Covalent Functionalization for Multi-walled Carbon Nanotube (f-MWCNT)-Folic Acid Bound Bioconjugate
Jacob M. Ngoy,
Sunny E. Iyuke,
Wilhelm E. Neuse
Clarence S. Yah
In the current concept, covalently functionalized multi-walled carbon nanotube (MWCNT) as a bioconjugate to folic acid (FA); an essential, bioavailable water soluble B-complex vitamin which is usually expressed on the surfaces of most tumoral cells was used. This was rendered possible through the design of a bioreversible binding water-soluble and biocompatible functionalized multi-walled carbon nanotubes (f-MWCNTs). The MWCNT was synthesized through the Chemical Vapor Deposition (CVD). The MWCNTs were covalently functionalized with sulphuric and nitric acids (3:1) at Room Temperature (RT), 50 and 100°C to generate the phenol and carboxyl groups. Furthermore, aspartic acid at 230°C was used to generate the carboxyl f-MWCNTs groups. The CNTs and f-MWCNTs were both characterized with the aid of a Transmission Electron Microscopy (TEM). The results showed a decreased in mol ratio (COOH/OH) of the f-CNTs from 80 to 20 nm as the temperature increases from RT to 100°C. The f-CNTs carboxyl were attached to 3-(N, N-dimethylamino) propylamine (DMP) and FA through 2-(1H-benzotrial-1-yl)-1, 1,3,3-tetramethylurium hexafluorophosphate (HBTU) to generate f-CNTs-FA conjugates. The results of the high resolution nuclear magnetic resonance (H1NMR) and infrared (IR) spectra showed CONH peak shifts bond bioreversible conjugation of FA at 94 and 101.3% and sizes 50 and 170 nm, respectively. The f-MWNT-FA moieties thereby have a greater versatility and can be used for the treatment and restoration of neoplasma cells.
March 04, 2011; Accepted: May 17, 2011;
Published: July 02, 2011
The search for new and effective drug delivery systems is a fundamental principle
to design and improve pharmacological and therapeutic profile of drug molecules
(Allen and Cullis, 2004; Fekri et
al., 2010; Alkhatib et al., 2010; Singh
et al., 2011; Warisnoicharoen et al.,
2011). Furthermore, a wide variety of drug delivery systems are currently
in existence (Pouton and Seymour, 2000). Current reports
by Ke et al. (2007) showed polymer-functionalization
CNTs as another important polymeric carbon nanocomposites delivery system. This
covalent technique allows the combination of different polymers with the CNTs
to create new compound classes with wide range of properties (Semwal
et al., 2010). To achieve this, it is therefore essential to functionalize
the CNTs with ideal polymers. Furthermore f-CNT can ease their penetration into
the cells, thus offering f-CNT as vehicles for drug delivery (Pantarotto
et al., 2004; Kam et al., 2004). In
fact f-CNT can carry one or more therapeutic agents with recognition capacity,
optical signal imaging and/or specific targeting, to treat diseases (Ferrari,
2005). For this purpose a new strategy for the multiple functionalization
of CNT with different types of molecules (Wu et al.,
2005) has been developed to ease disease treatment.
Pristine carbon nanotubes have high hydrophobic thus making them insoluble
in aqueous solutions. Apart from that they very conducive for the construction
of various electrochromic devices, depending on the specific applications in
need (Zambri et al., 2011).
The problem of insolubility or hydrophobicity can be achieved by functionalization
which is the attachment of functional groups on the surfaces of CNTs. For biomedical
applications, surface chemistry or functionalization is required to solubilize
CNTs, thereby rendering them biocompatible and low toxicity. Functionalization
generates functional groups at the surfaces of CNTs that react with other chemicals,
prepolymers and polymers, thus giving rise to a homogeneous dispersion or solubilization
of CNTs. Chemical reactions perform bonds on nanotube sidewalls resulting into
covalency properties. These covalencies can be created by chemical oxidation
with oxidizing agents such as nitric acid, sulfuric acid and potassium permanganate
(Niyogi et al., 2002; Rosca
et al., 2005; Zeng et al., 2008).
Others include 1, 3-dipolar cycoaddition with methine yides in the presence
of N, N-dimethyl formamide. During the process of functionalization, carboxyl
groups are formed at the ends and sidewalls of tubes. This type of functionalization
generates mainly the OH and COOH functional groups. Further attachments
can be made on the functional groups, thus increasing their solubility in organic
solvents (Xie et al., 2002). The carboxylic acid
groups often connect the CNTs with the amino-terminated sites present on the
biomolecules. During covalent modification, the carboxylic acids are usually
activated by thionyl or oxalyl chloride, carbodiimides or active esters, to
obtain highly reactive intermediate groups capable of linking the biomolecules
and CNTs in the presence of 2-(1H-benzotrial-1-yl)-1,1,3,3-tetramethylurium
hexafluorophosphate (HBTU). The covalent attachment assures the hydrophilic
properties of CNT.
Further, other anticancer therapies are well-known for their potency to cure
autoimmune disease (Wong, 2005). However, most do suffer
from low bioavailability and toxic side effects (Pignatello
et al., 2004). Therefore, an increased bioavailable targeted delivery
agent is highly desirable. However, most drugs require high dose concentrations
due to their low cellular uptake (Pignatello et al.,
2001). With functionalization bioavailable low dose concentration, low toxicity
drug targeting agents are possible (Pastorin et al.,
2006). Preliminary findings have shown that Methotrexate (MTX) an anti-cancer
drug when conjugated to CNT, its potency increases by 10 times in cell culture
(Prato et al., 2008). The problem faced is the
formation of an amide bond between the MTX and the CNTs reducing its efficacy.
The problem however, can be overcome by the introduction of a cleavage linker
or an enzymatically sensitive bond as demonstrated in dendrimers conjugates
(Quintana et al., 2002). The application of FA-
CNT conjugates as targeting moiety may render more efficacious translocation
to cancerous cells.
Folic acid conjugated CNTs on the other hand have been found necessary to operate
as receptor for targeting moiety during the uptake. Passive targeting approaches
are limited in their scope and thus, tremendous effort has been directed towards
the development of active approaches for drug targeting. Active targeting employs
specific modification of drug/drug-carrier nanosystems with active agents having
selected affinity for recognizing and interacting with the specific cell, tissue
or organ question (Vasir et al., 2005). Direct
coupling of drug to targeting ligand, restricts the coupling capacity to a few
drug molecules. In contrast, coupling of drug carrier nanosystems to ligands
allows import of thousands of drug molecules by means of one receptor targeted
ligand. Therefore, the current research was aimed at coupling MWCNT with folic
acid which can be used as a biocompatible molecule in the improvement of cancer
MATERIALS AND METHODS
Chemicals: The hydroxyamines and diamines were of analytical grade obtained from Adrich Chemie, Fluka AG, South Africa. Other solvents and reagents obtained from Adrich Chemie, Fluka AG, South Africa include: D,L aspartic acid, phosphoric acid, 3-Dimethylamino-1-Propylamide (DMP), Diethylenetriamine (DET), 2-2- (Ethylenedioxy)-Diethylamine (EDDA), 1,3 Diaminopropane (PDA), Dicyclohexylcarbodiimide (DCC), 2-(1H-Benzotriazol-1-yl)-1,1,3,3-Tetramethyluronium Fluorophosphates (HBTU), Triethylamine (TEA), ferrocene, folic acid, methotrexate, sodium hydroxide, Calcium chloride, chloridric acid, folic acid glacial acetic acid, ammonium hydroxide, sulphuric acid and nitric acid. Distilled water was used for all preparative work. The reaction solvent, N,N-Dimethylformamide (DMF), was distilled under reduced pressure with a fore-runs of around 10% being discarded and was dried over molecular sieves 4 D. All other solvents, Diethyl ether (Et2O), hexane, acetone and toluene were of laboratory grade, received from Adrich Chemie, Fluka AG, South Africa. The acetylene, argon and nitrogen gases were also of analytical grade obtained from Afrox South Africa for the CNT production.
The production multi-walled carbon nanotube (MWCNTs): The CNTs were
synthesized by the Chemical Catalytic Vapour Deposition (CCVD) which is made
up of a vertical silica plug flow reactor inside a furnace. It is a modification
of the chemical vapour deposition (Anreddy et al.,
2010). The furnace is connected to a swirled coiled mixer with a supply
of gas system of valves and rotameters. The upper part of the reactor is connected
to a condenser which leads to two delivery cyclones where the CNTs are collected.
The system is also connected to a temperature regulator and a pressure controller.
The valves control the flow of gases into the reactor. The CNTs were produced
according to Yah et al. (2011) and viewed using
the JEOL JEM-100S Transmission Electron Microscopy (TEM).
Functionalization of CNTs with sulphuric acid and nitric acid: The pristine MWCNTs were treated with hydrochloric acid, to remove impurities. One gram of MWCNTs was placed in a 500 mL round-bottom flask and 200 mL of HCL (30%) was added. The mixture was stirred using magnetic stirrer for 2 h, then diluted in water, filtered washed with deionised water and then dried overnight in a vacuum at 40°C to remove the iron impurities.
To functionalize the purify CNTs, 0.1 g of the purified MWCNTs were dispersed in 200 mL of acid (mixture of sulphuric acid 95% and nitric acid 95% of ratio 3:1) in a 500 mL round bottom flask equipped with a condenser. The dispersion was kept differently for 4 h at 100°C, 24 h at 50°C and 96 h at Room Temperature (RT). The resulting dispersion was diluted in water to neutral pH, filtered and the sample was dried in a vacuum at 40°C overnight. The modified CNTs (termed f-MWCNTS) were quantitatively analyzed by titration to determine the carboxylic acid (COOH) concentrations on the surface of treated CNTs. The f-MWCNTs were added into a 25 mL 0.04 M NaOH solution and stirred for 48 h to allow the solid CNT material to equilibrate with the NaOH solution. The mixture was then titrated with a 0.04 M HCl solution to determine the excess NaOH in the solution and the concentration of the carboxylates on CNTs.
Functionalization of CNTs with Aspartic acid: Approximately 0.15 g of MWCNTs were dispersed in a mixture of 3 g of Aspartic acid and 15 mL of N, N-Dimethyl Formamide (DMF) in a round-bottom flask of 250 mL equipped with a condenser and the reaction was controlled to 220°C for 6 h. After filtration, the solid was washed with water to neutral pH. The yield obtained was 183 mg.
Using titration formula:
where, Y = mmol of COOH on the surface of CNTs, X = The volume of HCl in mL.
The 1H NMR (29) analyses with D2O +NaOH showed CH2-COOH pick at chemical shift (3-2.3) expected. The TEM analysis were done according to the microscopic image obtained in Fig. 4 with nanotubes structure at d0 = 60 nm.
Functionalized carbon nanotubes (f-CNTs)-folic acid conjugates
A: f-CNTs (H2SO4+HNO3 at 100°C)
bound folic acid: Approximately 100 mg of f-CNTs (prepared at 100°C)
representing 0.38 mmol COOH was dispersed in 5 mL of DMF and 11.6 mg (0.114
mmol) of 3-Dimethylamino-1-Propylamine (DMP) representing 30% of molecular rate
was dissolved in 4 mL of DMF. After nitrogen flashing, the dispersion and the
solution were mixed together in a round bottom flask, stirred at 50°C. While
solution was still stirring, 75 mg (0.32 mmol) of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
fluorophosphates (HBTU was added and the pH was controlled to 7 using the triethylamine
(TEA) dropwise. After 24 h of reaction, a solution of 141 mg (0.32 mmol) 20%
excess of folic acid in 10 mL of DMF was prepared for 1 h and was added in the
main solution. Stirring was maintained for 6 h while the pH was kept to 7 with
TEA. The solid was collected by filtration and washed several times with Tetramethylurea
(TMU) to remove the excess FA and acetone. The solid was dried and kept in the
B: f-CNTs (Aspartic acid) bound folic acid (f-CNTs-DMP (40)-FA (60)): Also 100 mg of f-CNTs representing 40 mg (0.88 mmol) of CNT-COOH was dispersed in 5 mL of DMF and 36 mg (0.352 mmol) of 3-dimethylamino-1-propylamine (DMP) (40%) was dissolved in 4 mL of DMF. After nitrogen flashing, the dispersion and the solution were mixed together in a round-bottom flask, stirred at 50°C. During stirring, 206 mg (0.88 mmol) of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium fluorophosphates (HBTU) predissolved in 3 mL DMF was added dropwise and the pH was neutralised to 7 using the Triethylamine (TEA). After 2 h of reaction, a solution of 303 mg (0.7 mmol) of FA in 15 mL of DMF was prepared for 1 h and was added into the main solution. Stirring continued for 6 h while the pH was kept at 7 with TEA. The solid was washed several time with Tetramethylurea (TMU) and filtered to remove the excess FA and acetone. The solid was dried and kept in the desiccators.
The 1H NMR spectra and the Solid-state Infrared (IR) analysis:
The 1H NMR spectra were obtained at 300 and 400 MHz in DO2
solution. The chemical shifts, δ in ppm were referenced against sodium
3-(trimethylsily)-2, 2, 3, 3-d4 propionate. The pH values of the
sample in DO2 solution were adjusted to 10-11 by adding sodium hydroxide
in order to eliminate potential protonation effect. The Solid-state Infrared
(IR) spectra were recorded on KBr pellets over the region of 4000-6000 cm-1.
The attention was turned only on the significant bands. Samples for analysis
were dried using the Abderhalden apparatus and calcium chloride was done on
a HITACHI 2000 spectrometer, at a scan speed of 400 nm min-1.
RESULTS AND DISCUSSION
Synthesis of carbon nanotubes: The analysis of the Chemical Vapor Deposition
(CVD) product by TEM showed MWCNTs with outer diameter (do) of 20
nm and an inner diameter (di) of 10 nm as shown in Fig.
1. The MWCNTs were similar to those earlier produced and reported by Iyuke
et al. (2009) and Mohamed and Kou, 2011 where
the optimal temperature for production was found at 9000C with similar
diameter. The results were dissimilar from those produces by Mamba
et al. (2010) where they found MWCNTs as straight bundles of uniform
lengths, each bundles consisting of numerous closely packed MWCNTs ropes with
no indication of surface modifications.
The CNTs found could have a variety of applications because of their novel
structure, especially the small size and remarkable mechanical, thermal and
electrical properties (Zambri et al., 2011).
Small-diameter MWCNTs have ability to display enhanced reactivity relative to
larger-diameter nanotubes, due to increased curvature strain. However, because
of their novel structure of elemental carbon, CNTs are insoluble in all organic
solvents (Kostarelos, 2003).
Covalent functionalization of carbon nanotubes
Functionalization of CNTs with concentrated nitric acid and sulfuric
acid: (a) The MWCNTs were functionalized with H2SO4/HNO3
in mol ratio of 3:1 at 100, 50°C and Room Temperature, respectively
(RT). The TEM analysis of the f-MWCNTs showed variation at 100°C with outer
diameter (d0) = 20 nm and the inner diameter (din) = 12
nm (Fig. 2) while at 50°C do= 30 nm (Fig.
3) and at Room Temperature (RT) d0 = 80 nm and din=
40 (Fig. 4). In order to evaluate the variation of the size
of f-CNTs in terms of temperature, a graph of diameter versus temperature was
plotted as shown in Fig. 5. The water solubility of f-CNTs
was decreasing from 100°C to RT especially from pH = 9 and above.
The analysis of 1H NMR Spectra 1, 2, 3 done for f-CNTs obtained
at 100°C, 50°C, RT respectively showed peaks A and B in chemical shift
δ/ppm (1.5-2.5) for CH2COOH and (2.7-3.7) for CH2OH.
In order to calculate the quantity of COOH and OH, the integration of peak A
(CH2COOH) corresponding to 1 mol and was used as reference
to evaluate the peak B (CH2OH). After calculation, the following
results were obtained:
|1H NMR Spectra 3:
||f-CNTs: MWCNTs in H2SO4/HNO3 at
|| MWCNTS microscopic image (TEM)
|| f-CNTs (H2SO4/HNO3 at 100°C) microscopic image
|| f-CNTs (H2SO4/HNO3 at 50°C)
|| f-CNTs (H2SO4/HNO3 at RT)
|| Size of CNTs versus temperature
|H NMR Spectra 1:
||f-CNTs: MWCNTs in H2SO4 / HNO3
|1H NMR Spectra 2:
||f-CNTs: MWCNTs in H2SO4/HNO3
||The integration of peaks A and B in 1H NMR Spectra
1 for f-CNTs (100°C) shows after evaluation that 1 mol of COOH adsorbed
on the surface of MWCNTs, corresponded to 7.54 moles of OH
||The integration of peaks A and B in 1H NMR Spectra 2 for f-CNTs
(50°C) shows after evaluation showed 1mol of COOH absorbed on the surface
of MWCNTs, corresponded to 2.27 mol of OH
||The integration of peaks A and B in 1H NMR Spectra 3 for f-CNTs
(RT) after evaluation showed 1 mol of COOH absorbed on the surface of MWCNTs,
corresponded to 0.08 mol of OH
A sample of 200mg of f-CNTs was used for titration to evaluate the quantity of COOH in mmol incorporated on the surface of f-CNTs which through the mol ratio COOH/OH was calculated from different 1H NMR spectra. The integration was used to calculate the quantity of OH in mmol incorporated on the surface of f-CNTs. The experimental data from titration was used through the formula Y = 1-0.04 X to complete Table 1. The results in Table 1 were used to plot fraction of incorporation of COOH and OH versus the temperature as shown in Fig. 7 and the mol ratio COOH/OH versus the temperature as shown in the Fig. 8.
Different peaks including A, B, C, D and E were shown in the infrared (IR)
investigation as presented in Fig. 6.
||A:O-H stretch (3331.44 cm and 3334.86 cm) intermolecular hydrogen
bonded B:C-H stretch; (2892.90 and 2902.20 cm)
||C:C = O, stretch; (1693.04 and 1716.56 cm)
||D:C-O (1158.92-1028.01 cm-1) and (1156.39-1.1030.67 cm-1),
||E: Ring C = C, bend (663.67-574.56 cm-1), this confirm the
CNTs the graphene structure with sp2-bond carbon
|| Infrared Peaks of f-CNTs (H2SO4/HNO3)
||Fraction of COOH and OH group incorporation versus temperature
The results in Fig. 5 are elaborated from the TEM analyses
in Fig. 2-4 which showed increase in size
of nanotubes which were relative to decrease in temperature. This might have
been due to self-diffusion of molecules inside of CNTs thereby allowing the
CNTs to form a well-ordered nanoporous membrane (Casavant
et al., 2003; Hinds et al., 2004)
which can be incorporated in a macroscopic structure (Srivastava
et al., 2004) for separation devices. The study of self-diffusivity
can explain the non parabolic influence of nanotube sizes and temperature. Jobic
and co-workers had examined this effect in more detail for diffusion (Jobic
et al., 2009) using molecular dynamics simulations.
|| Mol rate COOH/OH versus temperature
||Fraction incorporation of COOH and OH on the surface of CNTs
with variable temperature
|2nH: Number of Hydrogen atom, 3X: The
volume of HCl in mL, 4Y: The quantity of COOH in mmol incorporated
on the surface of f-CNTs
They termed this behaviour the Levitation effect. If the guest
molecule fits perfectly in the window of the zeolite, the molecule appears to
be floating; increasing or decreasing the diameter of the guest molecules as
well as reducing the diffusion coefficient. This effect is temperature dependent
(Derouane et al., 1988). A similar effect has
been observed by Cannon and Hess (2010) for carbon nanotubes.
With regard to Fig. 5, the self-diffusivity was subsequently increasing with decreasing of temperature. Since the self-diffusion explains the incorporation of guest molecule in CNTs through nanoporous membrane, the diameter of CNTs has to increase in the tendency where the self-diffusion will increase. According to the objective of the study, attention was given to the trend where the surface adsorption (surface phenomenon) was possible, rather than where absorption was evident. The increase temperature which led to decrease in diameter, resulted in higher collision frequencies and faster motion. This enhances the particle probability to bond folic acid. The f-MWCNTs at 100°C were selected, due to their small size and the adsorption capabilities.
The 1H NMR Spectra 1, 2 and 3 were obtained with D2O
as solvent. Therefore COOH and OH groups directly attached on the surface of
CNTs were shifted in δ/ppm (4.5-5.5) for D2O peaks.
||f-CNTs (H2SO4/HNO3) structure
|| f-CNTs (aspartic acid) structure
The 1H NMR analysis for all f-CNTs made at different temperatures
(100, 50°C and RT) shown the presence of CH2COOH and CH2OH
due to the fact that amorphous carbon was removed purification by HCL (Monthioux
et al., 2010).
In addition, the infrared analysis from Fig. 6 and the 1H
NMR Spectra 1, 2 and 3 have confirmed that f-CNTs were produced and the entire
MWNTs was covered with CH2OH, CH2COOH, OH and COOH as
shown in Scheme 1.
In comparison with the result from Fig. 7 and 8,
it was found that both fractions of OH and COOH incorporation in f-CNTs were
increasing with increasing temperature. At room temperature, the fraction of
OH group incorporation was much neglected and the mol ratio COOH/OH was 32.4,
these were rapidly reduced to 1.1 at 50°C and sharply down to 0.35 at 100°C.
This shows that when working at room temperature or below there is possibility
of getting f-CNTs covered only with COOH. It was found that f-CNTs at 100°C
were more soluble than at RT and 50°C due to high presence of OH groups
on the CNTs surfaces. Thus, the abundance of OH and COOH groups on the surface
will facilitate more covalent attachment to polymers or drugs according to their
different electronegativities. This granted the f-CNTs made at 100°C to
be more useful than those made at low temperatures. The scope of this reaction
was very broad and produces f-CNTs that possess high solubility in a wide range
of different solvents. This was preferentially selected for the next phase of
the drug binding to carbon nanotubes.
Where Series 1: Fraction incorporation of carboxyl group (COOH) on the surface of CNTs and Series 2: Fraction incorporation of phenol group (OH) on the surface of CNTs.
Functionalization of CNTs with Aspartic acid: The functionalized carbon
nanotubes (f-CNTs) were also obtained by heating MWNTs with aspartic acid an
amino acid in DMF at 220°C. This resulted to the structure as shown in Scheme
3. The results of f-CNTs by aspartic are shown in Fig. 9
where 183 mg of f-CNTs by aspartic acid were soluble in water at pH of 10.
The 1H NMR Spectra 4 showed peaks A in chemical shift (δ/ppm)
3.050-2.200 for CH2COOH and B in chemical shift (δ/ppm)
4.40-4.70 for = N-CH CH2 (COOH)2 as expected. The different
peaks was obtained from infrared (IR) investigation (A, B, C, D, E, F and G)
are shown in Fig. 10.
||A: N-H stretch, at 3331.44 cm-1; B: C-H stretch
in the region 3015.95-2902.20cm-1 C: Overtone region usually
contains a prominent band in the region 2337.23-2119.11cm-1 D:
C = O bond stretch, at 1738.63 cm-1; E: C-N stretch, at 1436.30
cm-1 F: C-O (acetate) stretch, in the region 1228.93-1216.70
cm-1; G: O-H out of plane bend, 1011.82-912.05 cm-1
Using the titration formula Y = 1-0.04 X (Y: quantity of COOH in mmol incorporated
on the surface of f-CNTs and X: volume of HCl in mL) when working with 200 mg
of f-CNTs 0.88 mmol of COOH corresponding to 20% of COOH was incorporated on
the surface of CNTs after neutralization with excess NaOH and 3 mL of HCl. The
carboxyl group incorporated in the f-CNTs was intended to be bound with the
solubilizing group and the drug. The quantity of the solubilizing group and
drug were evaluated with 20% of fraction of COOH-f-CNTs at pH of 10. The TEM
analysis showed f-CNTs-aspartic acid diameter at 60 nm which was larger than
the diameter obtained for MWNTs in sulfuric and nitric acids at 100 and 50°C
and to a lesser extend at RT. According to the findings from Fig.
5, the diameter was decreasing with increase in temperature which was similar
to those earlier reported by Hunger et al. (2002),
Jakobtorweihen et al. (2006). The investigation
of peaks in 1H NMR Spectra 4 and infrared (IR) as shown in Fig.
10 which confirm the structure elaborated in Scheme 2.
|| f-CNTs (MWNTs +Aspartic acid in DMF at 220°C)
|| IR f-CNTs (aspartic acid)
Overall, the use of strong acid and amino acid to functionalize the carbon
nanotubes generates carboxylic groups (Scheme 1, 2)
which increase their dispersibility in aqueous solutions (Tasis
et al., 2006; Kim et al., 2007). Solubility
under physiological conditions is a key prerequisite to make CNT biocompatible
which can be linked to a wide variety of active molecules.
Functionalized carbon nanotubes (f-CNTs) bound Folic acid conjugates:
The folic acid was functionalized with the MWCNTs by the aid of 3-dimethylamino-1-propylamine
(DMP) as a strong base. This gave the amino group a positive charge enabling
the FA to bind. This was similar to those reported by Zeng
et al. (2006) were they prepared functionalized CNTs with biodegradable
poly (e-caprolactone) (PCL). They found that the presence of CNTs did not affect
the biodegradability of PCL. From HNMR results it was found that FA was attached
to surface of the MWCNTs. Furthermore functionalized CNT-PCL polymer matrix
could display enormous potential as a structural support material in tissue
f-CNTs (H2SO4+HNO3 at 100°C) bound
folic acid conjugates: The estimation of the percentage of -COOH in f-CNTs
was proved by the titration. The f-CNTs was achieved by the reaction between
COOH and NH2 via the coupling agent 0-benzotiazol-1-yl-tetramethylurorium
hexafluorophosphate (HBTU). Thus, the coupling led to the yield of 163 mg f-CNT-FA
similar to the chemical structure presented in Scheme 3. The
TEM analysis of f-CNTs (H2SO4+HNO3 at 100°C)
bound folic acid conjugates are shown in Fig. 11 with d0
= 50 nm and din = 20 nm and soluble at pH 6-7. The 1H
NMR Spectra 5 were similar to peaks shift shown in Scheme 3
as illustrated in Table 2.
With the size of 50 nm as outer diameter and 20 nm as inner diameter the conjugate
can be easily tracked into the cytoplasm and the nucleus. With regard to the
1H NMR Spectra 5, the fraction of incorporation of folic acid was
calculated by using the integration of A that is 1 and the sum of integration
of aromatic group E, F and G which is 1.64. With 30% of DMP and 70% of folic
acid, the mol ratio was 3/7 and the number of H expected was 20 H for A and
35H for the sum of E, F and G as it is shown in the structure (Scheme
3). Referring to A as 20H calculated with integration 1, the sum of E, F
and G with integration of 1.64 gave 32.8H calculated which corresponded to 94%
f-CNTs (Aspartic acid) bound folic acid conjugates: The estimation of
the percentage of COOH in f-CNTs was also evaluated by the titration which
was achieved by the reaction between COOH and NH2 via the coupling
agent 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium fluorophosphates (HBTU).
||f-CNTs (H2SO4/HNO3)-DMP (30)-FA
||f-CNTs (Aspartic acid)-DMP (40)-FA (60) Structure
|| f-CNTs (H2SO4/HNO3)-FA
|| HNMR spectra of f-CNTs (H2SO4/HNO3)-FA
|1Y is the quantity of COOH in mmol incorporated
on the surface of f-CNTs and X is the volume of HCl in mL
|| HNMR data f-CNTs (aspartic acid)-DMP (40)-FA (60)
|1Y is the quantity of COOH in mmol incorporated
on the surface of f-CNTs and X is the volume of HCl in mL
With the objective to raise the water solubility of f-CNTs, 40% of COOH
was evaluated using attached DMP as the solubilizing group. The folic acid was
used in excess with the objective to complete all the 60% of -COOH that did
not react with DMP. The result f-CNTs (Aspartic acid) bound folic acid conjugate
yielded 231 mg and the chemical structure is presented in Scheme
4. The TEM analysis is shown in Fig. 12 having diameter
of 170 nm. The product was soluble at pH of 6-7.
The 1H NMR Spectra 6 showed all expected chemical bands as illustrated
in Table 3. This therefore, confirms the reversible attachment
between f-CNTs with DMP and folic acid via amid bond formation. To calculate
the fraction incorporation of folic acid, the integration of peak A evaluated
to 1 was chosen as reference and corresponded to 4 H as expected and the integration
of aromatic peaks (E, F, G) evaluated to 3.8 was calculated 15.2 H while the
expected was 15 H as shown in Scheme 4 and then the fraction
incorporation of folic acid was 101.3%. The error was 1.3%.
The f-CNTs-drug conjugate from aspartic acid (Fig. 12) showed
the dark spot spread along the nanotubes while Fig. 11 related
to the f-CNTs of conjugate made from H2SO4/HNO3
at 100°C also dark spot at the end cap.
||f-CNTs (Aspartic acid)-DMP (40)-FA (60) microscopic image
|1H NMR Spectra 4:
||f-CNTs: MWCNTs+Aspartic acid in DMF at 230°C.
|1H NMR Spectra 5:
||f-CNTs (MWCNTs+H2 SO4+HNO3)-DMP
(30)-FA (70) 94% FA incorporation
The incorporation of COOH (17.1%) in f-CNTs with H2SO4/HNO3
at 100°C (Table 1) was lesser than the one (20%)
in f-CNTs with aspartic acid.
|1H NMR Spectra 6:
||f-CNTs (MWCNTs+Aspartic Acid)-DMP(40)-FA(60) Incorporation
The carbon nanotubes, due to its small size were synthesised as drug carrier target. The covalent functionalization was developed on the surface of MWCNT. The carboxyl groups reacted with HBTU as coupling agent DMP amines in one part and folic acid in other. The formation of CONH as bioreversible bond between the f-CNTs and folic acid was determined by 1H NMR and IR. The soluble f-MWCNTs TEM structures were conserved within the diameter range of 50 to 170 nm. The phenol (OH) and carboxyl (COOH) groups were attached on the surface of CNT making the rate COOH/OH to decrease with increasing temperature. The f-CNT covered surface with OH and COOH was more water soluble than the f-CNT covered COOH.
The authors acknowledge the financial support from the National Research Foundation (NRF) under the NRF Focus Area: NRF Nanotechnology flagship programme, DST/NRF Centre of Excellence. The student bursaries provided by the Wits University are much appreciated.
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