The CNTs (or CNOs) with concentrations of 0.05 wt.%, 0.10 wt.%, and 0.15 wt.% were mixed in deionized water and treated ultrasonically for 30 min to disperse as well as possible. 

The same volume of WPI fibril solution was added and mixed with magnetic stirring for 30 min. The mixture was then poured into the autoclave reactor (Hongchen, Xi'an, China) for hydrothermal reaction (80 ◦C, 20 h). 

Afterwards, the product was cooled down to room temperature the autoclave was opened and the mixture was taken out. The product was dried in an oven (60 ◦C) for 48 h.

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