Colloidal strategies for electrophoretic deposition of organic-inorganic composites for biomedical applications

Colloids and Surfaces A: Physicochem. Eng. Aspects 516 (2017) 219–225

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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa

Colloidal strategies for electrophoretic deposition of organic-inorganic composites for biomedical applications A. Clifford 1 , D. Luo 1 , I. Zhitomirsky ∗ Department of Materials Science and Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S4L7, Canada

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• Poly(styrene-alt-maleic acid) (PSM) • • • •

was used as a chelating dispersant for particles. PSM allowed for electrophoretic deposition (EPD) of biomaterials and composites. Hydroxyapatite, bioglass, TiO2 , Al2 O3 and carbon nanotubes were deposited. Methods for co-deposition of PSM with albumin and hemoglobin were developed. PSM adsorption, EPD mechanisms, film morphologies and applications are discussed.

a r t i c l e

i n f o

Article history: Received 28 September 2016 Received in revised form 20 December 2016 Accepted 21 December 2016 Keywords: Hydroxyapatite Bioglass Bioceramics Albumin Hemoglobin Biopolymer Electrophoretic deposition Film

a b s t r a c t Poly(styrene-alt-maleic acid) (PSM) exhibits a number of unique properties, which are important for diverse biomedical applications. A conceptually new strategy has been utilized for dispersion, charging and electrophoretic deposition (EPD) of advanced materials for biomedical applications, such as hydroxyapatite, bioglass, TiO2 and Al2 O3 using PSM. The approach is based on the use of chelating properties of maleic acid monomers, which created multiple bonds with surface atoms on the particle surface and allowed strong PSM adsorption and efficient particle dispersion. The deposition kinetics and mechanism have been investigated and the advantages of PSM have been discussed. Our new findings in the colloidal electrochemistry allowed for the co-deposition of PSM with hemoglobin and albumin, which have been utilized as model proteins for the development of the deposition mechanism. Another major finding was the possibility of efficient deposition of various composites, containing PSM, hydroxyapatite, bioglass, TiO2 , Al2 O3 , proteins and carbon nanotubes. Comprehensive electron microscopy data was used for the analysis of film morphologies at different experimental conditions. The results of this investigation paved the way for EPD of composite films, utilizing properties of different functional biomaterials. © 2016 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. E-mail address: [email protected] (I. Zhitomirsky). 1 The authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfa.2016.12.039 0927-7757/© 2016 Elsevier B.V. All rights reserved.

Electrophoretic deposition (EPD) is gaining increasing interest as a colloidal processing technique for a variety of technical applications [1,2]. This method can be combined with other

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Fig. 1. TEM image of HA particles, prepared by a chemical precipitation method. Inset shows a selected area electron diffraction pattern.

electrochemical strategies [3–5] for the deposition of composite films and coatings with advanced functionality. EPD offers many advantages for the deposition of films for biomedical applications [6–8]. Organic–inorganic biocomposite films have attracted substantial attention due to the possibility of combining the properties of organic and inorganic components [9]. Many investigations focused on the co-deposition of biocompatible polymers with inorganic particles [6,10]. The progress in EPD of biocompatible polymers and composites offers important opportunities in the development of advanced films and coatings for biomedical implants and devices [10–12]. Recently significant interest has been generated in the biomedical applications of poly(styrene-alt-maleic acid) (PSM) [13,14]. Nanoparticles of PSM were utilized as colloidal carries for anticancer agents. It was found that PSM and its derivatives allow for significant improvement in the pharmacological properties of various drugs [13,15]. The PSM formulated drugs showed high tumor targeting efficiency and low toxicity [13,14]. The relatively strong bonding of various drugs to PSM was beneficial for controlled drug release. Another important finding was the strong non-covalent bond between albumin and PSA, which allowed improved PSM particle stability in blood plasma [13]. The analysis of PSM interactions with phospholipids resulted in the synthesis of PSM-phospholipid complexes, which showed promising properties for various biomedical applications [16]. PSM was used for the immobilization of hemoglobin and the fabrication of biosensors [17]. Ag loaded PSM composites with antiseptic properties have been developed for biomedical devices [18]. PSM interactions with proteins showed that PSM is beneficial for the surface modification of various biomaterials [19]. Of particular interest are the important structural features of PSM, which the literature has not paid sufficient attention to. The structure of the maleic acid monomer includes two carboxylic groups (Fig. 1A), which make PSM a strong complexing agent. The two adjacent carboxylic groups form complexes with metal ions in solutions [20,21]. The chelating PSM polymer has been used for the removal of heavy ions from water [22,23]. It has also been reported that the PSM complexes exhibit valuable luminescence properties [24]. The chelating properties of PSM were utilized for the synthesis of inorganic materials. Particles with different morphologies were prepared by chemical precipitation methods in the presence of PSM [25–27]. A polymer mediated mineralization method was developed using the chelating PSM polymer for the fabrication of mesoscale-organized particles with unusual superstructures [28]. PSM has been utilized for the fabrication of one-dimensional nanostructures by hydrothermal synthesis [29]. New techniques

for template synthesis have been developed based on the chelating properties of PSM [30,31]. The chelating properties and biocompatibility of PSM are of particular interest for application in EPD of biomaterials. Particles of inorganic biomaterials must be well dispersed and charged in suspensions for EPD [10,32]. The adsorption of a dispersant is important for the fabrication of stable suspensions, because a non-adsorbed ionic dispersant promotes particle coagulation and sedimentation [33]. Previous investigations [33] showed the advantages of small organic dispersant molecules, containing chelating groups. Such molecules adsorbed on inorganic particles by chelating bonding to the metal atoms on the particle surface. However, the single chelating groups provided relatively weak bonding to the particle surface. In contrast, substantial improvement in adsorption and dispersion may be possible using chelating polymers, such as PSM. The chelating monomers of PSM can provide multiple adsorption sites for strong PSM adsorption on inorganic particles. Moreover, compared to small dispersant molecules, PSM offers the advantages of electrosteric stabilization. The goal of this investigation was EPD of biomaterials using PSM as a charging and dispersing agent. We targeted the deposition of films of advanced inorganic materials for biomedical implants, such as hydroxyapatite, bioglass, TiO2 and Al2 O3 . The analysis of experimental data provides an insight into the influence of PSM structure on the polymer adsorption, particle dispersion and EPD yield. The results presented below indicated that the use of PSM allows for EPD of inorganic materials at high deposition rates. Moreover, we report that PSM can be used for the deposition of other functional biomaterials, such as albumin and hemoglobin. We demonstrate that the problem of protein charge reversal, related to pH changes at the electrode surface, can be avoided and propose a mechanism, explaining EPD of albumin and hemoglobin. Another important finding was the possibility of co-deposition of different materials using PSM as a co-dispersant and formation of composite films. Comprehensive electron microscopy data was used for the analysis of film morphologies at different experimental conditions.

2. Experimental procedures Poly(styrene-alt-maleic acid) (PSM), TiO2 (anatase, 25 nm), human hemoglobin (Hb), bovine serum albumin (BSA), Ca(NO3 )2 ·4H2 O, (NH4 )2 HPO4 , NH4 OH (Aldrich, Canada), Al2 O3 (0.13 ␮m, Baikowski, USA), multiwalled carbon nanotubes (MWCNT, ID 4 nm, OD 13 nm, length 1–2 ␮m, Bayer,Germany) ® were used in this work. Bioactive glass powder (45S5 Bioglass ) of composition (wt%): 45% SiO2 , 24.5% Na2 O, 24.4% CaO and 6% P2 O5 and average particle size ∼5 ␮m was supplied by MO-SCI Corporation, USA. The procedure for the preparation of stoichiometric hydroxyapatite (HA) nanoparticles for EPD was based on that described in a previous work. Precipitation was performed at a temperature of 70 ◦ C by a slow addition of 0.6 M (NH4 )2 HPO4 solution into 1.0 M Ca(NO3 )2 solution. The pH of the solutions was adjusted to 11 with NH4 OH. Stirring was performed for 8 h at 70 ◦ C and 24 h at room temperature. The precipitate was washed with water and finally with isopropyl alcohol. It has been previously reported that this method resulted in the formation of crystalline HA. Fig. 1 shows a TEM image of HA particles, which exhibit a needle-like morphology with a typical length of 150 nm and aspect ratio of about 8. The electron diffraction pattern (Fig. 1, inset) confirms the HA crystallinity. The electrochemical cell for EPD included a stainless steel substrate and Pt counter electrode. The distance between the substrate and counter electrodes was 15 mm. The deposition voltage was varied in the range of 5–30 V. The deposition time was varied in

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Fig. 3. Deposit mass versus deposition time for 10 g L−1 suspensions of (a) HA, (b) bioglass, (c) TiO2 and (d) Al2 O3 , in ethanol-water solvent (60% ethanol), containing 1 g L−1 PSM at a deposition voltage of 30 V.

Fig. 2. (A) Chemical structure of PSM, (B) adsorption of PSM on an inorganic particle, involving complexation of a metal atom (M) on the particle surface.

the range of 1–4 min. EPD of HA, bioglass, TiO2 and Al2 O3 was performed using suspensions in mixed ethanol–water (40% water) solvent, containing dissolved PSM. The use of mixed ethanol–water solvent offered the advantage of reduced gas evolution at the electrode surface during EPD. However, water was used as a solvent in all deposition experiments involving BSA or Hb in order to avoid reactions of BSA or Hb with ethanol. The deposition yield was studied for the films deposited on stainless steel substrates. Electron microscopy investigations were performed using a JEOL JSM-7000F scanning electron microscope (SEM) and FEI Tecnai Osiris transmission electron microscope (TEM). FTIR studies were performed on Bruker Vertex 70 spectrometer. 3. Results and discussion PSM is a copolymer of styrene and maleic acid (Fig. 2A). The negative charge and chelating properties of PSM are related to the maleic acid monomer. It was hypothesized that chelating

properties can be beneficial for the PSM adsorption on inorganic nanoparticles. The analysis of the literature data on the chelation of ions in PSM solutions indicated that two carboxylic groups of the maleic acid monomers are involved in the complex formation [21,31]. A similar mechanism can provide PSM adsorption on particles. In this mechanism metal atoms on the particle surface are involved in the complex formation with two carboxylic groups of the maleic acid monomers (Fig. 2B). Our experimental results showed that PSM adsorbed on advanced materials for biomedical applications, such as HA, bioglass, TiO2 and Al2 O3 . The addition of PSM to HA, bioglass, TiO2 and Al2 O3 allowed for the fabrication of stable suspensions. The results indicated that anionic PSM adsorbed on the particles and allowed for their dispersion and colloidal stability. The maleic acid monomers provide multiple sites for the PSM adsorption on the particle surface. Compared to small organic molecules [33], containing a single chelating group, the multiple chelating monomers of PSM can potentially allow for stronger adsorption. Moreover, PSM offers the advantage of electrosteric stabilization. The EPD experiments provided further evidence of PSM adsorption on the inorganic particles. It was found that HA, bioglass, TiO2 and Al2 O3 particles were negatively charged in the suspensions

Fig. 4. SEM images at different magnifications of films, prepared from (A,B) 10 g L−1 HA, (C,D) 10 g L−1 bioglass and (E,F) 5 g L−1 HA and 5 g L−1 bioglass suspensions in an ethanol-water solvent (60% ethanol), containing 1 g L−1 PSM at a deposition voltage of 30 V, arrows in (E,F) show bioglass particles.

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Fig. 5. SEM images of films, prepared from (A) 10 g L−1 TiO2 , (B) 10 g L−1 Al2 O3 and (C) 1 g L−1 MWCNT suspensions in an ethanol-water solvent (60% ethanol), containing 1 g L−1 PSM at a deposition voltage of 30 V and (D) 1 g L−1 Hb, (E) 1 g L−1 Hb and 1 g L−1 MWCNT, (F) 1 g L−1 BSA suspensions, containing 1 g L−1 PSM in water at a deposition voltage of 10 V.

and deposited on anodic substrates. Fig. 3 shows deposit weight versus deposition time dependences for anodic deposits, prepared from the suspensions of the inorganic particles, containing PSM as a dispersant. The deposit mass increased with increasing deposition time. Therefore, the amount of the deposited material can be varied and controlled. The decrease of the deposition yield with time was related to the formation of the insulating deposit layer on the electrode and corresponding reduction of the voltage drop in the bulk of the suspension [9]. Fig. 3 indicates that relatively high deposition yield can be achieved. The films, prepared by EPD, were studied by SEM. Fig. 4(A,B) shows SEM images of the HA films. The EPD method allowed for the fabrication of continuous, crack free films (Fig. 4A). The SEM image at higher magnification (Fig. 4B) indicated that the film contained non-agglomerated HA particles. The needle shape of the particles observed in the SEM images is in agreement with the TEM data (Fig. 1). The deposited film showed porosity. The pore size was comparable with the size of the particles. Such porosity resulted mainly from the packing of the particles. The SEM image, presented in Fig. 4C indicated the formation of continuous bioglass films. The deposited film contained relatively large particles (Fig. 4D) in agreement with particle size data provided by the powder manufacturer. The EPD literature indicates that the dispersion and EPD of large particles presents difficulties due to particle sedimentation [1,34]. However, strong adsorption of PSM on the bioglass particles allowed for efficient electrosteric dispersion and charging. As a result, the bioglass films were successfully deposited on conductive substrates. The EPD of HA and bioglass can be used for the fabrication of biocompatible coatings for biomedical implants. The chemical composition of HA is similar to that of the mineral part of natural bone [10]. It is known that bioglass is a bioactive material, which readily reacts with physiological fluids, forming HA and creating tenacious bonds to hard and soft tissue [10]. Recently, interest has been generated in the fabrication of HA-bioglass composites with high biocompatibility and bioactive properties [10]. The use of PSM as a co-dispersant for HA and bioglass allowed for co-deposition of both materials and the formation of composite films by EPD. Fig. 4E,F shows SEM images of the composite films, which include needle shape HA particles and

larger bioglass particles of irregular shape. The EPD method allowed for the fabrication of continuous films of HA-bioglass composites. Deposition of advanced bioceramics, such as TiO2 and Al2 O3 , was also achieved by EPD. The SEM images (Fig. 5A,B) show porous microstructure of the deposited films. Moreover we discovered the possibility of dispersion and EPD of MWCNT films (Fig. 5C) using PSM. It is suggested that the PSM adsorption on MWCNT involved the ␲-␲ interactions of the styrene monomers of PSM with MWCNT. Another major finding of this study was the possibility of codeposition of PSM with proteins. Hb and BSA were used as model proteins for the development of the deposition mechanism. It is important to note that individual materials, such as PSM, Hb and BSA cannot be deposited by EPD. Previous investigations showed

Fig. 6. FTIR spectra of (a) as received PSM, and materials, deposited by EPD from aqueous solutions of (b) 1 g L−1 Hb and (c) 1 g L−1 BSA, containing 1 g L−1 PSM at a deposition voltage of 10 V.

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Fig. 7. Schematic of the EPD co-deposition mechanism of Hb or BSA with anionic PSM. The deposition mechanism involves electrophoretic transport of negatively charged proteins and PSM toward the anode in the basic bulk solutions, their accumulation at the electrode surface, charge reversal of the proteins, electrostatic coagulation and film formation.

Fig. 8. SEM images of films prepared from aqueous suspensions of (A) 0.5 g L−1 HA, (B) 1 g L−1 HA, (C) 0.5 g L−1 bioglass, (D) 1 g L−1 bioglass, containing 1 g L−1 PSM and 1 g L−1 BSA at a deposition voltage of 10 V.

that EPD involves two processes: electrophoresis and film formation [34]. Electrophoresis results in the accumulation of charged particles at the electrode surface, then the particles must coagulate to form a film. The difficulties related to EPD of PSM films result from the mutual electrostatic repulsion of the PSM molecules at the electrode surface, which prevents film formation. Another problem is related to EPD of proteins. The total charge of Hb and BSA represents the sum of pH-dependent charges of all ionisable cationic and anionic groups. It is known that Hb and BSA have isoelectric points (pI) of ∼7 and ∼5, respectively [35,36]. In aqueous solutions at pH > pI the Hb and BSA molecules acquire a negative

charge and move toward anode under the influence of an electric field. However, the pH decrease at the anode surface due to the reaction 2H2 O → O2 + 4H+ + 4e−

(1)

results in the charge reversal of the protein molecules. In this case the electrostatic repulsion forces prevent the deposition of positively charged proteins at the anode. We found that Hb and BSA can be successfully co-deposited with PSM. Relatively thick films with thickness of 1–10 ␮m can be obtained by the variation of the deposition voltage in the range

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Fig. 9. SEM images of films prepared from aqueous suspensions of (A) 0.5 g L−1 Al2 O3 , (B) 1 g L−1 Al2 O3 , (C) 0.5 g L−1 TiO2 , (D) 1 g L−1 TiO2 , containing 1 g L−1 PSM and 1 g L−1 BSA at a deposition voltage of 10 V.

of 5–10 V and deposition time in the range of 1–10 min. Fig. 5D shows an SEM image of a film, deposited from 1 g L−1 Hb solution in water, containing 1 g L−1 PSM. The EPD method allowed for the fabrication of continuous and crack free films. Moreover, MWCNT can be incorporated into the Hb-PSM films (Fig. 5E). The comparison of the SEM images, shown in Fig. 5C and E indicated that films prepared without Hb were porous, whereas the addition of Hb to the PSM solutions, containing dispersed MWCNT, resulted in the formation of relatively dense films. The SEM image presented in Fig. 5F shows that continuous, dense and crack free BSA-PSM film can be prepared by EPD. The deposited films were removed from the substrates and studied by FTIR. The FTIR spectrum of as-received PSM has been analyzed for the comparison. The spectrum of PSM (Fig. 6(a)) showed absorptions at 1401 cm−1 , attributed to the C O vibrations [20]. The C C stretching vibrations of the aromatic ring of a styrene monomer [20] contributed to absorptions at 1555, 1492, 1453 cm−1 . Similar absorptions at 1496 and 1453 cm−1 were found in the spectra of the deposited materials. The deposited materials showed absorptions at 1645 cm−1 for Hb (Fig. 6b) and 1647 cm−1 for BSA (Fig. 6c), attributed to amide I band [37,38]. Moreover, the deposits showed absorptions at 1525 and 1531 cm−1 related to amide II band [37,38] of Hb and BSA, respectively. Therefore, the FTIR studies of the deposited materials provided evidence of the formation of composite Hb-PSM and BSA-PSM films. Fig. 7 presents a schematic of the suggested deposition mechanism. In basic solutions at pH = 8 the electrophoretic motion of PSM and Hb or BSA (Fig. 7) resulted in the accumulation of the PSM polymer and proteins at the anode surface, where pH change resulted in the protein charge reversal. The electrostatic coagulation of positively charged proteins and negatively charged PSM resulted in electrostatic coagulation and film formation. It is known [39–42] that BSA forms insoluble complexes with anionic polyelectrolytes at pH below the pI. The formation of such complexes can promote the film deposition. The suggested mechanism was also confirmed

by the analysis of mixed Hb-PSM and BSA-PSM solutions, which showed precipitation at pH ∼ 3. The composite films, containing Hb and MWCNT can be used for application in biosensors [43], utilizing catalytic properties of Hb and conductivity of MWCNT. Methods of surface modification of biomaterials with BSA are currently under intensive investigation for the development of advanced surfaces with improved biocompatibility [44], reduced bacterial adherence [45] and improved anti-thrombogenic properties [46,47]. Therefore, the method for the deposition of BSA-PSM films has been further extended to the deposition of composites, containing HA, bioglass, TiO2 and Al2 O3 . Fig. 8 shows SEM images of the composite films, containing HA and bioglass in the BSA-PSM matrix. The analysis of the SEM images indicated that the increase in the particle concentration in suspensions resulted in increasing concentration of the particles in the deposited material. The films, prepared using BSA and PSM were relatively dense, compared to the films prepared without BSA (Fig. 4). Fig. 9 shows SEM images of the composites, containing Al2 O3 and TiO2 in the BSA-PSM matrix. The BSA-PSM material filled the voids between the inorganic particles, resulting in relatively dense films, compared to the films prepared without BSA (Fig. 5). The analysis of the SEM images indicated that the composition of the films can be varied by the variation of the concentration of the inorganic particles in the suspension. The increase in the concentration of the particles in the suspension resulted in their larger concentration in the film. 4. Conclusions PSM showed strong adsorption on particles of advanced inorganic materials for biomedical applications, such as HA, bioglass, TiO2 and Al2 O3 . The chelating monomers of maleic acid provided multiple adsorption sites by complexation of atoms on the surface of inorganic particles, which make PSM a versatile dispersing and charging agent for EPD of various materials. The deposition

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yield can be controlled by the variation of deposition time at a constant voltage. The use of PSM as a co-dispersant allowed for codeposition of materials and formation of composites, containing different inorganic particles. Further development and application of new chelating polyelectrolytes is a promising approach in EPD and other colloidal technologies. The chemical structure of PSM was beneficial for PSM adsorption on MWCNT, which allowed for MWCNT dispersion and EPD. PSM, Hb and BSA cannot be deposited by EPD individually, however the method developed in this investigation allowed for the fabrication of PSM–Hb and PSM–BSA films by EPD. The approach developed in this investigation can be further utilized for the EPD of other zwitterionic biomaterials, such as proteins, enzymes and drugs. We found that composite materials, containing PSM, BSA, Hb, MWCNT, HA, bioglass, TiO2 and Al2 O3 can be prepared by EPD for diverse biomedical applications in biosensors and biomedical implants.

Acknowledgement The authors gratefully acknowledge the Natural Sciences and Engineering Research Council of Canada for the financial support

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