Received date: March 28, 2016; Accepted date: April 27, 2016; Published date: May 05, 2016
Citation: Sierra JEB, Cornelio JAC, García AG, Osorno JB, Palacio LMH (2016) Modification of Carbon Nanotubes with Electronegativity Molecules to Control the Adhesion of Low Density Lipoprotein. Biochem Physiol 5:203. doi:10.4172/2168-9652.1000203
Copyright: © 2016 Sierra JEB, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Atherosclerosis is a cardiovascular disease that causes accumulation of lipoproteins, which leads to vascular injury and may even cause Acute Myocardial Infarction (AMI). The interaction of vascular endothelium with low - density lipoproteins (LDL) was modified by using two distinct groups of carbon nanotubes (CNTs). The first group was doped with aluminum sulfate (Al2(SO4)3) and boric acid (H3BO3), and the second group was functionalized by chemical route with carboxylic acid (COOH) and glucosamine (C6H13NO5). The catalysts used to grow the CNTs were Nickel (Ni) 50%, Cobalt (Co) 50% and Cobalt Iron (Fe - Co) 10% - 40%, by sol - gel route. Scanning electron microscopy (SEM), Raman, and contact angle were used to characterize CNTs. The Raman spectra of multi - wall carbon nanotubes showed three bands, which are called D (disorder), G (graphitization) and G’ (second harmonic order) which caused by the D band. It is observed that the intensity ratio ID / IG increases for functionalized CNTs. CNTs grown from nickel and functionalized by chemical route with glucosamine showed low wettability contact angle for the 2h and 18h oxidized LDL samples. CNTs grown from nickel and doped with aluminum sulfate showed an angle of contact with low wettability for the 2h oxidized LDL sample. An association of the sulphate groups in the density of the load and the cooperativity of the load with arginine and lysine rich peptides of the LDL sample were observed. The CNT catalyzed with (Fe - Co) and doped with boric acid evidenced a low strength of adhesion and greater surface tension for the 18h high degree oxidized LDL; resulting in a repulsion of residues of lysine and arginine of the altered structure of ApoB 100 of the LDL. The obtained CNT structures are presented as a possible devise coating with therapeutic potencial to avoid the progression of atherosclerosis.
Carbon nanotube; Low - density lipoproteins (LDL); Functionalization; Electronegativity molecules
Cardiovascular diseases are the main cause of death in Western societies, among which atherosclerosis stands out [1,2]. This is a chronic inflammatory process that is characterized by morphological and functional changes in the vascular wall. Resulting in the reduction of the blood flow, associated with a phenomenon of platelet aggregation and thrombosis [3-6], where low density lipoproteins (LDL) are special markers that can be recognized and accepted by endothelium which leads to arterial hardening and occlusion.
Nanotechnology offers new alternatives to find a solution to this disease [7-9]. New biocompatible materials possess nano - metric scales that prevent vascular restenosis, reducing the atheroma [10-12]. CNTs have received enormous attention in biomedical applications such as drug delivery agents, biosensors, bone material scaffolds and even as neuronal growth substrate [13-15]. CNTs can be modified by adding functional groups to enhance their hydrophobicity and improve their compatibility with any target tissue [16-18].
A well - established and efficient method to functionalize the nanotubes is to subject them to prolonged sonication at room temperature, in a mixture of concentrated sulfuric acid and nitric acid. The reaction starts at end caps of the CNTs, due to strong pressure from the hexagon - heptagon pairs, the end caps are quickly removed, leaving open end tubes functionalized with carboxylic acid groups (COOH) .
Modification of CNTs doped with molecules of aluminum sulfate (Al2(SO4)3), boric acid (H3BO3), and functionalized by chemical route with carboxylic acid (COOH) and glucosamine (C6H13NO5) are described in this article. Characterization of the obtained CNT structures was achieved by SEM, Raman, and contact angle analysis.
Carbon nanotube synthesis
CNTs were synthesized by chemical vapor deposition (CVD). A quartz tube was used to grow CNTs and a furnace equipped with a high precision automated computer temperature controller, which allowed to achieve the desired reaction temperature (700°C). Acetylene was used as the carbon source. The catalysts were nickel (50%), cobalt (50%) and Iron - Cobalt (10% - 40%) for the production of CNTs and the gas mixture was composed of 80 cc/ min nitrogen, 20 cc/ min acetylene and 15 cc/ min hydrogen. The processing sequence included reduction time of 20min, acetylene time of 30min and cooling time of 60 min [20-28].
Functionalization by doping: Doping of the CNTs (Figure 1) was carried out during the synthesis process by using an ultrasonic vaporizer attached to quartz tube of CVD devise. The vaporizer was loaded with a known amount of the precursor. The precursor solution contains the doping electronegativity molecules, either aluminum sulfate (Figure 1a) or boric acid (Figure 1b) both 2% w / v. The vaporized precursor is injected into the reaction zone through pulsated nitrogen gas (N2) carrying a flow of 0.2 L / min.
Carboxylic acid functionalization (COOH): The functionalization of CNT with carboxylic acid is based on the research of Matthew W. Marshall et al. 2006. The functionalization process was achieved by using 1mL of a concentrated solution of H2SO4 and HNO3 (3:1 v / v) for each 2mg of CNT (Figure 1c). This mixture was sonicated for 20 minutes in a water bath sonicator at a temperature of 20˚C, ice was used to control the water temperature; thus avoiding the breakdown of CNTs and preserving their original lengths. The functionalized CNT were washed with deionized water to remove any excess of acids, until a pH of 5 was reached. The final product was dried in a sealed oven at 60˚C .
Glucosamine functionalization (C6H13NO5): Functionalization with glucosamine (C6H13NO5) in a controlled atmosphere of nitrogen was based on the reports by Leize Zhu et al. . In order to add glucosamine to the CNTs, these must be previously functionalized with carboxylic acid. Initially, 1ml of distilled water, 0.5 ml ethanol was used as solvent followed by 195 μl of a solution of potassium bromide (10 g of KBr in nitric acid at 10%) were mixed per each 75 mg of CNTs (Figure 1d). The described process is known as halogenation and happens so that the diameter and the ion of bromine energy, tends to de - saturate the hydrogen atoms, therefore the hydroxyl group becomes available and is engaged and released; is there then when bromine enters the network.
Subsequently, the sample was placed in a desiccator with controlled atmosphere of nitrogen per 24 h, then the sample was washed 3 times with distilled water and ethanol. After the final wash 134.2 mg of glucosamine was added per each 75 mg of CNT. The sample was washed once again in a desiccator with controlled atmosphere of nitrogen during 72 hrs to avoid product contamination with any grafted polymer or leftover reactive. Once again the sample was washed 3 times with deionized water and left to dry under vacuum [30,31].
Based on the research by Evangelia Chnari , three different LDL was tested: unmodified LDL (natural state) and mildly and highly oxidized LDL. LDLs were obtained from porcine plasma with a concentration of 10 μg / mL.
The mildly oxidized LDLs were achieved by incubating 50 μg / ml of LDL with 10 μg of copper sulfate (Cu2SO4) at 37°C for 2 hours. Similarly, the highly oxidized LDL was obtained by exposing it to air and increasing the incubation time to 18 hours. In all cases the oxidation was completed with an aqueous solution of 0.01% w / v ethylenediamine tetra - acetic acid (EDTA).
Characterization of the samples was performed in order to know the internal structure, verify the effectiveness of the functionalization and confirm if the functional groups were properly adhered. Methods such as scanning electron microscopy (SEM), Raman spectroscopy, and measurements of contact angle were used.
A scanning electron Microscope JEOL JSM 5300, the beam of electrons was worked with a power of 3 to 5 keV, the electron mode was secondary and retro dispersed a stream of issuance of 75-85 μA and a working distance of 6 mm. The CNT sample was adhere to a microscope sample holder with a carbon double - sided tape. The micro - Raman spectrometer was used with a He - Ne laser (red line) having an approximately energy of 1.92 eV (632.8 nm). Samples were focused with a 50X objective, a grid of 600r / mm was used, the diameter of the irradiated area was 2 μm at room temperature and the integration time was 60 seconds.
Functionalized CNTs were molded into pills to perform analysis of wettability with the natural state and oxidized LDL to establish the contact angle. The measurements were taken at an ambient temperature of 25°C, and results were shown after one second (24 squares). The contact angle was measured using a Dataphysics OCA 15 Number series device, using a syringe with 1ml capacity to apply a drop on the surface of the CNT sample pill.
CNT doping with (Al2(SO4)3) and (H3BO3)
SEM characterization: The CNTs grown from the nickel (Ni) catalyst and doped with aluminum sulfate (Figure 2a) showed a greater productivity, with tangled growth of structures, and less presence of the catalyzer on the silica deposition base. CNTs grown from the Iron - Cobalt (Fe - Co) catalyst and doped with boric acid (Figure 2c), showed a higher productivity, growth tangled structures, a smaller diameter and less catalyzer presence.
Raman characterization: The Raman spectrum of CNTs grown from the nickel catalyst, and doped with aluminum sulfate (Figure 2b), and CNTs grown from the Iron - Cobalt catalyst and doped with boric acid (Figure 2d), showed the D band approximately at 1350 cm-1, indicating defects in the nanotube network and disorder of graphite. The G band can be observed at 1560 cm-1 is attributable to graphitic materials; therefore the ID / IG index is 0.83, greater than the ID / IG for CNTs doped with boric acid which was equivalent 0.77. Such values indicate deposition of aluminum sulfate on CNTs, and the presence of boric acid in smaller quantities. The result obtained by Hoyos shows widening of the bands caused by the amount of material defects, with shifts toward higher frequencies .
Contact angle measurement: The contact angle measurements performed on the CNT pills doped with Al2(SO4)3 (Table 1) and H3BO3 (Table 2) shows the results of the measurement taken at 9 seconds of exposure to the 0, 2 and 18 hour oxidized LDLs.
|Contact Angle Measurements|
|a) Ni–CNT-Al||b) Ni–CNT-Al||c) Ni–CNT-Al|
|Time(s)||Angle(°)||Time (s)||Angle (°)||Time(s)||Angle (°)|
Table 1: CNTs doped with Al2(SO4)3 pills to measure contact angle at different times: a) 0 hours, b) 2 hours, c) 18 hours.
|Contact Angle Measurements|
|a) Fe–CO-CNT-H3BO3||b) Fe–CO-CNT-H3BO3||c) Fe–CO-CNT-H3BO3|
|Time(s)||Angle (°)||Time (s)||Angle (°)||Time (s)||Angle (°)|
Table 2: CNTs doped with H3BO3 to measure contact angle at different times: a) 0 hours, b) 2 hours, c) 18 hours.
Less strength of adhesion and greater surface tension was observed for CNTs - Al2(SO4)3, with a contact angle of 179.8° for the two hour oxidized LDL. Material coating properties can be attributed to the CNTs - Al2(SO4)3 since it modifies the interaction with the mildly oxidized LDL; avoiding the LDL to reach a complete adherence. Lower bond strength and higher surface tension was observed for CNTs - H3BO3 when tested with the highly oxidized (18 hours) LDL with a contact angle of 134.6°. CNTs - H3BO3 it is a material that as a coating modifies the surface of the CNT, retaining the highly oxidized LDL, therefore this material should be considered as a therapeutic option by conducting previous studies of biocompatibility.
Carboxylic acid (COOH) and Glucosamine (C6H13NO5) functionalization
Characterization (SEM): The CNTs grown from cobalt (Co) catalyst and functionalized by chemical route with carboxylic acid (COOH) (Figure 3a), and CNTs grown on nickel (Ni) and functionalized by chemical route with glucosamine (C6H13NO5) (Figure 3c) presented a lower productivity, a smaller diameter, silica accumulations, and an increased of nonlinear tangled structures.
Raman characterization: Raman spectroscopy of CNTs grown from cobalt and carboxylic acid functionalized (Figure 3b), illustrate that the first band, D band, is at about 1400 cm-1, indicating disorder in the graphite layer. An increase of the G band is observed approximately at 1650 cm-1 belonging to the first order fundamental vibration, which refers to the tangential elongation. In this case the intensity ratio ID / IG is 1.07, indicating the presence of carboxylic acid on the functionalized CNTs. The results are comparable to those obtained by Pradip Kar et al. There was an increment in the ratio of intensity with the increase of functionalized CNT .
The Raman spectroscopy results of CNTs grown from nickel catalyst and functionalized by chemical route with glucosamine (Figure 3d), it can be observe that the first D band is at approximately 1350 cm-1, indicating graphite defects. The G band, located at approximately 1650 cm-1 corresponds to graphitable material. The high ratio ID / IG is 1.16 greater than the ratio ID / IG 1.07 of carboxylic acid functionalized CNTs; the introduction of glucosamine on CNTs is confirmed by band broadening.
Contact angle measurement: Contact angle measurements done with CNTs grown from nickel and functionalized with glucosamine are shown in Table 3. CNTs catalyzed with cobalt and functionalized with carboxylic acid Table 4. The results show the measurements taken at 9 seconds of exposure to the LDL at 0,2 and 18 hours of oxidation.
|Contact Angle Measurements|
|a) Ni-CNT-C6H13NO5||b) Ni-CNT-C6H13NO5||c) Ni-CNT-C6H13NO5|
|Time (s)||Angle(°)||Time (s)||Angle (°)||Time (s)||Angle (°)|
Table 3: Contact angle measurement pills for CNTs functionalized by chemical route with Ni-CNT-C6H13NO5 at a) 0 hours, b) 2 hours, c) 18 hours.
Ni - CNTs - C6H13NO5 (Table 3) show a low wettability observed with a contact angle after of 115.6° for the 2 hour oxidized LDL, which showed surface modification of functionalized CNTs by high degree of sulfation, increasing the charge density and augmenting LDL binding.
Co - CNTs - COOH (Table 4) presented lower bond strength and higher surface tension at 0 hours with a contact angle of 121.3°. The functionalized CNT with carboxylic acid acts on the native LDL without any structural modification
|Contact Angle Measurements|
|a) Co–CNT-COOH||b) Co–CNT-COOH||c) Co–CNT-COOH|
|Time (s)||Angle (°)||Time (s)||Angle (°)||Time (s)||Angle (°)|
Table 4: Contact angle measurement pills for CNTs functionalized by chemical route with Co-CNT-COOH at a) 0 hours, b) 2 hours, c) 18 hours.
The aim of this research for future work will be to perform cytotoxic and biocompatibility analysis as well as in vivo experimentation in order to assess the dynamic behavior of the functionalized CNT with the lipoproteins LDL.
The presence of aluminum sulfate and boric acid on the CTNs was confirmed by Raman spectroscopy. Evidence of higher productivity of doped CNTs with tangled structure growth and less presence of catalyzer was observed by SEM micrography. Also, for CNT functionalized by chemical route, lower productivity and presence of silica accumulation were observed.
Electronegativity molecules of the CNTs nickel - aluminum, nickel - glucosamine, cobalt - carboxylic showed a contact angle with a low wettability to the 2h oxidized LDL. Sulfate and carboxylic groups exhibit an increase in load density and load cooperativity at the union of the rich lysine - arginine peptides of LDL.
The iron - cobalt - H3BO3 and nickel - glucosamine CNTs are electronegative molecules that showed less strength of adhesion and greater surface tension at 18h oxidized LDL. Interaction of lysine - arginine residues with the altered structure ApoB 100 of LDL was also observed. These electronegative molecules possess therapeutic potential for atherosclerosis treatment in the retention of LDL.
The authors are grateful to Universidad Pontificia Bolivariana for technical and financial support to carry out this investigation.