Insight into the fabrication, characterization, and in vitro cytotoxicity studies approaches of halloysite-based functional anhydride containing polymer nanocomposites

Abstract This study aims to design, synthesize, and characterization of maleic anhydride-vinyl acetate and its halloysite nanotubes and to evaluate the cytotoxic effects of nanostructures on human gingival fibroblasts. Poly(maleic anhydride-alt-vinyl acetate) [Poly(MA-alt-VA)] was synthesized via charge transfer complex-radical polymerization as potential dental fillings, adhesives, or bone cement materials. ATR-FTIR, XPS, and XRD spectroscopic methods were used to enlighten the structure–property of nanomaterials. Determining surface area and pore volume BET was used. Morphological characterization was viewed by HRTEM. Cytotoxic effects of nanocomposites on cell viability were evaluated by MTT assay for different incubation periods and concentrations for human gingival fibroblasts. Graphical Abstract


Introduction
Polymeric material optimization by the addition of a small amount of filler in nanometer-scale dimension has been the focus of many industrial and academic aspects.Adding filler to polymeric materials has significantly increased the mechanical, barrier, thermal, and electrical properties [1,2] .Using polymer and clay is a way to prepare nanocomposites and the performance of polymer/clay nanocomposites depends on some factors, such as the clay content and dispersion state [3] .Nanocomposites have a widespread biomedical nanotechnology application due to their exceptional properties, especially in several new nanostructured materials, such as nanotubes, nanocontainers, and nanorods [4,5] .Polymer/clay nanocomposites have attracted intense research interest in the unique physical and chemical properties resulting from the combined use of organic and inorganic materials in one compound [5][6][7] .An eco-friendly composite based on natural halloysite nanotube (HNT) enables the design of smart composite materials synergistically for the controlled release of drugs [8] .Halloysite can be found in a variety of morphologies, such as short tubular, spheroidal, and platy clays-kaolin as well as montmorillonite, the most common type of elongated tubes [9] .The diverse chemical composition of the inner (aluminol) and outer (siloxane) surfaces of HNT assign for the selective functionalization of the lumen and exterior, to optimize the properties.The inner and outer surface properties of HNT, make this material suitable for the design of nanocomposites for biomedical applications [10] .HNT can be useful for easy modification/reaction of many synthetic and biological components because of the larger area of the surface, positively charged inner surface (Al-OH groups), and negatively charged outer surface (Si-OH and Si-O-Si groups) [11,12] .Important applications of HNT can be seen in the development of nanomaterials using nanoelectronics for applications, such as thin-film transistors, wearable electronics, artificial skin and muscle, and solar panels [13,14] .For life science applications, from drug delivery, via oral or topical administration, to tissue scaffolds and regenerative medicine, halloysite clay nanotubes featured potential uses while assessing their cellular internalization, stability, biosafety, and biocompatibility [15][16][17] .
The synthesis approach of polymer/HNT nanocomposites can be referred to as the reversible insertion of guest species into a host matrix while maintaining the structural feature of the host.Preparation of nanocomposites was done by using in situ polymerization of functional monomers, such as N-vinylcarbazole [18] , methyl methacrylate [19] , N-butylmaleimide [20] , and e-caprolactone [21] .Although many organic monomers are used in situ intercalative polymerization, binary or ternary, the intercalative radical copolymerization of monomer systems in the presence of clay has been rarely investigated.The application has gained great importance.For the development of these types of materials especially dental restorative hybrid materials; anhydride-containing polymer/clay nanocomposites were planned to design and synthesis, as well as cell viability tests, were studied for this purpose [22,23] .
Cell viability assays are commercially used to test materials' safety and biocompatibility.These studies are crucial to determining the clinical use of materials [24,25] .Tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphnyltetrazolium bromide (MTT) assay is a well-accepted technique for testing material's toxicity.Basically, MTT is reduced by mitochondrial succinate dehydrogenase to yield a blue formazan product, which does not cross the plasma membrane, and accumulates in cells then shows the viability of cells [26,27] .Maleic anhydride and vinyl acetate copolymers are used as potential dental adhesives polymers because of their bioadhesive, antibacterial and mucoadhesive properties [22,23,28] .For this purpose, we choose human gingival fibroblasts to evaluate the cytotoxicity of nanocomposites.
The objective of the present work is to study of complex formation between maleic anhydride (MA, electron-acceptor monomer) and vinyl acetate (VA, electron-donor monomer) binary copolymerization and to assess the cytotoxic effects of these nanomaterials on gingival fibroblasts [29] .The functional copolymers, having a combination of rigid/flexible linkages and an ability of complex-formation with halloysite (HNT) and their nanocomposites have been synthesized by interlamellar complex-radical copolymerization of intercalated monomer complexes of MA and VA monomer mixtures.These polymeric nanocomposites are designed for potential dental applications, such as dental resin, dental adhesive, filler, etc.For this purpose, the cytotoxic effects of the nanocomposites were also determined by MTT assay and AO/PI staining on human gingival fibroblasts.Poly(MA-alt-VA) pristine and its halloysite-based nanocomposites were characterized by using ATR-FTIR, and XPS XRD spectroscopic methods to enlighten the structureproperty of synthesized materials.The surface area and pore volume were studied by Brunauer-Emmett-Teller (BET) method.Morphological characterization of polymer/HNT (PHNT) nanotubes was the High-Resolution Transmission Electron Microscope (HRTEM) method.

Materials and chemical reagents
Maleic Anhydride (MA: m.p. 52.8 C; Sigma-Aldrich) was purified before use by recrystallization from anhydrous benzene and sublimation in a vacuum.Vinyl acetate (VA) was supplied from Fluka.The benzoyl peroxide (BPO) (Fluka) was recrystallized twice from the methanol solution.HNT was supplied by ESAN, Eczacıbas ¸ı, Other reagents, including organic solvents, were purified by ordinary methods.

Synthesis of the copolymers
Poly(MA-alt-VA) copolymer and copolymer/HNT nanocomposites were synthesized via the method of in situ charge transfer complex (CTC) polymerization.The synthesis was conducted by the implementation of in situ solution copolymerization method.Copolymerization was carried out with BPO as an initiator at 75 C in degassed tubes in methyl ethyl ketone (MEK) under a nitrogen atmosphere.Poly(MA-alt-VA) was synthesized by the use of 1:1 mol ratios of monomers feed.Monomers, BPO, and MEK were suspended at 75 C under a nitrogen atmosphere in a glycerin bath for 6 h.After the reaction took place for a given time, the contents of the tubes were poured into a large amount of n-hexane to precipitate the copolymer.The obtained powder-like products were separated by ultracentrifugation and redeposited by filtration.Synthesized polymers with this method were dried at 40 C to a constant weight.

Preparation of polymer-halloysite nanocomposites
(P/HNT) Poly(MA-alt-VA)/halloysite nanocomposites were synthesized in the same copolymerization conditions in the presence of the various HNT amounts.Halloysite, accounting for 1, 3, 5, and 20% (w/w) of the monomers (MA and VA) in copolymer were added into 5 mL MEK at room temperature and stirred for 1 h in reaction tubes placed in a thermostat with glycerin bath to prepare the intercalated complex of monomer and clay without adding initiator.
After the reaction occurred within a given time, monomers were added (6 mL) stirring at room temperature for 3 h.Then, the initiator BPO and the rest of the MEK (2 mL) were added stirring at 75 C for 6 h under a nitrogen atmosphere.The obtained copolymer nanocomposites were reprecipitated in n-hexane at 5 C (Scheme 1).

Attenuated total reflectance-Fourier transform infra-
red (ATR-FTIR) spectroscopy Synthesized polymer and its HNTs-based nanocomposites ATR-FTIR spectrums were taken by Thermo Nicolet IS 10 ATR-FTIR spectrophotometer between 4,000 and 400 cm À1 range, 64 scans were taken at a 4 cm À1 .

X-ray diffraction (XRD) spectroscopy
The space between the layers of samples, (d), was investigated using X-rays diffraction (XRD) Rigaku DMAX 2200 (Japan) X-Ray diffractometers (XRD) (Cu-Ka and Ni filter k ¼ 1.54059A ) at a scanning speed of 10/min, a tension of 40 kV and a current of 30 mA.Bragg Equation 1 was used to calculate the interlayer spacing, (d).
In Equation 1, n stands for the degree of refractory while h symbolizes its angle.

X-ray photoelectron spectroscopy (XPS)
X-ray photoelectron spectroscopy (XPS) was used for surface analysis and composition of HNT, copolymer, and copolymer/HNT nanocomposites by using PHI 5000 Versa Probe.200 mm X-ray (source Al Ka) spot was applied.The pass energy was set at 187.8 eV (general scan) and 58.7 eV (partial scan) for hydrocarbon C1s line from adventitious carbon.

Brunauer, Emmett, and Teller (BET) surface
area analyzer Halloysite, copolymer, and its nanocomposites nitrogen adsorption/desorption isotherms were measured at 77 K with a Quantachrome NOVA 2200e surface area and pore volume analyzer.The specific surface area was calculated by the multipoint Brunauer-Emmett-Teller (BET) method.The total pore volume was evaluated at a P/P0 close to 0.995.BJH method was used to calculate the pore volume by using the desorption branch of the isotherm.
Briefly, for each incubation period, all media was removed and media containing MTT were added to each well and incubated for 4 h.After 4 h the media containing MTT was removed and 100 mL of acidic isopropanol (0.05 N HCl in absolute isopropanol) was added to each well.A Scheme 1. Synthesis of copolymer/HNTs nanocomposites via in situ charge transfer complex (CTC) polymerization.
microplate reader (EZ Read 400 Microplate Reader, Biochrom, UK) was used for measuring absorbance (OD) at 570 nm.Data were expressed as mean ± standard deviation of six replicates.GraphPad Prism (v9.1.2) was used for statistical analyses.Unpaired Student's t-tests were selected for comparing the treated and untreated groups.A value of p < 0.05 was considered significant.
To identify morphological changes in gingival fibroblasts, cells were examined under an inverted microscope (IX70 Olympus, Japan).To visualize cell viability after treatment an Acridine orange/Propidium iodide (AO/PI) staining was performed.Briefly, for each incubation period, cells were fixed with methanol, and staining was done with AO/PI (v:v ratio of 1:1) for 20 s.Fluorescence attachments of an inverted microscope were used to visualize cells.Acridine orange-stained cells were observed filter in green color (520-560 nm) while propidium iodide-stained cells were in red color (510-560 nm) (IX70 Olympus, Japan).(halloysite nanotube) and synthesized poly(MA-alt-VA) copolymer, H1-P(MA-alt-VA), H3-(MA-alt-VA), H5-P(MAalt-VA), and H20-P(MA-alt-VA) copolymer/halloysite nanocomposites are given in Figure 1.Stretching of inner surface hydroxyl groups (-OH) of the HNT belonging to Al-OH of pure halloysite absorption bands can be seen at 3,691 and 3,625 cm À1 as two characteristic vibration bands.The peak observed at 3,440 cm À1 can be explained as the -OH deformation caused by water on the surface or between the layers due to the moisture-attracting feature of halloysite [32,33] .The shoulders and broadness of the structural -OH band are mainly due to contributions of several structural -OH groups that can exist in the HNT.HNT nanotubes of Si-O-Si unit stretching bands are observed at 1,118   and 1,003 cm À1 (Figure 1).The vibration band of the internal Al-OH groups of halloysite is seen at 907 cm À1 and the band at 750 cm À1 is ascribed to the Al-O-Si deformation (Figure 1).When the ATR-FTIR spectrum of the poly(MA-alt-VA) copolymer is examined, the partial hydrolysis of the anhydride unit results in a -OH band at 3,058 cm À1 can be observed [34] .Maleic anhydride unit of copolymer at the wavelengths of 1,894 and 1,703 cm À1 show the -C¼O vibrations of symmetric and asymmetric, respectively (Figure 1).A partially hydrolyzed carbonyl unit of maleic anhydride, -C¼O and -OH complex peak can be seen at around 1,600-1,500 cm À1 .The -CH bending stretching of the vinyl acetate unit was observed at 1,219 cm À1 , and the other characteristic peaks of VA; -CH 3 at 1,373 cm À1 , and -CH 2 strain at 1,434 cm À1 are observed in Figure 1b.When the band comments obtained from the ATR-FTIR spectrum are compared with the literature, it is seen that the copolymer has been synthesized successfully [34][35][36] .When the spectrum of nanocomposites is examined (Figures 1a and 1b), a formed bond between halloysite nanotube and the copolymer can be observed that the characteristic bands of -OH groups observed in the spectrum with the increase in the amount of halloysite by the mass, minimal shift toward low frequency is observed.This result can be explained as the increase in the halloysite-copolymer compatibility with the increasing amount of clay and the formation of stronger hydrogen bonding [29] .

Results and discussion
The shifts occurred as a result of the interactions of the carbonyl/carboxyl units of the copolymer and the Al-OH and Si-OH groups on the nanotube.To compare the specific FTIR bands belonging to HNT, copolymer, and nanocomposites, they are all given in Table 1.Two characteristic bands observed at 3,691 and 3,625 cm À1 belonging to the -OH groups of halloysite disappeared after the copolymer/HNT nanocomposite formation; due to the water loss resulting from the bond between halloysite and copolymer [37][38][39] .Particularly, vibration bands of halloysite and copolymer are clearly observed in H20-P (MA-alt-VA) sample which contains the most halloysite by mass (Table 1).When the ATR-FTIR spectra results are evaluated in general, changing the shape and displacement of the -OH bands (intermolecular or/and intramolecular H-bonding) can be explained as an interaction between HNT and copolymer increment depending on the amount of % HNT in copolymer/halloysite nanotubes (P/HNT).Structural features explained by ATR-FTIR spectroscopy were detailed and verified by XRD and XPS techniques.

X-ray diffraction (XRD) spectroscopy studies
The X-ray diffraction (XRD) method allows the interpretation of whether the polymer/clay nanocomposite material intercalated or exfoliated structure taking into account the distance between the layers (d) (basal space).XRD diffractograms HNT, Poly(MA-alt-VA), and its nanocomposites can be seen in Figures 2(a-f).The characteristic peak of halloysite at 2h ¼ 12 given in Figure 2a confirms that the halloysite clay mineral has a multi-walled structure and the gap between the layers (d001) was found at 0.719 nm using the Bragg equation (nk ¼ 2dsinh), and this value corresponds to the dehydrated form of halloysite [30].The presence of halloysite in the tubular form is also was determined by the presence of a peak at 2h ¼ 20 .The dehydrated state was also confirmed with the presence of the (d002) basal reflection at 24.5 2h.The first peak is attributed to internal/ surface pores, including spaces between the overlaps of folded HNTs sheets and the second peak is attributed to the central lumen of the tubes.HNTs have relatively few tubetube interactions due to chemical and geometrical aspects and do not tend to agglomerate [39,40] .
XRD pattern of the Poly(MA-alt-VA), can be seen in Figure 2b.Inter-and intramolecular hydrogen bonding in the copolymer can cause a semi-crystalline character.After the polymerization between halloysite, maleic anhydride, and vinyl acetate, the diffraction peaks of the composite material are in accordance with the combination of halloysite peaks and the Poly(MA-alt-VA)'s diffuse scattering peak [29] .By using XRD, they would be simply defined as intercalated, in that there was an observed increase in the dspacing as compared to the original clay d-spacing (Table 2).
The diffraction patterns of the halloysite and the copolymer disappear or are weakened in the composite material, with the position of some peaks changing and the strength of the peaks reduced [41] .It is noticed that the d(001) basal plane peak of halloysite in the composite material is shifted negatively which correspondent with the halloysite basal spacing.This result may reveal that the layers of halloysite were enlarged during the copolymerization process.
In the interlamellar copolymerization, HNT was used different amounts.As the HNT amount is increased in the nanocomposite; specific peaks of the HNT begin to appear  (Figures 2c-e).When the XRD patterns have gone through from H1-P(MA-alt-VA) to H20-P(MA-alt-VA), the native of the X-ray pattern became similar to the virgin HNT.In the designed and synthesized nanostructures, there is not a great difference in XRD patterns because of the interaction and reaction with the HNT surface.X-ray photoelectron spectroscopy (XPS) was used to examine the surface reactions in detail and to the complementary method of XRD.

X-ray photoelectron spectroscopy (XPS) studies
X-ray photoelectron spectroscopy (XPS) is one of the most well-known and widely used methods for the analysis of solid-state materials.It measures the kinetic energy of emitted electrons by the photoelectric effect induced by Xray illumination based on Einstein's photoelectric effects [42] .The survey spectrum shows which elements exist in a given sample and their relative proportions, i.e., the atomic percent (Figure 3).The peak positions of elements of typical interest identified in organic polymer nanocomposites are approximately C(1s) at 285 eV, N(1s) at 398 eV, O(1s) at 531 eV, and Si(2p) at 99 eV.Figures 3(a-e) show the XPS spectrum of HNT, copolymer, and its nanocomposites at various amounts of HNT.When the XPS spectrum of HNT is examined, the O(1s) band at 529 eV shows Al-O and Si-O bonds in HNT and copolymer at 529 eV.Si 2 s/2p and Al 2 s/2p bands and 166-66 houses were observed.At 284 eV, carbon bands are observed [43] .As the % HNT amount increases in the synthesized nanostructures, the characteristic Si 2 s/2p and Al 2 s/2p bands of HNT begin to become more prominent in the range of 155-75 eV.This shows that halloysite has been placed in nanocomposites and copolymer/halloysite nanotubes have been successfully synthesized [44,45] .Si and Al bands were prominent in the H20-P(MA-alt-VA) copolymer/clay nanocomposite with the highest mass % of HNT (Table 3).Signals from Si, Al, and O in element mapping were attributed to the HNTs and the uniform distribution in the nanostructures.When we compare pristine HNT; with the 20% HNT-containing nanocomposite, the decrease in the binding energy of Si 2p is due to the hydrogen bond formed between halloysite and polymer.

Brunauer, Emmett, and Teller (BET) surface area analysis
BET surface area analysis is investigated on HNT, pristine copolymer, and nanocomposites by using nitrogen adsorption/desorption isotherms and are treated according to the Brunauer-Emmett-Teller (BET) theory.Halloysite nanotubes (HNTs) are a natural aluminosilicate clay mineral with a large specific surface area, low cost, and abundant storage [45] .The multi-point BET surface area, S BET obtained for the HNT sample, is 91.77m 2 g À1 (Figure 4).The regular tubular morphology, bulk structure, rich mesoporous, high aspect ratio, small dimension, and high strength suggest that HNTs have potential uses in high-performance polymeric nanocomposites [46] .Geometrically, the tube-like morphologies with a proper aspect ratio generate few opportunities for large-area contact between tubes [7] .HNT pore size and pore volume are also determined by Density Functional Theory Method (DFT) and Barrett-Joyner-Halenda Method (BJH) methods are listed in Table 4. BET analysis was also employed to configure the surface area, pore volume, and pore size of the nanocomposites.The copolymer surface area found as near zero.When the HNT amount increased in nanocomposite structures, the surface area is also increased and max was found H3-P(MAalt-VA) as 7.386 m 2 /g.The best adsorption capacities should be achieved by a large specific surface area of the adsorbent material.This increase in the surface area might be due to the proposed synthetic method for nanocomposites as in situ charge transfer complex (CTC) method which made the nanocomposites more porous structure.

High-resolution transmission electron microscope (HRTEM) analysis
A transmission electron microscope at high-resolution mode called HRTEM describes the diffraction pattern and layer orientation in the structure.Different layers structures and deposition of different materials could be distinguished apparently from the micrograph.To confirm the state of dispersion of nanotubes in the copolymeric matrix, HRTEM analysis for nanocomposite with the highest content of the HNT of the H20-P(MA-alt-VA) hybrid system was also carried out (Figure 5).It can be seen that the dispersed halloysite nanotubes are long and short forms in the matrix.Probably, some of the HNTs tubular forms were broken during compounding [47,48] .On the HRTEM images, a few agglomerates of nanotubes can be seen.At the higher magnification on the TEM image, it can be seen that the individual and entangled agglomerates of HNTs were embedded in the organic copolymeric matrix [29] .

Cell viability and MTT assay
Cytotoxicity of nanostructures was assessed by MTT assay.Human gingival fibroblasts were treated with different concentrations of (25, 50, and 500 mg/mL) poly(MA-alt-VA), H1-P(MA-alt-VA), H3-(MA-alt-VA), H5-P(MA-alt-VA), and H20-P(MA-alt-VA) for 24, 48, and 72 h.Results indicated that nanocomposites did not show any cytotoxic effects on human gingival fibroblasts for all incubation periods and the difference between the treatment group and control is not statistically significant (p > 0.005).Nanocomposites treated groups had the same cell viability as the control group.The difference between the nanocomposites treated groups and the control group was not significant for all concentrations (25, 50, and 500 mg/mL) (p > 0.005).Figure 6 represents cell viability results of 48 (Figure 6), 24, and 72 h treatments also did not show cytotoxic effects on gingival fibroblasts (Supplementary Figures S1 and S2).
We also visualize cell morphology and viability with AO/ PI staining which is another independent method to test cell viability.Living cells stained with AO/PI fluoresce green under darkfield fluorescence microscopy, while nonviable cells fluoresce orange [49] .Parallel to the MTT assay results, the AO/PI staining of cells showed that at the highest concentration (500 mg/mL) of treatment with all nanocomposite groups, the viable cell number (stained green) is close to the control group (Figure 7).
For all nanocomposite treatment groups, viable cells observed green alike control group.These results also supported our MTT assay results.Determine a cell type for an in vitro cytotoxicity assay was crucial to show the safety of the materials.The cell type can be a cell line that is widely used or a primary cell line according to the scope of study [50,51] .The dental cement contains a polymerized form of maleic anhydride (MA) and vinyl acetate (VA) [52,53] .Therefore, it is important to measure the cytotoxic effects of nanocomposites on gingival fibroblasts.Our study showed that despite the highest concentration of nanocomposites there is no cytotoxicity on gingival cells.These results revealed the biosafety and the potential of nanocomposites for dental applications.

Conclusion
Poly(maleic anhydride-alt-vinyl acetate) and its halloysite nanostructures containing anhydride groups were synthesized via charge transfer complex (CTC) radical polymerization designed for potential functional polymeric dental fillings or bone cement materials.All synthesized materials peculiarities were characterized and enlightened by spectroscopic methods by ATR-FTIR, XRD, and XPS.The structure-property relationships were clarified in detail by comparing HNT, copolymer, and nanocomposites with the used spectroscopic methods used.
Surface area, pore volume, and pore size were determined by Brunauer-Emmett-Teller (BET) method.Morphological characterization of polymer/HNT (PHNT) nanotubes was done the High-Resolution Transmission Electron Microscope (HRTEM) method.The evidence of successfully synthesized nanostructures can be given as an increment of the surface area of nanocomposites and also TEM images.
The biological activity of synthesized copolymer and its nanocomposites showed no cytotoxicity for gingival cells.Because of their chemical properties and nontoxic effects on cells, these nanocomposites are novel materials for treating dental injuries, repair, restoration, and replacement.