Facile Method to Prepare pH-Sensitive PEI-Functionalized Carbon Nanotubes as Rationally Designed Vehicles for Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Delivery

: A new pH-sensitive system designed for drug-delivery purposes and based on functionalized multiwall magnetic carbon nanotubes (Mag-CNTs) was synthesized for the effective incorporation of non-steroidal anti-inflammatory drugs (NSAIDs), aiming at drug release in characteristic acidic conditions close to the actual conditions of inflamed tissues. Cationic hyperbranched polyethyleneimine (PEI) was immobilized on the surface of Mag-CNTs via electrostatic interactions between the positively charged protonated amines within the polymer and the carboxyl groups on the chemically oxidized Mag-CNT surface. The addition of the NSAID with a carboxylate donor, Naproxen (NAP), was achieved by indirect coupling through the amino groups of the intermediate linker PEI. FT-IR, Raman, and UV–vis spectroscopy were employed to fully characterize the synthesized nanocarrier and its functionalization procedure. The interaction of the designed nanocarrier with bovine serum albumin (BSA) was studied in vitro by fluorescence emission spectroscopy while its in vitro interaction with calf-thymus (CT) DNA was monitored by UV–vis spectroscopy and viscosity measurements and via competitive studies with ethidium bromide. The calculated binding constants were compared to those of free NAP revealing a higher binding affinity for BSA and CT DNA. Finally, drug-release studies were performed, revealing that the electrostatic linkage ensures an effective release of the drug in the acidic pH typical of inflamed cells, while maintaining the multiwall nanotubes (MWNTs)–drug conjugates stable at the typical bloodstream.


Introduction
A fascinating category of nanomaterials possessing unique physical, chemical, and physiological properties is carbon nanotubes (CNTs) [1][2][3][4][5][6].Their chemical modification has attracted increasing attention over recent years in order to improve their solubility and compatibility, tailor their structures and properties, and open a whole new world of novel CNT-based nanostructures and 2 of 18 nanomaterials [7][8][9][10][11].In a parallel manner, the use of nanotechnology in the field of pharmaceutics and drug-delivery has remarkably grown over the last few years, giving "birth" to a new term developed on the basis of this technology-nanopharmaceuticals.As a matter of fact, CNTs possess many intriguing features that make them attractive drug-delivery carriers such as: (a) An enhanced permeability and retention (EPR) effect.Nanocarriers such as nanoparticles (NPs), liposomes, and CNTs, experience a higher accumulation in tumor tissues as compared to normal tissues because of the poorly formed blood and lymphatic vessels that supply rapidly proliferating tumors [12].The EPR effect empowers CNTs to transport chemotherapeutic agents preferentially to tumor sites [13].(b) Trans-membrane penetration and intracellular accumulation through the needle-like shape of CNTs via the "nanoneedle" mechanism [14] and/or energy-dependent endocytic pathways [15].(c) Extraordinary ability for drug-loading and efficient attachment onto the surface or within the interior core of CNTs [16][17][18].The functionalization of CNTs involves three different approaches: (i) the attachment [19,20] of molecules through covalent bonding; (ii) the functionalization of molecules on the graphitic surface by an adsorption mechanism [21]; (iii) the encapsulation of molecules in the empty cavity of the nanotubes.
Considering the non-covalent functionalization of CNTs, molecules containing aromatic groups are adsorbed on the conjugated surface of the nanotubes mainly through π-π interactions [22].CNTs are excellent candidates for the role of carriers mainly because their inner space can be filled with nanoparticles or molecules while the graphitic wall can be further functionalized in order to enrich biocompatibility, to succeed biodegradation [23], and to convey novel functions for targeting [24] or imaging purposes [25][26][27].In comparison with the non-covalent functionalization of CNTs, the covalent one is clearly more accurate, better controllable and robust.The conjugates of non-covalent functionalized CNTs are easily dissociated in biological fluids, creating more concerns about potential hazardous effects.One of the main reasons for this is the undesired exchange of the detached molecules with serum proteins after administration.Nevertheless, it seems that, for certain cases, the non-covalent functionalization of CNTs is more advisable since the experimental conditions are ideal to maintain the physical properties (structural, electrical and optical) of the CNTs as well as the properties of the adsorbed molecules.On the other hand, the covalent grafting of various substances on the graphitic sidewalls dramatically changes the conjugated π-electron framework of CNTs mainly due to the induced rehybridization (from sp 2 to sp 3 ) of the derivatized carbon atoms accompanied by a drastic change of their optical-electrical properties.
In special cases, CNTs can also reveal fascinating magnetic properties (Mag-CNTs) which derive from the metal catalyst impurities entrapped at the CNT extremities during their manufacture.These Mag-CNTs can be used as magnetic resonance imaging (MRI) contrast agents even with low metal impurities (>2% iron, nickel), having a significant effect on the observed 1 H transverse (1/T 2 ) relaxation rate of water [28].Mag-CNTs can offer a plethora of capabilities in clinical uses such as: (a) contrast agents in MRI; (b) drug carriers effectively surpassing certain limitations that drugs undergo mainly because of the decreasing efficacy and rather poor cytotoxicity and/or targeting [29].
The main objective of designing "smart" Mag-CNTs drug-carriers is the selective targeting of specific biological entities by avoiding "barriers" inside the body.These in vivo barriers: (a) shield the body against external species intrusion; (b) limit the movement of Mag-CNTs; (c) enter severe changes to the surface and their magnetic properties; (d) result in an early uptake by cells after biochemical signaling induces a negative host response before the Mag-CNTs reach the target tissue [30,31].A representative example is the blood-driven agglomeration of Mag-CNTs when entering the body through intravascular administration.Until recently, intermediate biocompatible coatings using polyethyleneglycol (PEG) co-polymers, polyethylene imine (PEI) molecules, dextranes, liposomes, etc. have been studied in depth regarding case of the indirect drug addition on as-prepared Mag-CNTs [29].
Non-steroidal anti-inflammatory drugs (NSAIDs) are perhaps the most prescribed drug groups [32][33][34][35][36] widely used in drastic inflammatory conditions and chronic inflammatory diseases.Despite their wide usage, it has been shown that the long-term use of NSAIDs is accompanied by a high risk of gastrointestinal bleeding causing a significant reduction in current therapies mainly C 2020, 6, 62 3 of 18 because drugs must frequently be administered at suboptimal doses to reduce cytotoxicity.A plethora of strategies have been employed over the last 50 years for the development of NSAIDs that spare the gastrointestinal tract [32][33][34][35][36] without any promising results.A possible answer to this problem is the use of nanoplatforms as controlled release systems, developed the last decade due to advances made in nanoscience and nanotechnology.The main advantages of these systems are the following: (a) they maintain the drug concentration in the body releasing only in the injured cells; (b) increase the overall therapeutic efficiency of the drug and (c) all physical properties such as size, porosity, geometry and surface functionalization can be controlled at the nanoscale level.
Herein, we present the effective attachment of Naproxen (NAP), a carboxylate NSAID, commonly used for the treatment of inflammations and pain of various wounds on the surface of PEI-functionalized carboxylated Mag-CNTs.A facile method is employed for the preparation of the Mag-CNTs@PEI@NAP carriers based on the indirect coupling of NAP through the amino groups of the intermediate linker PEI.The interaction of the produced nanocarrier with bovine serum albumin (BSA) was studied in vitro along with its interaction to bind to calf-thymus (CT) DNA.The calculated binding constants revealed a higher binding affinity for BSA and CT DNA than free NAP.Finally, drug-release studies were performed, revealing an effective release of the drug in acidic conditions.

Shortening and Functionalization of Mag-CNTs (NL002)
Mag-CNTs were shortened employing an ultrasonic processor (24 kHz, 400 W, UP400S Hielscher).A total of 3.0 g of Mag-CNTs was suspended in 400 mL 3DH 2 O employing ultrasonication in full power and a time duration close to 4 h.The sample was filtered under vacuum using a 0.47 µm nylon membrane filter (Isopore Millipore) and dried in the oven for 12 h at 110 • C. Scanning electron microscopy (SEM) of the Mag-CNTs revealed a direct relation of the ultrasonication time and the final length of the carbon material.Ultrasonication in full power for 4 h showed a length range between 500 and 700 nm, while for 2 h the length range was between 1.5 and 2.0 µm.
For the carboxylated Mag-CNTs, 2.0 g of Mag-CNTs was dispersed in 100 mL HNO 3 65% employing an ultrasonic processor for 10 min, 180 W and the solution was stirred on a hot plate (mechanically) at 90 • C for 3.5 h.Afterwards, the heating was switched off and the solution was continuously stirred overnight.The sample was filtered under vacuum using a 0.47 µm nylon membrane filter under 3DH 2 O, reflux up to a pH between 7 and 8 and dried in the oven for 12 h at 110 • C.
For the deprotonation of the carboxylic groups, a two-step procedure was carried out.Firstly, the sample was dispersed in 80 mL of an aqueous solution NaOH 10% w/w under ultrasonication (5 min at 210 W).Then, the solution was vacuum-filtered and, under stirring, dispersed in 100 mL of an aqueous solution of NaOH 10% w/w, and stirred for 30 min.This procedure was repeated one more time and finally the sample was vacuum-filtered using a 0.45 µm polycarbonate membrane filter under 3DH 2 O maintaining the pH between 7 and 8, and dried overnight at 110

PEI-Functionalization of Carboxylated Mag-CNTs (NL003)
A total of 40 mg of the carboxylated Mag-CNTs (sample name: NL002) was suspended in 20 mL of chloroform.This mixture was then placed in a bath sonicator for 30 min to break up any of the larger aggregates.Concurrently, 4 mL of the PEI solution (1.08 g/mL) was added to 16 mL of 3DH 2 O to a total volume of 20 mL at a concentration of 200 mg of PEI/mL.These two solutions were then combined in a suitable beaker.This beaker was then covered and placed under the horn sonicator at 20% power for 3 h while undergoing slight magnetic stirring.After this period, the mixture was heated on a hotplate to evaporate any excess chloroform, taking care not to raise the temperature above 80 • C. Once the chloroform had been taken off, the Mag-CNTs@PEI (code name: NL003) was subjected to centrifugation to remove large aggregates and the supernatant vacuum was filtered through a 0.45 micrometer PTFE membrane, while being continuously washed with ethanol.The centrifugation step is very important since the agglomeration of carbon nanotubes as a result of the van der Waals interactions tends to increase cytotoxicity.After 3 washes with ethanol, the membranes were left to dry overnight in a warm oven.

Indirect Coupling of NAP on Mag-CNTs@PEI (NL004)
A total of 20 mg of Mag-CNTs@PEI (sample name: NL003) was suspended in 6 mL of 3DH 2 O and placed in a water bath sonicator for 30 min.During a second step, a fresh solution of 48 mg of EDC, 108 mg NAP, and 0.1 mL of Et 3 N to 5 mL of chloroform was prepared.These two solutions were then combined and placed under the horn sonicator for 4 min at 40% power to ensure good dispersion.This mixture was then covered and magnetically stirred for 24 h.After stirring, the solution was again heated to remove any excess of chloroform.As before, care was taken not to let the solutions temperature rise above 80 • C and was then filtered in the same way through a 0.45 micrometer PTFE membrane while being washed with ethanol.The filter paper was then removed and left to dry before collecting the Mag-CNTs@PEI@NAP (NL004) product.

FT-IR/UV-Vis Spectra
Infrared spectra (400-4000 cm −1 ) were recorded on a Nicolet FT-IR 6700 spectrometer with samples prepared as a KBr disk while the Attenuated Total Reflection (ATR) spectra were recorded on a "Bruker Optics" Alpha-P Diamond ATR Spectrometer of Bruker Optics GmbH".UV-vis spectra were recorded in solution at concentrations in the range 10 −5 -10 −3 M on a Hitachi U-2001 dual beam spectrophotometer.Viscosity experiments were carried out using an ALPHA L Fungilab rotational viscometer equipped with an 18 mL LCP spindle.Fluorescence spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer.The C, H, and N elemental analysis was performed on a Perkin-Elmer 240B elemental analyzer.

Raman Measurements
The acquisition of the Raman spectra was obtained with a Renishaw InVia Reflex at 785 nm (1.58 eV).The laser power was kept below 1.6 mW in order to minimize the laser-heating effects on the probed materials while the laser was focused on the sample using a ×100 objective.

Drug-Release Protocol
The emission characterization of NL004 was recorded on a Cary Eclipse emission spectrophotometer.The drug-release behavior was investigated according to the following procedure: 1 mg of NL004 was dispersed in 5 mL of three buffered solutions (pH = 4.1, 6.8 and 10.1) under stirring at room temperature.At predetermined time intervals the solution was transferred in a cuvette and the emission intensity of NAP at 357 nm was recorded.The cumulative release (%) was expressed as: C 2020, 6, 62 5 of 18 where W t and W drug indicate the weight of drug released from the hybrid material at time t, and the total amount of loaded drug, respectively [37].W t and W drug were calculated from the calibration curve of free naproxene emission intensity at various concentrations, under the same excitation and emission wavelengths.

Preparation of CT DNA Solution for DNA-Binding Studies
The stock DNA solution was prepared by diluting CT DNA to buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0); the resultant solution was stirred at 4 • C for three days, and kept at 4 • C up to two weeks.The UV spectrum of the stock CT DNA solution was recorded giving a ratio of UV absorbance at 260 and 280 nm (A 260 /A 280 ) equal to 1.89, showing that the DNA was sufficiently free of protein [38].The concentration of the CT DNA solution was determined via the UV absorbance at 260 nm with a 1:20 dilution using ε = 6600 M −1 cm −1 [39].

Albumin and DNA-Binding Studies
In order to evaluate the biological behavior of NL004, a DMSO solution was used.Mixing this solution with the aqueous buffer DNA/albumin-containing solutions used in the studies never exceeded 5% DMSO (v/v) in the final solution, which was needed due to low aqueous solubility.The biological behavior of free NAP has been previously reported [40,41].Control experiments with DMSO were performed and no changes in the spectra of the albumins or CT DNA were observed.All studies were performed at room temperature.
The albumin-binding was studied by tryptophan fluorescence quenching experiments using bovine serum albumin (BSA, 3 µM) in buffer (containing 15 mM trisodium citrate and 150 mM NaCl at pH 7.0).The quenching of the emission intensity of tryptophan residues of BSA at 343 nm was monitored using NL004 as the quencher [42].The fluorescence emission spectra of BSA in the presence of the solution were recorded in the range 300-500 nm with an excitation wavelength (λ ex ) of 295 nm.The fluorescence emission spectra of NL004 in DMSO/buffer solutions did not exhibit any appreciable fluorescence emission band under the same experimental conditions (i.e., λ ex = 295 nm).The influence of the inner-filter effect on the measurements [43] was evaluated by Equation (S1) and did not affect the measurements.The Stern-Volmer and Scatchard equations (Equations (S2)-(S4)) and graphs were used in order to study the interaction of NL004 with BSA and calculate the Stern-Volmer constant K SV (in M −1 ), the SA-quenching constant k q (in M −1 s −1 ), the SA-binding constant K (in M −1 ) and the number of binding sites per albumin n [44].
The interaction of NL004 with CT DNA was studied directly by UV-vis spectroscopy and viscosity measurements and indirectly via EB-competitive studies.UV-vis spectroscopy was employed to investigate the possible binding modes to CT DNA and to calculate the DNA-binding constant (K b ).Therefore, the UV-vis spectra of CT DNA were recorded for a constant DNA-concentration in the presence of NL004 at increasing [compound]/[DNA] mixing ratios (=r).The DNA-binding constant of NL004, K b (in M −1 ), was determined by the Wolfe-Shimer equation (Equation (S5)) [45] and the plots [DNA]/(ε A -ε f ) versus [DNA] when the UV-vis spectra of the compound were recorded in the presence of DNA for diverse r values.
The viscosity of DNA ([DNA] = 0.1 mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) was measured in the presence of increasing amounts of NL004 (up to the value of r = 0.40).All measurements were performed at room temperature.The obtained data are presented as (η/η 0 ) 1/3 versus r, where η is the viscosity of DNA in the presence of the compound, and η 0 is the viscosity of DNA alone in buffer solution.
The competitive study of NL004 with EB was investigated by fluorescence emission spectroscopy in order to examine the ability of NL004 to displace EB from its DNA-EB conjugate.The DNA-EB conjugate was prepared by adding 20 µM EB and 26 µM CT DNA in buffer (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).The possible intercalating effect of NL004 was studied by adding a certain amount of NL004 stepwise into a solution of the DNA-EB complex.The influence of the C 2020, 6, 62 6 of 18 addition of NL004 to the DNA-EB solution was obtained by recording the changes of fluorescence emission spectra with λ ex = 540 nm.NL004 did not show any fluorescence emission bands at room temperature in solution or in the presence of DNA or EB under the same experimental conditions (λ ex = 540 nm); therefore, the observed quenching was attributed to the displacement of EB from its EB-DNA conjugate.The value of the Stern-Volmer constant (K SV , in M −1 ) was calculated according to the linear Stern-Volmer equation (eq.S2) [46] and the plot Io/I versus [Q].Taking τ o = 23 ns as the fluorescence lifetime of the EB-DNA system, ref. [47] the quenching constant (k q , in M −1 s −1 ) for NL004 was calculated according to eq.S3.

Synthetic Considerations
Mag-CNTs could be functionalized with groups containing oxygen, such as -OH and -COOH, mainly at the outer sidewall ends where several defects are introduced during the oxidation procedure.These oxygen-containing groups could be used for the reaction to conjugate other functional groups (Figure 1).The functionalization of the Mag-CNTs was confirmed by FT-IR spectroscopy (Figure S1) where characteristic absorption peaks at 3400 cm −1 , and (1615, 1117) cm −1 corresponded to the hydroxyl and carbonyl groups conjugated to the C=C and the C-O stretching vibration, respectively.In our experimental protocol, PEI, a cationic polymer in branched form, was used to provide a high density of cations on the surface of the carbon nanotubes.Despite its extensive applications, PEI is cytotoxic and chemical modification is required to improve its properties as a transfection reagent [23,[26][27][28].The direct functionalization of the carboxylated carbon nanotubes with PEI would not only increase their biocompatibility but also reduce the toxicity of the PEI.In spite of this, the conclusions on the toxicity and transfection efficiency of PEI-functionalized carbon nanotubes in comparison to pure PEI that have appeared in the literature are quite contradictory [48,49].In our study, the branched PEI1 .8 kDa readily adsorbed on the surface of the carbon nanotubes through a biphasic procedure (Figure 1) and rendered a hydrophilic surface (it is possible for every third atom of the polymer backbone to be positively charged).In the FT-IR spectrum of the PEI-functionalized carbon nanotube (NL003), the characteristic intense peak at 3360 cm −1 was assigned to the N-H of PEI (Figure S1).Additionally, the band at 1650 cm −1 of the NL003 resulted from the bending of primary amine groups (-NH 2 ), which was incorporated into a broad band at 1580 cm −1 in PEI [50].The amino groups of PEI were further exploited for the covalent attachment of the carboxylate drug (NAP) through an amide formation.
After the reaction with NAP, the cytotoxicity of PEI can be safely assumed to be decreased due to the reduction in PEI's cationic density.In the FT-IR spectrum of the PEI-NAP-functionalized carbon nanotube (NL004) the characteristic vibrations of the amide formation between the -NH 2 of the PEI and the -COOH of the NAP were observed (~1640 cm −1 ) and proved the successful reaction [51].

Introduction
A fascinating category of nanomaterials possessing unique physical, chemical, and physiological properties is carbon nanotubes (CNTs) [1][2][3][4][5][6].Their chemical modification has attracted increasing attention over recent years in order to improve their solubility and compatibility, tailor their structures and properties, and open a whole new world of novel CNT-based nanostructures and nanomaterials [7][8][9][10][11].In a parallel manner, the use of nanotechnology in the field of pharmaceutics and drug-delivery has remarkably grown over the last few years, giving "birth" to a new term developed on the basis of this technology-nanopharmaceuticals.As a matter of fact, CNTs possess many intriguing features that make them attractive drug-delivery carriers such as: a) An enhanced permeability and retention (EPR) effect.Nanocarriers such as nanoparticles (NPs), liposomes, and CNTs, experience a higher accumulation in tumor tissues as compared to normal tissues because of the poorly formed blood and lymphatic vessels that supply rapidly proliferating tumors [12].The EPR effect empowers CNTs to transport chemotherapeutic agents preferentially to tumor sites [13].b) Trans-membrane penetration and intracellular accumulation through the needle-like shape of CNTs via the "nanoneedle" mechanism [14] and/or energy-dependent endocytic pathways [15].c) Extraordinary ability for drug-loading and efficient attachment onto the surface or within the interior core of CNTs [16][17][18].The functionalization of CNTs involves three different approaches: i) the attachment [19,20] of molecules through covalent bonding; ii) the functionalization of molecules on the graphitic surface by an adsorption mechanism [21]; iii) the encapsulation of molecules in the empty cavity of the nanotubes.
Considering the non-covalent functionalization of CNTs, molecules containing aromatic groups are adsorbed on the conjugated surface of the nanotubes mainly through π-π interactions [22].CNTs are excellent candidates for the role of carriers mainly because their inner space can be filled with nanoparticles or molecules while the graphitic wall can be further functionalized in order to enrich biocompatibility, to succeed biodegradation [23], and to convey novel functions for targeting [24] or imaging purposes [25][26][27].In comparison with the non-covalent functionalization of CNTs, the covalent one is clearly more accurate, better controllable and robust.The conjugates of non-covalent

Magnetic Properties
The magnetic hysteresis curve at 4 K and in the field range from -6 to 6 Tesla and the ZFC-FC magnetic susceptibility measurements of the magnetic NL004 hybrid material are shown in Figure 2. In the inset of the same Figure is shown a magnification of the low field magnetization data in the form of M/Ms where the hysteresis loop of the hybrid material with a coercive field ca.0.15 T is clearly resolved.The value of saturation magnetization, M s , is 3.15 emu/g, due to the low percentage of ferromagnetic Fe/Ni catalyst residues (as it is already confirmed by manufacturer) while the value of M r is close to 1.26 emu/gr.Having a value of M r /M s close to 0.5 (ca.0.4), the magnetic nanoparticles are defined as a single-domain with a uniaxial anisotropy and randomly distributed easy axis at temperature well below the blocking temperature [52,53].The temperature dependence of the magnetic susceptibility data of NL004, in the experimental mode of ZFC-FC with a very low external magnetic field (50 Oe), is also shown in Figure 2. The linear temperature dependence of the ZFC susceptibility data is related to the domain wall pinning processes [54] and the negative values at low temperatures are a direct consequence of the existence of magnetic interactions, possible crystal structure disorders and intrinsic negative spin polarization [55,56].
temperature well below the blocking temperature [52,53].The temperature dependence of the magnetic susceptibility data of NL004, in the experimental mode of ZFC-FC with a very low external magnetic field (50 Oe), is also shown in Figure 2. The linear temperature dependence of the ZFC susceptibility data is related to the domain wall pinning processes [54] and the negative values at low temperatures are a direct consequence of the existence of magnetic interactions, possible crystal structure disorders and intrinsic negative spin polarization [55,56].

Raman Studies
In Figure 3 is shown the Raman spectra of raw and functionalized Mag-CNTs excited with the 785 nm laser line.The main features of these spectra are the so called: (a) D-band at ~1303 cm 1 ; (b) Gband at ~1580 cm 1 ; (c) D' at ~1601 cm 1 .The origin of the D-and D' bands is the double resonance Raman scattering process and are mainly disorder-induced features [57].Their frequencies shift to higher values with the increase in the energy of the excitation laser while the intensity of the D' significantly decreases [58].The pair of D and D' bands are usually attributed to the presence of amorphous or disordered carbon while the structural disorder is due to the finite or nanosized graphitic planes, defects on the nanotube walls and possibly other forms of carbon products.The G band originates from in-plane tangential stretching of the C-C bonds in the graphene sheets and it is doubly degenerate.
The profile characteristics of all Raman bands along with the intensity ratio ID/IG were deduced by Lorentzian fits on the Raman spectra (Figure 3) and summarized in Table 1.The high value of ID/IG for the pristine Mag-CNTs samples implies that pristine material is highly disordered or contains amorphous carbon (Table 1).The modification treatment of the pristine material either by COOH groups (NL002) or the PEI@NAP (NL004) results in an increase in the ID/IG ratio by about 21% and 16%,

Raman Studies
In Figure 3 is shown the Raman spectra of raw and functionalized Mag-CNTs excited with the 785 nm laser line.The main features of these spectra are the so called: (a) D-band at ~1303 cm 1 ; (b) G-band at ~1580 cm 1 ; (c) D' at ~1601 cm 1 .The origin of the D-and D' bands is the double resonance Raman scattering process and are mainly disorder-induced features [57].Their frequencies shift to higher values with the increase in the energy of the excitation laser while the intensity of the D' significantly decreases [58].The pair of D and D' bands are usually attributed to the presence of amorphous or disordered carbon while the structural disorder is due to the finite or nanosized graphitic planes, defects on the nanotube walls and possibly other forms of carbon products.The G band originates from in-plane tangential stretching of the C-C bonds in the graphene sheets and it is doubly degenerate.
The profile characteristics of all Raman bands along with the intensity ratio I D /I G were deduced by Lorentzian fits on the Raman spectra (Figure 3) and summarized in Table 1.The high value of I D /I G for the pristine Mag-CNTs samples implies that pristine material is highly disordered or contains amorphous carbon (Table 1).The modification treatment of the pristine material either by COOH groups (NL002) or the PEI@NAP (NL004) results in an increase in the I D /I G ratio by about 21% and 16%, respectively.The position of the G band in the NL004 sample shifts to lower values (~4 cm −1 ) with respect to the pristine CNTs suggesting that the treatment induces mechanical strain on the CNTs.

Interaction with BSA
Serum albumin (SA) is among the most common proteins in blood plasma; SA has the role of transporting drugs, metal ions and/or complexes towards cells and tissues through the bloodstream.The intense fluorescence emission band of the BSA solution (3 µM) at 342 nm, when excited at 295 nm, is mainly attributed to the tryptophan residues found at positions 134 and 212 [44].The inner-filter effect, as calculated with eq.S1, was negligible and slightly affected the measurements.
The addition of NL004 into the BSA solution resulted in a low-to-moderate quenching of ~30% of the initial fluorescence intensity of BSA fluorescence (Figure 4 and Figure S2).The observed intensity decrease in the fluorescence emission band at λ = 342 nm could be probably assigned to possible changes in the protein secondary structure leading to changes in the tryptophan environment of BSA stemming from the binding of NL004 to BSA [44].
The constants K sv and k q for NL004 were calculated by the Stern-Volmer quenching equation (eq.S2 and S3) and the corresponding Stern-Volmer plot (Figure S3) and are summarized in Table 2.More specifically, the derived k q constant (=2.05(±0.10)× 10 12 M −1 s −1 ) is much higher than the value of 2.0 × 10 10 M −1 s −1 showing the existence of a static quenching mechanism [42].The constant K of NL004, as calculated by the Scatchard equation (eq.S4) and the corresponding Scatchard plot (Figure S4), is relatively high (=7.84(±0.35)× 10 4 M −1 ) and within the range found for metal-NAP compounds [40,41,59,60].Comparing the behavior towards BSA of NL004 containing functionalized NAP with that of free NAP, we may conclude that functionalized NAP in NL004 presents higher quenching and binding constants than free NAP (Table 2).The constants Ksv and kq for NL004 were calculated by the Stern-Volmer quenching equation (eq.S2 and S3) and the corresponding Stern-Volmer plot (Figure S3) and are summarized in Table 2.More specifically, the derived kq constant (=2.05(±0.10)× 10 12 M −1 s −1 ) is much higher than the value of 2.0 × 10 10 M −1 s −1 showing the existence of a static quenching mechanism [42].The constant K of NL004, as calculated by the Scatchard equation (eq.S4) and the corresponding Scatchard plot (Figure S4), is relatively high (=7.84(±0.35)× 10 4 M −1 ) and within the range found for metal-NAP compounds [40,41,59,60].Comparing the behavior towards BSA of NL004 containing functionalized NAP with that of free NAP, we may conclude that functionalized NAP in NL004 presents higher quenching and binding constants than free NAP (Table 2).In total, the K constant of NL004 to BSA seems high enough to suggest its attachment to BSA in order to become transported.It is also much lower than the value of 10 15 M −1 (= the binding constant of avidin with diverse compounds; these constitute the strongest known non-covalent binding); therefore, the binding of NL004 to BSA seems reversible, proposing the releasing ability upon arrival at potential biological targets [61].Table 2. Comparison of the spectroscopic data (% of BSA fluorescence quenching), the quenching and the binding constants of NL004 and NAP for BSA (k q , K).In total, the K constant of NL004 to BSA seems high enough to suggest its attachment to BSA in order to become transported.It is also much lower than the value of 10 15 M −1 (= the binding constant of avidin with diverse compounds; these constitute the strongest known non-covalent binding); therefore, the binding of NL004 to BSA seems reversible, proposing the releasing ability upon arrival at potential biological targets [61].

Interaction with DNA
The interaction of double-stranded DNA with diverse compounds may take place via either covalent (replacement of a labile ligand of the complex by a nitrogenous DNA base, as in the case of cisplatin) or non-covalent fashion (intercalation, electrostatic interactions and groove-binding) [62].Furthermore, the interaction of compounds containing NSAIDs, such as NAP with double-stranded DNA, may be connected to potential anticancer, antioxidant and/or anti-inflammatory activities.Therefore, the in vitro DNA-binding of NL004 was studied by UV-vis spectroscopy and viscosity measurements and via the ability to displace the typical DNA-intercalator EB, and was compared to the behavior of free NAP which has been already reported [40,41].
UV-vis spectroscopy is usually used to study the DNA-binding mode and to calculate the DNA-binding strength.Any changes of the DNA UV-band or the intraligand transition bands of the compounds may be due to their interaction and may reveal their possible mode.The UV-vis spectra C 2020, 6, 62 11 of 18 of a CT DNA solution (0.18 mM) were recorded in the presence of increasing amounts of NL004; the DNA UV-band at 258 nm exhibited hypochromism (Figure 5A) suggesting the interaction of NL004 with CT DNA [63].In the UV-vis spectra of NL004, diverse bands were observed with most of them being attributed to the nanotube and band located at 324 nm, which are attributed to NAP (Figure 5B).Upon addition of CT DNA, all these UV-bands of NL004 exhibited considerable hypochromism (up to ~40%) and were followed by a slight red-shift or were eliminated (Table 3).
C 2020, 6, 62 10 of 17 measurements and via the ability to displace the typical DNA-intercalator EB, and was compared to the behavior of free NAP which has been already reported [40,41].UV-vis spectroscopy is usually used to study the DNA-binding mode and to calculate the DNAbinding strength.Any changes of the DNA UV-band or the intraligand transition bands of the compounds may be due to their interaction and may reveal their possible mode.The UV-vis spectra of a CT DNA solution (0.18 mM) were recorded in the presence of increasing amounts of NL004; the DNA UV-band at 258 nm exhibited hypochromism (Figure 5A) suggesting the interaction of NL004 with CT DNA [63].In the UV-vis spectra of NL004, diverse bands were observed with most of them being attributed to the nanotube and band located at 324 nm, which are attributed to NAP (Figure 5B).Upon addition of CT DNA, all these UV-bands of NL004 exhibited considerable hypochromism (up to ~40%) and were followed by a slight red-shift or were eliminated (Table 3).The DNA-binding constant Kb of NL004 was calculated by the Wolfe-Shimer equation (eq.S4) [44] and the corresponding plot [DNA]/(εA-εf) versus CT DNA (Figure S5).The Kb constant of NL004 (= 8.66(±0.07)× 10 4 M −1 ) is approximately three times higher than that of free NAP (Table 3) suggesting that the functionalization of NAP in NL004 may result in an enhanced affinity for CT DNA.In addition, the Kb of NL004 is in the range reported for metal-NAP complexes [36,51,52] and is close to the Kb constant of the classical intercalator ethidium bromide (EB) (Kb = 1.23(±0.07)× 10 5 M −1 ), as calculated by Dimitrakopoulou et.al. [64].

Spectroscopic Data and Constants
The DNA-binding constant K b of NL004 was calculated by the Wolfe-Shimer equation (eq.S4) [44] and the corresponding plot [DNA]/(ε A -ε f ) versus CT DNA (Figure S5).The K b constant of NL004 (= 8.66(±0.07)× 10 4 M −1 ) is approximately three times higher than that of free NAP (Table 3) suggesting that the functionalization of NAP in NL004 may result in an enhanced affinity for CT DNA.In addition, the K b of NL004 is in the range reported for metal-NAP complexes [36,51,52] and is close to the K b constant of the classical intercalator ethidium bromide (EB) (K b = 1.23(±0.07)× 10 5 M −1 ), as calculated by Dimitrakopoulou et al. [64].
The features from the UV-vis spectroscopic titrations may suggest the binding of functionalized NAP to CT DNA; the observed hypochromism may reveal the possibility of an intercalative interaction mode, although significant steric hindrance may exclude it.Therefore, in order to conclude the DNA-binding fashion of NL004, DNA-viscosity measurements and EB-competitive studies were carried out [63,65].The study of DNA-viscosity is an essential technique in order to clarify/verify the DNA-interaction mode of a compound, because of the sensitiveness of the DNA-viscosity to relative DNA-length changes [66].As it is known, in the case of classic intercalation of a compound in-between the DNA base pairs, the DNA base pairs' separation distance at intercalation sites, and consequently the relative DNA-length, will increase so that the bound compound is hosted inducing thus an increase in DNA-viscosity.When non-classical intercalation (including groove-binding and electrostatic interaction) occurs, the relative DNA-length remains the same or decreases slightly because of the DNA-helix bending; thus, the relative DNA-viscosity will remain stable or will present a slight increase [66].The viscosity of the CT DNA solution (0.1 mM) was measured by adding increasing amounts of NL004 and a slight decrease in the relative DNA-viscosity was observed (Figure 6).Therefore, the changes of the relative DNA-viscosity may suggest the absence of a classical intercalation between NL004 and CT DNA; taking also into consideration the steric hindrance induced by the nanotube, we may suggest that the interaction of functionalized NAP in NL004 probably takes place via the external interaction mode.
The features from the UV-vis spectroscopic titrations may suggest the binding of functionalized NAP to CT DNA; the observed hypochromism may reveal the possibility of an intercalative interaction mode, although significant steric hindrance may exclude it.Therefore, in order to conclude the DNA-binding fashion of NL004, DNA-viscosity measurements and EB-competitive studies were carried out [63,65].
The study of DNA-viscosity is an essential technique in order to clarify/verify the DNAinteraction mode of a compound, because of the sensitiveness of the DNA-viscosity to relative DNAlength changes [66].As it is known, in the case of classic intercalation of a compound in-between the DNA base pairs, the DNA base pairs' separation distance at intercalation sites, and consequently the relative DNA-length, will increase so that the bound compound is hosted inducing thus an increase in DNA-viscosity.When non-classical intercalation (including groove-binding and electrostatic interaction) occurs, the relative DNA-length remains the same or decreases slightly because of the DNA-helix bending; thus, the relative DNA-viscosity will remain stable or will present a slight increase [66].The viscosity of the CT DNA solution (0.1 mM) was measured by adding increasing amounts of NL004 and a slight decrease in the relative DNA-viscosity was observed (Figure 6).Therefore, the changes of the relative DNA-viscosity may suggest the absence of a classical intercalation between NL004 and CT DNA; taking also into consideration the steric hindrance induced by the nanotube, we may suggest that the interaction of functionalized NAP in NL004 probably takes place via the external interaction mode.EB is a typical indicator of intercalation [67].The DNA-intercalation of EB occurs via the insertion of the planar EB-phenanthridine ring in-between adjacent DNA bases resulting in the formation of an EB-DNA conjugate which exhibits an intense fluorescence emission band at 592 nm.The addition of intercalating compounds into the EB-DNA solution will obviously result in a significant quenching of the EB-DNA emission band because of the displacement of EB in the EB-DNA conjugate by the inserting compounds which can bind to DNA equally or even more strongly than EB.The fluorescence emission spectra of an EB-DNA solution ([EB] = 20 μM, [DNA] = 26 μM) were recorded in the absence and presence of increasing amounts of NL004 (Figure 7).The addition of NL004 resulted in a moderate decrease (up to 50%, Figure S6) in the intensity of the emission band at 592 nm.The observed fluorescence quenching (Table 3) is rather moderate and may indicate a relatively low competition of the NL044 with EB in its binding to DNA, although the DNA-binding constant of NL044 is close to that of EB.Consequently, we may suggest that the intercalating ability of NL044 is rather low [52,53] and the intercalation between NL004 and CT DNA should not be expected, in accordance to DNA-viscosity measurements.The linear Stern-Volmer equation (eq.S2) and the corresponding Stern-Volmer plot of EB-DNA (R = 0.99, Figure S7) proved that the EB-DNA fluorescence emission quenching might be a result of the partial displacement of EB in EB-DNA by NL004.The calculated Stern-Volmer constant (Ksv) and quenching constant (kq, as calculated from EB is a typical indicator of intercalation [67].The DNA-intercalation of EB occurs via the insertion of the planar EB-phenanthridine ring in-between adjacent DNA bases resulting in the formation of an EB-DNA conjugate which exhibits an intense fluorescence emission band at 592 nm.The addition of intercalating compounds into the EB-DNA solution will obviously result in a significant quenching of the EB-DNA emission band because of the displacement of EB in the EB-DNA conjugate by the inserting compounds which can bind to DNA equally or even more strongly than EB.The fluorescence emission spectra of an EB-DNA solution ([EB] = 20 µM, [DNA] = 26 µM) were recorded in the absence and presence of increasing amounts of NL004 (Figure 7).The addition of NL004 resulted in a moderate decrease (up to 50%, Figure S6) in the intensity of the emission band at 592 nm.The observed fluorescence quenching (Table 3) is rather moderate and may indicate a relatively low competition of the NL044 with EB in its binding to DNA, although the DNA-binding constant of NL044 is close to that of EB.Consequently, we may suggest that the intercalating ability of NL044 is rather low [52,53] and the intercalation between NL004 and CT DNA should not be expected, in accordance to DNA-viscosity measurements.The linear Stern-Volmer equation (eq.S2) and the corresponding Stern-Volmer plot of EB-DNA (R = 0.99, Figure S7) proved that the EB-DNA fluorescence emission quenching might be a result of the partial displacement of EB in EB-DNA by NL004.The calculated Stern-Volmer constant (K sv ) and quenching constant (k q , as calculated from eq.S3 for fluorescence lifetime of the EB-DNA system, τ o = 23 ns [42]) of NL004 (Table 3) are significantly lower than those of free NAP, thus showing a lower ability to displace EB. eq.S3 for fluorescence lifetime of the EB-DNA system, τo = 23 ns [42]) of NL004 (Table 3) are significantly lower than those of free NAP, thus showing a lower ability to displace EB.

Drug-Release Studies
The emission spectra of free NAP solution and the suspension of NL004 are presented in Figure 8.No emission was detected for the raw carboxylated Mag-CNTs (NL002) and the PEI-functionalized Mag-CNTs (NL003).Comparison between the spectra of the free NAP solution (λmax,em = 357 nm) and NL004 (λmax,em = 405 nm) reveals a significant broadening and a red-shift in the maximum emission wavelength for the latter one.Both these features can be attributed to changes in the chemical environment surrounding the drug molecule due to the location of this drug in NL004 [68,69].These findings confirm a successful incorporation of NAP in NL004.The characteristic shoulder observed at the spectrum of the suspension at 357 nm comes from free NAP.It is well known that acidic conditions are typical of inflamed tissues.In order to check if we have a pH-sensitive targeted drug-delivery, three representative pH values (acidic, neutral and basic) were chosen [68].The release of the drug from NL004 was tested at pH 4.1, 6.8 and 10.1.

Drug-Release Studies
The emission spectra of free NAP solution and the suspension of NL004 are presented in Figure 8.No emission was detected for the raw carboxylated Mag-CNTs (NL002) and the PEI-functionalized Mag-CNTs (NL003).Comparison between the spectra of the free NAP solution (λ max,em = 357 nm) and NL004 (λ max,em = 405 nm) reveals a significant broadening and a red-shift in the maximum emission wavelength for the latter one.Both these features can be attributed to changes in the chemical environment surrounding the drug molecule due to the location of this drug in NL004 [68,69].These findings confirm a successful incorporation of NAP in NL004.The characteristic shoulder observed at the spectrum of the suspension at 357 nm comes from free NAP.
C 2020, 6, 62 12 of 17 eq.S3 for fluorescence lifetime of the EB-DNA system, τo = 23 ns [42]) of NL004 (Table 3) are significantly lower than those of free NAP, thus showing a lower ability to displace EB.

Drug-Release Studies
The emission spectra of free NAP solution and the suspension of NL004 are presented in Figure 8.No emission was detected for the raw carboxylated Mag-CNTs (NL002) and the PEI-functionalized Mag-CNTs (NL003).Comparison between the spectra of the free NAP solution (λmax,em = 357 nm) and NL004 (λmax,em = 405 nm) reveals a significant broadening and a red-shift in the maximum emission wavelength for the latter one.Both these features can be attributed to changes in the chemical environment surrounding the drug molecule due to the location of this drug in NL004 [68,69].These findings confirm a successful incorporation of NAP in NL004.The characteristic shoulder observed at the spectrum of the suspension at 357 nm comes from free NAP.It is well known that acidic conditions are typical of inflamed tissues.In order to check if we have a pH-sensitive targeted drug-delivery, three representative pH values (acidic, neutral and basic) were chosen [68].The release of the drug from NL004 was tested at pH 4.1, 6.8 and 10.1.It is well known that acidic conditions are typical of inflamed tissues.In order to check if we have a pH-sensitive targeted drug-delivery, three representative pH values (acidic, neutral and basic) were chosen [68].The release of the drug from NL004 was tested at pH 4.1, 6.8 and 10.1.Figure 9 summarizes the results.At pH 4.1, the release profile indicates a rapid release up to a maximum of 42% NAP within the first 50 min.This corresponds to 0.38 mg of released NAP.Moreover, the release experiment conducted at pH 6.8 and 10.1 indicated a much lower percentage of NAP release (19% and 16%, respectively).

Conclusions
A facile and effective way for the attachment of Naproxen (NAP), a carboxylate NSAID, on the surface of PEI-functionalized carboxylated Mag-CNTs was presented based on the indirect coupling of NAP through the amino groups of the intermediate linker PEI.The interaction of the designed nanocarrier with BSA was studied in vitro by fluorescence emission spectroscopy while its in vitro interaction to bind to CT DNA was monitored by UV-vis spectroscopy and viscosity measurements and via competitive studies with ethidium bromide.The calculated binding constants were compared to those of free NAP revealing a higher binding affinity for BSA and CT DNA.Finally, drug-release studies were performed, revealing that the electrostatic linkage ensures an effective release of the drug in the acidic pH typical of inflamed cells, while maintaining the MWNT-drug conjugates stable at the typical bloodstream.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Figure S1: FT-IR spectra.Figures S2-S7: The Stern-Volmer and Scatchard graphs from interaction with serum albumins and interaction with CT-DNA.
Author Contributions: V.T. contributed in the project administration, conceptualization, visualization, and writing-reviewing of original draft.N.L. (Nikolia Lalioti) contributed in the methodology, supervision of the synthetic methodology and revised the paper.J.P. contributed in Raman measurements and revised the paper.N.L. (Nathan Langford) contributed in the synthetic methodology.E.V.-J.contributed in resources, Project administration funding and revised the paper.C.K. contributed in the biological studies.G.P. contributed in the methodology and supervision of the biological studies and revised the paper.V.B. contributed in photoluminescence and drug release measurements and revised the paper.All authors have read and agreed to the published version of the manuscript.

Figure 2 .
Figure 2. Field-dependent magnetization hysteresis curve of NL004 at 4 K.The blue-shaded area is shown for clarity reasons at the right part of the figure where the y-axis is given in the form of M/Ms with Ms = 3.15 emu/gr (saturation value of magnetization for NL004).At the right part is also shown the ZFC-FC temperature dependence of the DC susceptibility data of NL004 at external magnetic field of 50 Oe.

Figure 2 .
Figure 2. Field-dependent magnetization hysteresis curve of NL004 at 4 K.The blue-shaded area is shown for clarity reasons at the right part of the figure where the y-axis is given in the form of M/Ms with M s = 3.15 emu/gr (saturation value of magnetization for NL004).At the right part is also shown the ZFC-FC temperature dependence of the DC susceptibility data of NL004 at external magnetic field of 50 Oe.

Figure 3 .
Figure 3. Indicative Raman spectra of the NL001, NL002, NL004 products where the characteristic D, G and D' Raman bands are shown.
Figure 9 summarizes the results.At pH 4.1, the release profile indicates a rapid release up to a maximum of 42% NAP within the first 50 min.This corresponds to 0.38 mg of released NAP.Moreover, the release
pH 6.8 and 10.1 indicated a much lower percentage of NAP release (19% and 16%, respectively).

Table 1 .
The profile characteristics of D, G, D' Raman bands the intensity ratio (I D /I G ).

Table 2 .
Comparison of the spectroscopic data (% of BSA fluorescence quenching), the quenching and the binding constants of NL004 and NAP for BSA (kq, K).