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poly ε-caprolactone antibacterial nanocomposite blends for medical applications
Accepted Manuscript Title: Interface modified Polylactic acid/Starch/Poly caprolactoneAntibacterialNanocompositeBlendsforMedicalApplications
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Author: Seyed Mohammad Davachi Behzad Shiroud Heidari Iman Hejazi Javad Seyfi Erfan Oliaei Arman Farzaneh Hamid Rashedi PII: DOI: Reference:
S0144-8617(16)30972-9 http://dx.doi.org/doi:10.1016/j.carbpol.2016.08.037 CARP 11462
To appear in: Received date: Revised date: Accepted date:
18-7-2016 11-8-2016 11-8-2016
Please cite this article as: Davachi, Seyed Mohammad., Heidari, Behzad Shiroud., Hejazi, Iman., Seyfi, Javad., Oliaei, Erfan., Farzaneh, Arman., & Rashedi, Hamid., Interface modified Polylactic acid/Starch/Poly -caprolactone Antibacterial Nanocomposite Blends for Medical Applications.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.08.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interface modified Polylactic acid/Starch/Poly ε-caprolactone Antibacterial Nanocomposite Blends for Medical Applications Seyed Mohammad Davachia,*, Behzad Shiroud Heidaria, Iman Hejazia, Javad Seyfib, Erfan Oliaeia, Arman Farzaneha, Hamid Rashedic a
Applied Science Nano Research Group, ASNARKA, P.C. 1619948753, Tehran, Iran. Department of Chemical Engineering, Shahrood Branch, Islamic Azad University, P.O. Box 36155-163, Shahrood, Iran. c Department of Biotechnology, School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran b
*
Corresponding Author. Email address: [email protected] (S.M. Davachi) Tel/Fax: +98 -21 – 77655928
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Graphical Abstract
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Highlights
Antibacterial nanocomposite blends with encapsulated drug prepared via melt blending. Increase of nHA enhances hydrophilicity, antibacterial activity and drug release. Adverse effect of antibacterial drug, was eliminated due to the presence of nHA. 3% nHA showed good adjustment between hydrolytic degradation and release profile. Electrospun microfibers of optimum blend demonstrated an improved cell attachment.
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Abstract In this study, an optimized interface-modified ternary blend with antibacterial activity based on polylactic acid/starch/poly ε-caprolactone (PLASCL20), mixed with nano hydroxyapatite (nHA) via melt blending. This method results in a homogeneous nanocomposite blend in which the addition of 3% nHA improves the overall properties such as hydrolytic degradation, hydrophilicity, antibacterial activity and the drug release comparing to PLASCL20. Moreover, the simultaneous use of nHA and encapsulated triclosan (LATC30) compensated the negative effect of triclosan through increasing the possible cell attachment. According to the contact angle results, nHA was thermodynamically driven into the interface of PLA and PCL/Starch phases. The addition of 3% nHA showed a good adjustment between the hydrolytic degradation and the release profile, therefore, their electrospun microfibers demonstrated an improved fibroblast (L929) cell attachment. The aforementioned nanocomposite blend is a suitable antibacterial candidate for many medical applications with minimum side effects due to the controlled release of triclosan.
Keywords: PLA, PCL, Starch, Interface modified blend, Antibacterial, Nano hydroxyapatite.
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1. Introduction: Biodegradable polymers, and polymers derived from renewable resources, currently entice a good deal of interest because of their environmental friendliness, since they offer a proper combination of biodegradability, compostability, and compatibility (Mofokeng & Luyt, 2015). Polylactic acid (PLA) is a semi-crystalline thermoplastic which can be obtained from renewable resources such as starch. PLA is being used in a wide variety of applications in diverse fields because of its extremely good tensile strength and high modulus. However, this polymer is very brittle having serious physical aging issues for the duration of its service life (Seyed Mohammad Davachi & Kaffashi, 2015a) and has hydrophobic character limiting its use in medical and scientific applications due to the reduced level of cellular attachments (Torabinejad, Mohammadi-Rovshandeh, Davachi, & Zamanian, 2014). Therefore, blending or copolymerizing PLA with a polymer with a softer nature would be a great alternative for modifying its brittleness (Mofokeng & Luyt, 2015; Navarro-Baena et al., 2016; Torabinejad et al., 2014). Poly ε-Caprolactone (PCL) is a semi-crystalline thermoplastic polyester which provides high flexibility, low melting point, thermal stability and excellent compatibility with other polymers, even though its high elongation, low modulus, and higher price in comparison to the conventional polymers could be regarded as limitations to its applications (Mittal, Akhtar, & Matsko, 2015). Starch is doubtlessly a beneficial material for biodegradable plastics due to its value and natural abundance. Nevertheless, it has high water absorption and weak mechanical properties such as brittleness as compared with the alternative thermoplastic polymers. Blending starch with PLA increased PLA’s hydrophilicity (Mohammadi-Rovshandeh et al., 2014) and blending it with PCL expanded the biodegradation rate of PCL (Mittal, Akhtar, & Matsko, 2015). An alternative to conquer PLA and PCL drawbacks could be mixing them collectively with starch (Carmona, Corrêa, Marconcini, & Mattoso, 2015). There are several studies that have investigated the ternary 5
blends of PLA, PCL and thermoplastic starch (TPS) (Carmona et al., 2015; Liao & Wu, 2009; Mittal, Akhtar, Luckachan, & Matsko, 2015; Mittal, Akhtar, & Matsko, 2015; Sarazin, Li, Orts, & Favis, 2008), however in our recent study, a ternary blend of PLA, PCL and starch with an interface change of PCL and starch turned into organized (Davoodi et al., 2016). Sarazin et al. (Sarazin et al., 2008) produced the binary and ternary blends of PLA, PCL, and TPS using two different types of starch in a single-step extrusion process and demonstrated their superior physical properties. TPS grades with glycerol contents of 24 and 36 % have been used, and the obtained blends alleviated the weaknesses of each polymer. Liao et al. lowered the price of PLA/PCL blends by adding starch into these blends. In fact, the problem of poor compatibility between PLA/PCL blend and starch was surmounted by grafting acrylic acid to the PLA/PCL blend. A chemical reaction took place between –OH group of starch and –COOH group of AA-gr-PLA/PCL(Liao & Wu, 2009). Mittal et al. studied the structural characterization and time-dependent morphological modifications in addition to the mechanical, thermal, rheological and morphological properties of binary and ternary blends of PLA, TPS and PCL (Mittal, Akhtar, Luckachan, et al., 2015; Mittal, Akhtar, & Matsko, 2015). Carmona et al. modified this blend by adding 2 wt% of Methylene diphenyl diisocyanate (MDI), which increased the melt viscosity of the ternary blend by the urethane linkages which was formed with TPS and it also was efficient to acquire notable enhancements in tensile strength and ductility of the ternary blend (Carmona et al., 2015). The mentioned researches had been conducted with an objective of enhancing the properties of PLA. The mentioned biomaterials possess no intrinsic antibacterial properties, and consequently, to prevent the formation of micro-organisms, anti-infection modifications of polymers were implemented (Seyed Mohammad Davachi & Kaffashi, 2015b; Seyed Mohammad Davachi, Kaffashi, Zamanian, Torabinejad, & Ziaeirad, 2016; Khakbaz et al., 2015, 2016). Within the last decade, several compounds have been used as antibacterial
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retailers, from which silver and triclosan had been widely employed in clinical programs. Triclosan is a famous commercial and a Food and Drug Administration (FDA) authorizedsynthetic-non-ionic-broad-spectrum antimicrobial agent that was chosen as the antibacterial agent in this study (Makarovsky, Boguslavsky, Lellouche, Banin, & Lellouche, 2012). It is chemically stable and may be heated up to 200°C for up to two hours while showing no degradation, so it is suitable for incorporation into diverse bolstered plastic substances (Suller & Russell, 2000). Nanohydroxyapatite (nHA) is the major inorganic component of hard tissue and is an ideal bioactive material for orthopedic applications exhibiting a bone bonding capacity under in-vivo situations (Dadbin & Naimian, 2014; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Hule & Pochan, 2007). nHA have mechanical properties similar to the natural bone, and because of its small size and the large specific surface area, it showed a massive growth in protein adsorption and osteoblast cellular adhesion comparing to the micron sized ceramics (Sui et al., 2007). However, the brittleness, migration from the implanted sites and loose agglomeration are the primary troubles of nHA which could be overcome by incorporation into polymer matrices (Dadbin & Naimian, 2014; Hule & Pochan, 2007; Sarvestani & Jabbari, 2006). In our preceding work, PLLA/TC nanoparticles with the composition of 70/30% (LATC30) were prepared by the emulsification–diffusion technique which demonstrated encapsulation performance, high molecular dispersion, and good release profiles (Seyed Mohammad Davachi & Kaffashi, 2015b). LATC30 was added to a PLLA with higher molecular weight through melt mixing to enhance the overall properties and the antibacterial activity, which 5% of LATC30 showed surest effects, therefore, the actual dosage of TC is 1.5% since the nanoparticles consist of 30% TC (Seyed Mohammad Davachi & Kaffashi, 2015b; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). Hybrid nanocomposites of PLLA, LATC30, and nHA were also studied, and by the simultaneous use of nHA and LATC30, the detrimental impact of
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triclosan on the cells was eliminated (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). In our recent publication, an interface-modified ternary blend based on PLA/PCL/starch was prepared via melt blending (Davoodi et al., 2016). It needs to be mentioned that no modification was done on the starch unlike the previous studies, and the addition of LATC30 imparted antibacterial property to the blend. The interfacial affinity among hydrophilic starch and hydrophobic polyesters was enhanced by interactions between triclosan –Cl groups and PCL ester groups (Kweon, Kawasaki, Nakayama, & Aiba, 2004), while the PCL and starch were chemically attached due to the inclusion complexes of starch (Lay Ma, Floros, & Ziegler, 2011; Polaczek, Starzyk, Maleńki, & Tomasik, 2000). In the current study, the optimum PLASCL blend of our previous work (PLASCL20) (Davoodi et al., 2016) was used and nHA with various quantities was added via an internal mixer to prepare the blend nanocomposites. The main novelty of the current work is the simultaneous application of nHA and encapsulated triclosan in a novel interface modified PLASCL blend (ternary blend) that has not been addressed so far, to eliminate the unfavorable impact of triclosan by increasing the possible cell attachment due to the existence of nHA making the blend more suitable for medical applications. 2. Materials and methods 2.1. Materials PLLA was synthesized from the purified L-lactide prepared by L-lactic acid (S. M. Davachi, Kaffashi, & Roushandeh, 2012; S.M. Davachi, Kaffashi, Roushandeh, & Torabinejad, 2012) via ring opening polymerization reactive extrusion method which has been fully described elsewhere (Seyed Mohammad Davachi & Kaffashi, 2015b). The weight average molecular weight (Mw) and polydispersity index (PDI) of the PLLA were 120,000 g.mol-1 and 1.78, respectively. Poly (ɛ-caprolactone) with a molecular weight (Mn) of 80,000 purchased from Sigma-Aldrich (Germany). The technical grade corn starch supplied by Glucosan Co. (Iran) 8
used in this study. The moisture content of the starch measured by drying the starch in an oven heated up to 105°C for 24h was 12 wt%. Triclosan (TC) was a generous gift from KAF Co. (Branch of DAROUGAR group, Iran) originally was Dekaben TC Premium, Jan Dekker, Netherlands. Nano hydroxyapatite or nHA (KF-HAP04) with a size ranging from 20 to 40 nm and 99% purity purchased from Kinfon Pharma, China. The PLLA/TC nanoparticles (LATC30) were prepared according to the procedure mentioned in our previous work (Seyed Mohammad Davachi & Kaffashi, 2015b), based on which the PLLA/TC nanoparticles were synthesized by the modified emulsification–diffusion process, and the sample containing 30% TC and 70% PLLA demonstrated the highest encapsulation efficiency with an approximate size of 100–300 nm. From then on, PLLA is referred as PLA. All the other chemical and solvents were reagent grades procured from Merck (Darmstadt Germany).
2.2. Sample preparation Prior to the blending process, the PLA, LATC30 and starch granules were vacuum dried at 80°C for 4h, and PCL granules were also vacuum dried at 30oC for 12h. Afterwards, they were melt-blended via an internal mixer (Brabender 50 EHT at a rotational speed of 60 rpm for 20 min). The components were sequentially loaded into the mixing chamber with various compositions. The stock temperature of the internal mixer maintained at 170°C. The obtained blends were molded into 1-2 mm sheets by hot-press at 170°C applying 14 bar pressure for 5 min and then cooled to the room temperature (Seyed Mohammad Davachi et al., 2015). Eventually, the prepared samples were sterilized by ethylene oxide. The samples were named as PLASCL followed by a three-digit number. The first two numbers account for the weight percentage of PCL, which is the optimized composition of the ternary blend (20wt%) according to our previous study (Davoodi et al., 2016). The third number belongs to the nHA ranging from 1 to 7%. Based on the preceding research, 5 wt% LATC30 was added to the 9
100 wt% of the blends (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). The compositions of nanocomposites blends are listed in Table 1 and they were generally coded as PLASCL20X. Table 1. Composition of PLLA/LATC30/nHA nanocomposites Samples PLASCL20 PLASCL201 PLASCL203 PLASCL205 PLASCL207
PLA (wt.%) 50 50 50 50 50
Starch (wt.%) 30 30 30 30 30
PCL (wt.%) 20 20 20 20 20
LATC30 Content (wt.%) 5 5 5 5 5
nHA Content (wt.%) 1 3 5 7
For contact angle measurements, PLA, SCL20 (60wt% starch, 40wt% PCL and a 5% of LATC30 similar to PLASCL20) were separately prepared using an internal mixer. Furthermore, 0.2 g of both TC and nHA powders were pressed at room temperature for 1 min underneath 50 bar loading to prepare flat tablets for contact angle measurement. 2.3. Melt Electrospinning A custom-made melt electrospinning device was used to prepare nonwoven microfibers. PLASCL203 was fed into the vertical steel cylinder of the melt electrospinning device and heated via an electrical heating system to 180oC for about 15 min to ensure complete melting of the sample. The resulted melt pushed out from a 0.58 mm diameter spinneret at a flow rate of 50 µL/h through pressure from a mechanically regulated piston and a voltage of 20 kV was applied. The distance from the spinneret to the collector was 10 cm. All the parameters were optimized according to Hutmacher and Dalton (Hutmacher & Dalton, 2011). 2.4. Characterization The infrared spectroscopy was performed using the FTIR-ATR, Bruker Equinox 55LS 101 series with the resolution of 4 cm−1 (averaging 50 scans) in the frequency range of 600–4000 cm-1, for determining the functional groups. DSC was performed by a Mettler Toledo DSC 1 10
Star device equipped with a low-temperature accessory. Heating scans were repeated to confirm the reproducibility. The temperature scale calibrated with the excessive-purity standards, and the measurements were carried out at a heating rate of 10 °C/min, in the nitrogen atmosphere and at temperatures ranging from -60 to 260°C. The glass transition temperatures (Tg) of samples were taken from the midpoint of the stepwise specific heat increment. All of the adjustments were according to ASTM D3418. The degree of crystallinity (Xc) for samples were determined according to our previous study ( ) (Davoodi et al., 2016), where ΔHm and ΔHc are the enthalpies of melting and cold crystallization, respectively. W and
are the weight fraction of poly(L-lactide) and
melting enthalpy of 100% crystalline poly(L-lactide) or PCL, respectively. The antibacterial activity of nanocomposite blends was investigated against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) bacteria, according to the disc diffusion method. This approach was carried out in a medium solid agar Petri dish. The samples were molded into disc shapes 2.5 cm in diameter, sterilized with ethylene oxide for 2h, and positioned on E. coli and S. aureus cultured agar plates. Then, they were incubated for 24h at 37oC, and the inhibition zones were recorded (Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). To observe the cell viability of the nanocomposites, the MTT assay was carried out on fibroblast cells for the disc shaped samples similar to the preceding studies. The MTT assay was based on the ability of living cells to reduce a tetrazulium-based compound (MTT) to a purplish formazan product (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). The UV–visible BEL SPECTRO LGS53 spectrophotometer (Italy) was used to determine the amount of TC release from the nanocomposite blends, and the release outcomes were verified with the Peppas-Korsmeyer, zero order, first order, Higuchi and Hixon-Crowell models (Costa & Sousa Lobo, 2001; Davoodi et al., 2016). The Kruss 11
G10 instrument (Germany) was employed for measurement of water contact angle to obtain hydrophilicity of the samples. The water contact angle of samples was measured by dropping droplets of ~5 μL at room temperature. The distance of vibrating syringe was ~5 cm, and all the contact angles were obtained at the initial stage (i.e., <10 s). The water contact angle (CA) measurements of each sample were conducted at least three times across the sample surface. Knowledge of thermodynamic equilibrium was helpful in detecting the location of nHA in the blends since the time of melt mixing was sufficiently long for the immigration. The surface free energy of every discrete component of samples were determined using dispersive and polar surface energy (𝛾d and 𝛾p) with a set of test liquids on a solid surface, where s and l represent the solid and liquid surfaces, respectively, and
ϴ
is the contact angle of liquid
droplet on the surface (Equation 1 and 2). The interfacial energy of every two components were obtained from their dispersive and polar parts of surface energies using harmonic-mean and geometric-mean equations reported in equation 3 and 4 where γ i is the surface energy of component i, γ12 is the interfacial energy of components 1 and 2, and d and p superscripts stand for dispersive and polar parts of surface free energy of the components, respectively (Davoodi et al., 2016). (1) (
)
√
√
(2)
Harmonic Mean:
(
Geometric Mean:
(√
) √
(3) )
(4)
Finally, the wetting coefficient (ωa) has been used to predict the thermodynamic equilibrium distribution of nano-fillers in the polymer blend as shown in equation 5. (5)
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For in-vitro hydrolytic degradation studies, the sheet specimens with the 1×1×0.15 cm3 surface area and weight of 0.15 gr were hot pressed and incubated in 50 ml phosphate buffer with pH 7.4 at 37oC while being stirred. The specimens were periodically removed, dried up, weighed and the weight loss percentage was calculated (S. M. Davachi et al., 2012; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). The SEM micrographs were obtained by Vega Tescan, Czech, on the fracture surface of samples in Liquid N2 to observe the dispersion of TC and nHA. 3. Results and discussion 3.1. ATR-FTIR Spectra Figure 1 depicts the FTIR-ATR spectra of PLASCL20X nanocomposites. The C=O stretching vibrations and vibrations of C-O bonds in ester groups (PLA and PCL) display peaks at 1751, 1181 cm-1. The peaks at 1362 and 1453 cm-1 are indicative of the CH3 and CH2 groups of PLLA and PCL, respectively, which appeared due to the physical dispersion of PCL in PLA matrix and also partial miscibility of these polyesters (Davoodi et al., 2016). The C-C peaks can be visible at 870 cm-1. In step with our previous studies, the C-Cl stretching bond shows a peak at 754 cm-1, while the CH stretching and C=C ring-related vibrations multiple bonds occur at ~2940 cm-1 both of which demonstrate the existence of TC (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016).
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Figure 1. FTIR-ATR spectra of PLASCL20X nanocomposites.
The nHA functional groups such as PO43- showed a series of peaks at 560, 594, 1008 and 1220 cm−1. The OH- absorption peaks at 634 and 3027 cm-1 of nHA also appeared with very low intensity. Subsequently, a broad peak observed in the range of 3000-3600 cm-1 indicates the presence of H2O in the nHA powder; however, because of the low intensity of the aforementioned peaks, the amount of H2O is rather insignificant (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). The intensity of the peaks was increased upon addition of nHA. As a result of nHA incorporation, the PO43- and C-O peaks emerged at 1042~1129 and 1453 cm-1, respectively. The increase in the intensity of carbonyl groups at 1751 cm-1 was also observed due to the existence of this functional group in both polymer and nHA. The peaks of the hydroxyl group cannot be observed because of their low severity in nHA, or perhaps, hydrogen bonding with polymer carbonyl groups. It has been suggested that the hydroxyl groups might actively support the cellular attachment and increase the hydrolytic degradation rate in an in-vitro investigation (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). Based on our previous studies, the main structure of PLA and PCL has not been modified during the melt blending, and the results clearly state that 14
since PCL had a chemical reaction with starch and TC due to indexes complexes of starch (Davoodi et al., 2016), nHA could be homogeneously and physically dispersed throughout the blend. 3.2. Thermal Properties Thermal behavior of PLASCL20X nanocomposite blends was studied by DSC technique, as depicted in Figure 2. The important thermal properties of these materials are also reported in Table 2. As is observed, upon addition of nHA, melting temperature (Tm) of PCL phase in nanocomposites was increased comparing to PLASCL20. A slight growth in Tm was also noted, with growth in nHA content. In fact, such rise is quite negligible which suggests that nHA might exist in Starch-PCL phase (SCL) or near the mentioned phase, however, no sign of agglomeration was observed in this phase (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016). In contrast with the PLASCL blends which showed a peak with a small shoulder belonging to the Tg of PLA, such shoulder was not detected in these nanocomposites since they have utterly overlapped with Tm of PCL. In PLASCL samples, both Tm and Xc of PCL were decreased because the thermal histories were supposed to be removed via heating the samples, and due to slow rate of PCL crystallization, the chains had not enough time to form ordered structures (Jenkins & Harrison, 2006); however, upon addition of nHA, both of the mentioned parameters are increased due to the fact that nHA could act as nucleating agent. It was found that upon further enhancement in nHA content, the crystallinity of PCL shows a growing trend. The increased crystallinity has been reported for those systems containing PCL as the dispersed phase within the PLA phase at the matrix (Chavalitpanya & Phattanarudee, 2013; Davoodi et al., 2016; Noroozi, Schafer, & Hatzikiriakos, 2012). The reason for this phenomenon has been ascribed to the fact that the interface of such system can serve as a nucleating agent in the course of crystallization stage of PLA (Kusanagi, 15
Chatani, & Tadokoro, 1997). It was observed that as the nHA content increased, the PLA’s crystallinity was increased comparing to PLASCL20. Another possible reason for the observed growth in Xc is that SCL may disrupt the regularity of the chain structures, and the free space between chains was increased (Davoodi et al., 2016); however, upon addition of nHA, the free space decreased, and as a result, Xc increased. The exothermal peaks right before Tm of PLA phase belongs to the recrystallization of those imperfect crystals of PLA into α crystals of higher perfection, and upon addition of nHA, a slight growth was observed. The Tm shows nearly no change, and the results are in agreement with the previous reports (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016).
Table 2. Thermal and surface behavior of PLASCL20X samples PLASCL20 (Davoodi et al., PLASCL201 PLASCL203 PLASCL205 2016)
Contact Angle
Thermal Properties
Tm (oC) (PCL) Tg (oC) (PLLA) Tm (oC) (PLLA) ΔHm (J/g) (PCL) Xc (%)a (PCL) ΔHm (J/g) (PLLA) Xc (%)b (PLLA)
a,b
θm
53.7
53.63
54.17
50.1
54.33
PLASCL207 54.67
Overlapped with PCL Tm
163.0
163.03
163.04
163.06
163.34
14.8
14.93
15.12
15.48
16.13
53.1
53.59
54.27
55.56
57.89
21.5
22.08
24.13
24.58
24.91
46.2
47.48
51.89
52.86
53.57
68.37
67.11
65.42
62.89
60.18
By considering the melting enthalpy of 100% crystalline PCL as 139.3 J/g (Davoodi et al., 2016) and PLLA as 93 J/g (Seyed Mohammad Davachi & Kaffashi, 2015b)
Finally, it is also evident from Figure 2 that upon addition of nHA, the crystallization peaks become narrower, or the widths of the peaks are reduced, comparing to PLASCL20. Furthermore, the rate of crystallization was enhanced with an increase in nHA content. 16
Figure 2. DSC thermograms of PLASCL20X nanocomposites
3.3. Antibacterial activity, cell viability and in-vitro degradation of PLASCL20X nanocomposite blends To investigate the antibacterial activity of TC in the presence of nHA in PLASCL20X nanocomposite blends, PLASCL203 sample, as a representative, was placed on the seeded agar plates of E. coli and S. aureus to measure the effectiveness of TC. According to Figure 3a and 3b, the inhibition zone around the sample were 4 and 7 mm, respectively. Similar to PLASCL20 and our other studies, the inhibition zone against S. aureus was more pronounced rather than E. coli., which confirms antibacterial activity against both Gram-negative and Gram-positive bacteria, while it has greater activity against Gram-positive species (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016; Davoodi et al., 2016). According to Petersen, TC is able to decrease the bacterial growth and polymer adherences directly from the polymer surface with the
minimum
antimicrobial
release.
Moreover,
secondary bonding
between
the
microorganism and the polymer is considered to be interrupted by TC vibrational fluctuating molecular bond rotations as a possible mechanism to prevent the microbial surface
17
attachments (Petersen, 2016). The MTT assay within three and seven days after seeding was examined for investigation of in-vitro biocompatibility, the effect of nHA content variation on the discharge of TC, and the overall effect on fibroblast cell growth. As depicted in Figure 3c, with an increase in nHA content, the cell numbers are increased due to the better cell attachment on the surface of the nanocomposites (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Torabinejad et al., 2014). It was previously reported that upon addition of PCL, the number of attached cells were decreased due to the weaker attachment of the cells on the surface of the blends (Davoodi et al., 2016). The addition of TC facilitated the attachment of starch and PCL while the blend showed antibacterial activity, the destructive impact of TC decreased, despite the fact that, a slight reduction in MTT assay trend was observed.
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Figure 3. PLASCL20X nanocomposite blends (a) Antibacterial activity against E. coli (b) Antibacterial activity against S. aureus (c) MTT assay after 3 and 7 days (d) Hydrolytic Degradation at 37 oC (e) Accumulative release profile in 6 months
The addition of TC can kill the cells alongside the microbes and bacteria, which is a negative effect of using TC, however by encapsulation the negative effect reduced (Seyed Mohammad 19
Davachi & Kaffashi, 2015b; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016; Davoodi et al., 2016). It was seen that with the addition of nHA and increase in its content, the antimicrobial release was enhanced due to the increased degradation. However, an interesting result in PLASCL20X nanocomposite blends is that with an increase in nHA content, the adverse effect of TC on the cells is disappeared. In fact, the MTT assay results clearly show the cell growth in the presence of antibacterial agent alongside with nHA, which has been previously observed for nHA in PLA matrix (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). For in-vitro degradation investigation, the PLASCL20X samples were immersed in phosphate buffer at pH of 7.4 and temperature of 37oC. PLASCL20 showed a 15-month degradation time, which was faster than PLA and PCL due to the addition of hydrophilic starch (Davoodi et al., 2016). As can be seen in Figure 3d, upon addition of nHA, the blends exhibit faster degradation times comparing to PLASCL20, due to the hydrophilic nature of nHA. With the increase in nHA content, the degradation happens faster, wherein PLASCL201 and PLASCL207 were degraded after 13 and 10 months, respectively. 3.4. Drug release rate studies and kinetics According to the TC calibration curve in our previous work (Seyed Mohammad Davachi & Kaffashi, 2015b), the drug release of the PLASCL20X nanocomposite blends at 37 oC in buffer solution are shown in Figure 3e. The blends exhibit a burst release at the first 24 h which for PLASCL201 and PLASCL203 overall is less than 1 μg/ml, and is in agreement with the previous studies that used LATC30 (Seyed Mohammad Davachi & Kaffashi, 2015b; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016; Davoodi et al., 2016). The amount of release for the samples with different nHA contents is 0.12, 0.14, 0.16 and 0.18 μg/ml every day, which shows a growing trend with an increase in the nHA content. The minimum inhibitory concentration (MIC) of TC is between 0.025- 1 μg/ml for medical and 20
packaging applications, and the obtained results seem suitable (Davoodi et al., 2016; Suller & Russell, 2000). The drug release was monitored up to six months to examine the drug release mechanism for the long-term treatments. The degradation and release time need to show similar profiles since a continuous drug release during the application is desirable. Based on previous results, PLASCL20 degraded and released the drug in 15 months (Davoodi et al., 2016). PLASCL201 released the drug in 13.5 months; PLASCL203 released it in 12 months, which was near their degradation times while the other samples released the drug earlier than their degradation which is not desirable. Based on these results, only PLASCL203 releases the drug precisely within its degradation period. For medical applications, a slower release profile is preferable. The accumulative release profiles for PLASCL blends were analyzed by using the Peppas-Korsmeyer, zero-order, first-order, Higuchi, and Hixon–Crowell models, to investigate the TC release mechanism. Table 3 reports the regression coefficients (R2) as well as their equation constants calculated and fitted to the obtained data. According to the Peppas-Korsmeyer model, the drug delivery mechanism with the increase in nHA content shows different behavior, as it first changes from non-Fickian diffusion (anomalous transport) to Fickian diffusion up to 3% nHA, and with an increase in nHA amount, it again shows nonFickian behavior. The ‘n’ values around 0.5 have to be utilized for the Fickian diffusion of non-well-known pharmaceutical polymeric dosage, and as all the samples show the same range, the ideal result should be obtained at the lower amounts and PLASCL203 has the lowest ‘n’ among the other samples and with round approximation, it can be well fitted to Peppas-Korsmeyer equation (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). Table 3. Regression coefficients and constants of models fitted to the release of triclosan in PLASCL20X blends Peppas-Korsmeyer Zero Order First Order Higuchi Hixon-Crowell Samples R2 n K0 R2 K1 R2 KH R2 Ks R2 PLASCL20 0.9589 0.56 0.0045 0.9986 0.001 0.7331 0.2905 0.9424 -0.0005 0.9063 PLASCL201 0.9595 0.58 0.005 0.9974 0.001 0.7311 0.3052 0.9294 -0.0005 0.9103 PLASCL203 0.9616 0.54 0.0055 0.9986 0.001 0.7051 0.3403 0.9451 -0.0005 0.9239 PLASCL205 0.9561 0.60 0.0064 0.9987 0.001 0.6835 0.3922 0.9443 -0.0005 0.9293
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PLASCL207
0.9454
0.62
0.0073
0.9986
0.001
0.7285
0.4497
0.9395
-0.0006
0.9335
The release of a low-soluble drug in the release environment is verified as the regression coefficients for different blends showed an excellent fitting with the zero-order model. All the samples show relatively high regression coefficient with Higuchi model due to the absorption of TC in phosphate buffer solution. As the drug diffusion is faster than the matrix degradation, the drug release mainly happens due to the diffusion (Davoodi et al., 2016). Because of rather a long-time degradation of PLASCL blends, the release mechanism could not fit well in the first order and Hixon–Crowell models. However, with the increase in nHA content and faster degradation of the matrix, the regression coefficient of Hixon–Crowell is enhanced and displays reasonable regression coefficients due to the consistent amount of LATC30 in the PLASCL20X samples in comparison with the PLASCL20. According to the release studies and the regression coefficients of different models, the zero order, PeppasKorsmeyer and Higuchi models are the best models to describe the PLASCL20X nanocomposite blends release behavior. Overall, based on the release profiles and matrix hydrolytic degradation, PLASCL203 is recommended as the best candidate with the optimum results. 3.5. Morphological Studies and Surface Behavior The change in the hydrophilicity of nanocomposites surfaces was measured using the contact angle of distilled water and the polymer. The results of a water droplet sitting on the surface of PLASCL20 and nanocomposites are reported in Table 2. As expected, because of nHA hydrophilic nature, with increasing its content, the angles were reduced. As mentioned earlier, PLASCL20 consists of discrete spherical domains of starch–PCL, uniformly dispersed in the PLA matrix. Deionized water and diiodomethane were selected as the test liquids for measuring contact angles. The calculation of wetting coefficient in the previous work (ωa > 1) has shown TC nanoparticles were much more localized in the PLA phase 22
(Davoodi et al., 2016). Based on the equation 1-5 and knowing the deionized water and diiodomethane contact angles of PLA, nHA, and SCL20 tablets, the wetting coefficient for nHA was calculated as well.
Figure 4. (a) SEM image of PLASCL203 fracture surface (b) EDX mapping of Cl and Ca elements in PLASCL203 (c) SEM image of electrospun PLASCL203 fibers (d) Cell attachment on PLASCL203 fibers after 12 h.
Deionized water contact angles of PLA, nHA, and SCL20 were measured as 72.5, 60.5, and 54o and their diiodomethane contact angles were measured as 55.5, 12.3, and 37, respectively. Hence, the wetting coefficient for nHA was calculated to be -0.541 and -0.538 based on the harmonic mean and geometric mean equations, respectively. This result implies that nHA is more preferably located at the interface of PLA and SCL20 phases. Therefore, one could infer that nHA was thermodynamically driven into the interface of phases during
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the melt mixing process. Since SCL20 is finely dispersed in the PLA matrix, it indicates that nHA has been dispersed throughout the whole blend. The attained results were further approved by SEM images in Figure 4. To observe the dispersion of LATC30 and nHA in the ternary blends and prove the previous claims, SEM imaging with EDX (energy dispersive Xray) were taken from PLASCL203 as a representative and depicted in Figure 4a,4b. TC contains “Cl” groups (Green dots) and nHA contains ‘‘Ca’’ groups (Red dots) as was detected. As TC modified the interface of starch and PCL phase, the good distribution of Cl groups is a sign of good dispersion of SCL phase, while, the homogenous dispersion of Ca groups in the PLA matrix and alongside the Cl groups confirms that nHA has been dispersed in the whole blend. The SEM image of melt electrospun PLASCL203 fibers is also shown in Figure 4c. The diameter of the prepared samples ranges from 45 to 65 µm as they need to host the fibroblast (L929) cells with the size of 5-10 µm (Higuchi & Tsukamoto, 2004) (Figure 4c). The good cell attachment on PLASCL203 melt electrospun fibers after 12h can be seen in Figure 4d, and due to the presence of nHA, they may exhibit a higher extent of surface attachment comparing to PLA and PCL.
4. Conclusion An optimized interface-modified antibacterial ternary blend based on PLA/PCL/starch was melt-mixed with nHA at different concentrations. According to FTIR results, no chemical reaction occurred, and the main structure of PLA and PCL was not modified during the melt blending. Moreover, it was found that nHA has been physically dispersed throughout the whole blend. Unlike the PLASCL blends, both Tm and Xc of PCL were increased in the case of nanocomposites. The Xc showed the same trend for PLA phase, while the Tm has not changed, and Tg of PLA shows a complete overlapping with Tm of PCL. Moreover, with an increase in nHA content, the crystallization peaks became narrower confirming an increased
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rate of crystallization. The antibacterial properties of the prepared blends against Grampositive and Gram-negative bacteria was demonstrated as well. The MTT assay showed an increase in the number of cells in the presence of antibacterial agent accompanied with nHA making the nanocomposite blend a more suitable candidate for medical applications. It was also observed that by increasing nHA content, the hydrolytic degradation was increased comparing to PLASCL20. According to the contact angle results, it was proved that nHA was thermodynamically driven into the interface of PLA and SCL20 phases, and since SCL20 is homogenously dispersed throughout the PLA matrix, nHA was dispersed in the whole blend. Finally, microfibers of PLASCL203 were prepared via melt electrospinning process, and the attachment of fibroblast (L929) cells on these fibers were observed. It was revealed that the PLASCL203 shows a desirable drug release, excellent thermal and antibacterial properties, and the combined use of nHA alongside the encapsulated TC (only 1.5% triclosan) eliminates the adverse effects of the antibacterial drug. Therefore, PLASCL203 could be a suitable alternative for a wide range of applications especially in medical applications, and it also can be used in both hard and soft tissue engineering applications as well.
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Khakbaz, M., Hejazi, I., Seyfi, J., Jafari, S.-H., Khonakdar, H. A., & Davachi, S. M. (2015). A novel method to control hydrolytic degradation of nanocomposite biocompatible materials via imparting superhydrophobicity. Applied Surface Science, 357, 880–886. Kusanagi, H., Chatani, Y., & Tadokoro, H. (1997). An approach to the ab initio molecular orbital calculation of the stabilizing energy on polymer double-stranded helix. Polymer Journal, 29(1), 65–68. Kweon, D.-K., Kawasaki, N., Nakayama, A., & Aiba, S. (2004). Preparation and characterization of starch/polycaprolactone blend. Journal of Applied Polymer Science, 92(3), 1716–1723. Lay Ma, U. V., Floros, J. D., & Ziegler, G. R. (2011). Formation of inclusion complexes of starch with fatty acid esters of bioactive compounds. Carbohydrate Polymers, 83(4), 1869–1878. Liao, H.-T., & Wu, C.-S. (2009). Preparation and characterization of ternary blends composed of polylactide, poly (ɛ-caprolactone) and starch. Materials Science and Engineering: A, 515(1), 207–214. Makarovsky, I., Boguslavsky, Y., Lellouche, J., Banin, E., & Lellouche, J.-P. (2012). An Increased Efficiency of Triclosan Delivery by Novel Acrylate-Based Nanoparticles. Journal of Nanomedicine & Nanotechnology, 2012. Mittal, V., Akhtar, T., Luckachan, G., & Matsko, N. (2015). PLA, TPS and PCL binary and ternary blends: structural characterization and time-dependent morphological changes. Colloid and Polymer Science, 293(2), 573–585. Mittal, V., Akhtar, T., & Matsko, N. (2015). Mechanical, thermal, rheological and morphological properties of binary and ternary blends of PLA, TPS and PCL. Macromolecular Materials and Engineering, 300(4), 423–435. Mofokeng, J. P., & Luyt, A. S. (2015). Morphology and thermal degradation studies of melt-mixed poly
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−
Author: Seyed Mohammad Davachi Behzad Shiroud Heidari Iman Hejazi Javad Seyfi Erfan Oliaei Arman Farzaneh Hamid Rashedi PII: DOI: Reference:
S0144-8617(16)30972-9 http://dx.doi.org/doi:10.1016/j.carbpol.2016.08.037 CARP 11462
To appear in: Received date: Revised date: Accepted date:
18-7-2016 11-8-2016 11-8-2016
Please cite this article as: Davachi, Seyed Mohammad., Heidari, Behzad Shiroud., Hejazi, Iman., Seyfi, Javad., Oliaei, Erfan., Farzaneh, Arman., & Rashedi, Hamid., Interface modified Polylactic acid/Starch/Poly -caprolactone Antibacterial Nanocomposite Blends for Medical Applications.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.08.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Interface modified Polylactic acid/Starch/Poly ε-caprolactone Antibacterial Nanocomposite Blends for Medical Applications Seyed Mohammad Davachia,*, Behzad Shiroud Heidaria, Iman Hejazia, Javad Seyfib, Erfan Oliaeia, Arman Farzaneha, Hamid Rashedic a
Applied Science Nano Research Group, ASNARKA, P.C. 1619948753, Tehran, Iran. Department of Chemical Engineering, Shahrood Branch, Islamic Azad University, P.O. Box 36155-163, Shahrood, Iran. c Department of Biotechnology, School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran b
*
Corresponding Author. Email address: [email protected] (S.M. Davachi) Tel/Fax: +98 -21 – 77655928
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Graphical Abstract
2
Highlights
Antibacterial nanocomposite blends with encapsulated drug prepared via melt blending. Increase of nHA enhances hydrophilicity, antibacterial activity and drug release. Adverse effect of antibacterial drug, was eliminated due to the presence of nHA. 3% nHA showed good adjustment between hydrolytic degradation and release profile. Electrospun microfibers of optimum blend demonstrated an improved cell attachment.
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Abstract In this study, an optimized interface-modified ternary blend with antibacterial activity based on polylactic acid/starch/poly ε-caprolactone (PLASCL20), mixed with nano hydroxyapatite (nHA) via melt blending. This method results in a homogeneous nanocomposite blend in which the addition of 3% nHA improves the overall properties such as hydrolytic degradation, hydrophilicity, antibacterial activity and the drug release comparing to PLASCL20. Moreover, the simultaneous use of nHA and encapsulated triclosan (LATC30) compensated the negative effect of triclosan through increasing the possible cell attachment. According to the contact angle results, nHA was thermodynamically driven into the interface of PLA and PCL/Starch phases. The addition of 3% nHA showed a good adjustment between the hydrolytic degradation and the release profile, therefore, their electrospun microfibers demonstrated an improved fibroblast (L929) cell attachment. The aforementioned nanocomposite blend is a suitable antibacterial candidate for many medical applications with minimum side effects due to the controlled release of triclosan.
Keywords: PLA, PCL, Starch, Interface modified blend, Antibacterial, Nano hydroxyapatite.
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1. Introduction: Biodegradable polymers, and polymers derived from renewable resources, currently entice a good deal of interest because of their environmental friendliness, since they offer a proper combination of biodegradability, compostability, and compatibility (Mofokeng & Luyt, 2015). Polylactic acid (PLA) is a semi-crystalline thermoplastic which can be obtained from renewable resources such as starch. PLA is being used in a wide variety of applications in diverse fields because of its extremely good tensile strength and high modulus. However, this polymer is very brittle having serious physical aging issues for the duration of its service life (Seyed Mohammad Davachi & Kaffashi, 2015a) and has hydrophobic character limiting its use in medical and scientific applications due to the reduced level of cellular attachments (Torabinejad, Mohammadi-Rovshandeh, Davachi, & Zamanian, 2014). Therefore, blending or copolymerizing PLA with a polymer with a softer nature would be a great alternative for modifying its brittleness (Mofokeng & Luyt, 2015; Navarro-Baena et al., 2016; Torabinejad et al., 2014). Poly ε-Caprolactone (PCL) is a semi-crystalline thermoplastic polyester which provides high flexibility, low melting point, thermal stability and excellent compatibility with other polymers, even though its high elongation, low modulus, and higher price in comparison to the conventional polymers could be regarded as limitations to its applications (Mittal, Akhtar, & Matsko, 2015). Starch is doubtlessly a beneficial material for biodegradable plastics due to its value and natural abundance. Nevertheless, it has high water absorption and weak mechanical properties such as brittleness as compared with the alternative thermoplastic polymers. Blending starch with PLA increased PLA’s hydrophilicity (Mohammadi-Rovshandeh et al., 2014) and blending it with PCL expanded the biodegradation rate of PCL (Mittal, Akhtar, & Matsko, 2015). An alternative to conquer PLA and PCL drawbacks could be mixing them collectively with starch (Carmona, Corrêa, Marconcini, & Mattoso, 2015). There are several studies that have investigated the ternary 5
blends of PLA, PCL and thermoplastic starch (TPS) (Carmona et al., 2015; Liao & Wu, 2009; Mittal, Akhtar, Luckachan, & Matsko, 2015; Mittal, Akhtar, & Matsko, 2015; Sarazin, Li, Orts, & Favis, 2008), however in our recent study, a ternary blend of PLA, PCL and starch with an interface change of PCL and starch turned into organized (Davoodi et al., 2016). Sarazin et al. (Sarazin et al., 2008) produced the binary and ternary blends of PLA, PCL, and TPS using two different types of starch in a single-step extrusion process and demonstrated their superior physical properties. TPS grades with glycerol contents of 24 and 36 % have been used, and the obtained blends alleviated the weaknesses of each polymer. Liao et al. lowered the price of PLA/PCL blends by adding starch into these blends. In fact, the problem of poor compatibility between PLA/PCL blend and starch was surmounted by grafting acrylic acid to the PLA/PCL blend. A chemical reaction took place between –OH group of starch and –COOH group of AA-gr-PLA/PCL(Liao & Wu, 2009). Mittal et al. studied the structural characterization and time-dependent morphological modifications in addition to the mechanical, thermal, rheological and morphological properties of binary and ternary blends of PLA, TPS and PCL (Mittal, Akhtar, Luckachan, et al., 2015; Mittal, Akhtar, & Matsko, 2015). Carmona et al. modified this blend by adding 2 wt% of Methylene diphenyl diisocyanate (MDI), which increased the melt viscosity of the ternary blend by the urethane linkages which was formed with TPS and it also was efficient to acquire notable enhancements in tensile strength and ductility of the ternary blend (Carmona et al., 2015). The mentioned researches had been conducted with an objective of enhancing the properties of PLA. The mentioned biomaterials possess no intrinsic antibacterial properties, and consequently, to prevent the formation of micro-organisms, anti-infection modifications of polymers were implemented (Seyed Mohammad Davachi & Kaffashi, 2015b; Seyed Mohammad Davachi, Kaffashi, Zamanian, Torabinejad, & Ziaeirad, 2016; Khakbaz et al., 2015, 2016). Within the last decade, several compounds have been used as antibacterial
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retailers, from which silver and triclosan had been widely employed in clinical programs. Triclosan is a famous commercial and a Food and Drug Administration (FDA) authorizedsynthetic-non-ionic-broad-spectrum antimicrobial agent that was chosen as the antibacterial agent in this study (Makarovsky, Boguslavsky, Lellouche, Banin, & Lellouche, 2012). It is chemically stable and may be heated up to 200°C for up to two hours while showing no degradation, so it is suitable for incorporation into diverse bolstered plastic substances (Suller & Russell, 2000). Nanohydroxyapatite (nHA) is the major inorganic component of hard tissue and is an ideal bioactive material for orthopedic applications exhibiting a bone bonding capacity under in-vivo situations (Dadbin & Naimian, 2014; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Hule & Pochan, 2007). nHA have mechanical properties similar to the natural bone, and because of its small size and the large specific surface area, it showed a massive growth in protein adsorption and osteoblast cellular adhesion comparing to the micron sized ceramics (Sui et al., 2007). However, the brittleness, migration from the implanted sites and loose agglomeration are the primary troubles of nHA which could be overcome by incorporation into polymer matrices (Dadbin & Naimian, 2014; Hule & Pochan, 2007; Sarvestani & Jabbari, 2006). In our preceding work, PLLA/TC nanoparticles with the composition of 70/30% (LATC30) were prepared by the emulsification–diffusion technique which demonstrated encapsulation performance, high molecular dispersion, and good release profiles (Seyed Mohammad Davachi & Kaffashi, 2015b). LATC30 was added to a PLLA with higher molecular weight through melt mixing to enhance the overall properties and the antibacterial activity, which 5% of LATC30 showed surest effects, therefore, the actual dosage of TC is 1.5% since the nanoparticles consist of 30% TC (Seyed Mohammad Davachi & Kaffashi, 2015b; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). Hybrid nanocomposites of PLLA, LATC30, and nHA were also studied, and by the simultaneous use of nHA and LATC30, the detrimental impact of
7
triclosan on the cells was eliminated (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). In our recent publication, an interface-modified ternary blend based on PLA/PCL/starch was prepared via melt blending (Davoodi et al., 2016). It needs to be mentioned that no modification was done on the starch unlike the previous studies, and the addition of LATC30 imparted antibacterial property to the blend. The interfacial affinity among hydrophilic starch and hydrophobic polyesters was enhanced by interactions between triclosan –Cl groups and PCL ester groups (Kweon, Kawasaki, Nakayama, & Aiba, 2004), while the PCL and starch were chemically attached due to the inclusion complexes of starch (Lay Ma, Floros, & Ziegler, 2011; Polaczek, Starzyk, Maleńki, & Tomasik, 2000). In the current study, the optimum PLASCL blend of our previous work (PLASCL20) (Davoodi et al., 2016) was used and nHA with various quantities was added via an internal mixer to prepare the blend nanocomposites. The main novelty of the current work is the simultaneous application of nHA and encapsulated triclosan in a novel interface modified PLASCL blend (ternary blend) that has not been addressed so far, to eliminate the unfavorable impact of triclosan by increasing the possible cell attachment due to the existence of nHA making the blend more suitable for medical applications. 2. Materials and methods 2.1. Materials PLLA was synthesized from the purified L-lactide prepared by L-lactic acid (S. M. Davachi, Kaffashi, & Roushandeh, 2012; S.M. Davachi, Kaffashi, Roushandeh, & Torabinejad, 2012) via ring opening polymerization reactive extrusion method which has been fully described elsewhere (Seyed Mohammad Davachi & Kaffashi, 2015b). The weight average molecular weight (Mw) and polydispersity index (PDI) of the PLLA were 120,000 g.mol-1 and 1.78, respectively. Poly (ɛ-caprolactone) with a molecular weight (Mn) of 80,000 purchased from Sigma-Aldrich (Germany). The technical grade corn starch supplied by Glucosan Co. (Iran) 8
used in this study. The moisture content of the starch measured by drying the starch in an oven heated up to 105°C for 24h was 12 wt%. Triclosan (TC) was a generous gift from KAF Co. (Branch of DAROUGAR group, Iran) originally was Dekaben TC Premium, Jan Dekker, Netherlands. Nano hydroxyapatite or nHA (KF-HAP04) with a size ranging from 20 to 40 nm and 99% purity purchased from Kinfon Pharma, China. The PLLA/TC nanoparticles (LATC30) were prepared according to the procedure mentioned in our previous work (Seyed Mohammad Davachi & Kaffashi, 2015b), based on which the PLLA/TC nanoparticles were synthesized by the modified emulsification–diffusion process, and the sample containing 30% TC and 70% PLLA demonstrated the highest encapsulation efficiency with an approximate size of 100–300 nm. From then on, PLLA is referred as PLA. All the other chemical and solvents were reagent grades procured from Merck (Darmstadt Germany).
2.2. Sample preparation Prior to the blending process, the PLA, LATC30 and starch granules were vacuum dried at 80°C for 4h, and PCL granules were also vacuum dried at 30oC for 12h. Afterwards, they were melt-blended via an internal mixer (Brabender 50 EHT at a rotational speed of 60 rpm for 20 min). The components were sequentially loaded into the mixing chamber with various compositions. The stock temperature of the internal mixer maintained at 170°C. The obtained blends were molded into 1-2 mm sheets by hot-press at 170°C applying 14 bar pressure for 5 min and then cooled to the room temperature (Seyed Mohammad Davachi et al., 2015). Eventually, the prepared samples were sterilized by ethylene oxide. The samples were named as PLASCL followed by a three-digit number. The first two numbers account for the weight percentage of PCL, which is the optimized composition of the ternary blend (20wt%) according to our previous study (Davoodi et al., 2016). The third number belongs to the nHA ranging from 1 to 7%. Based on the preceding research, 5 wt% LATC30 was added to the 9
100 wt% of the blends (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). The compositions of nanocomposites blends are listed in Table 1 and they were generally coded as PLASCL20X. Table 1. Composition of PLLA/LATC30/nHA nanocomposites Samples PLASCL20 PLASCL201 PLASCL203 PLASCL205 PLASCL207
PLA (wt.%) 50 50 50 50 50
Starch (wt.%) 30 30 30 30 30
PCL (wt.%) 20 20 20 20 20
LATC30 Content (wt.%) 5 5 5 5 5
nHA Content (wt.%) 1 3 5 7
For contact angle measurements, PLA, SCL20 (60wt% starch, 40wt% PCL and a 5% of LATC30 similar to PLASCL20) were separately prepared using an internal mixer. Furthermore, 0.2 g of both TC and nHA powders were pressed at room temperature for 1 min underneath 50 bar loading to prepare flat tablets for contact angle measurement. 2.3. Melt Electrospinning A custom-made melt electrospinning device was used to prepare nonwoven microfibers. PLASCL203 was fed into the vertical steel cylinder of the melt electrospinning device and heated via an electrical heating system to 180oC for about 15 min to ensure complete melting of the sample. The resulted melt pushed out from a 0.58 mm diameter spinneret at a flow rate of 50 µL/h through pressure from a mechanically regulated piston and a voltage of 20 kV was applied. The distance from the spinneret to the collector was 10 cm. All the parameters were optimized according to Hutmacher and Dalton (Hutmacher & Dalton, 2011). 2.4. Characterization The infrared spectroscopy was performed using the FTIR-ATR, Bruker Equinox 55LS 101 series with the resolution of 4 cm−1 (averaging 50 scans) in the frequency range of 600–4000 cm-1, for determining the functional groups. DSC was performed by a Mettler Toledo DSC 1 10
Star device equipped with a low-temperature accessory. Heating scans were repeated to confirm the reproducibility. The temperature scale calibrated with the excessive-purity standards, and the measurements were carried out at a heating rate of 10 °C/min, in the nitrogen atmosphere and at temperatures ranging from -60 to 260°C. The glass transition temperatures (Tg) of samples were taken from the midpoint of the stepwise specific heat increment. All of the adjustments were according to ASTM D3418. The degree of crystallinity (Xc) for samples were determined according to our previous study ( ) (Davoodi et al., 2016), where ΔHm and ΔHc are the enthalpies of melting and cold crystallization, respectively. W and
are the weight fraction of poly(L-lactide) and
melting enthalpy of 100% crystalline poly(L-lactide) or PCL, respectively. The antibacterial activity of nanocomposite blends was investigated against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) bacteria, according to the disc diffusion method. This approach was carried out in a medium solid agar Petri dish. The samples were molded into disc shapes 2.5 cm in diameter, sterilized with ethylene oxide for 2h, and positioned on E. coli and S. aureus cultured agar plates. Then, they were incubated for 24h at 37oC, and the inhibition zones were recorded (Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). To observe the cell viability of the nanocomposites, the MTT assay was carried out on fibroblast cells for the disc shaped samples similar to the preceding studies. The MTT assay was based on the ability of living cells to reduce a tetrazulium-based compound (MTT) to a purplish formazan product (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). The UV–visible BEL SPECTRO LGS53 spectrophotometer (Italy) was used to determine the amount of TC release from the nanocomposite blends, and the release outcomes were verified with the Peppas-Korsmeyer, zero order, first order, Higuchi and Hixon-Crowell models (Costa & Sousa Lobo, 2001; Davoodi et al., 2016). The Kruss 11
G10 instrument (Germany) was employed for measurement of water contact angle to obtain hydrophilicity of the samples. The water contact angle of samples was measured by dropping droplets of ~5 μL at room temperature. The distance of vibrating syringe was ~5 cm, and all the contact angles were obtained at the initial stage (i.e., <10 s). The water contact angle (CA) measurements of each sample were conducted at least three times across the sample surface. Knowledge of thermodynamic equilibrium was helpful in detecting the location of nHA in the blends since the time of melt mixing was sufficiently long for the immigration. The surface free energy of every discrete component of samples were determined using dispersive and polar surface energy (𝛾d and 𝛾p) with a set of test liquids on a solid surface, where s and l represent the solid and liquid surfaces, respectively, and
ϴ
is the contact angle of liquid
droplet on the surface (Equation 1 and 2). The interfacial energy of every two components were obtained from their dispersive and polar parts of surface energies using harmonic-mean and geometric-mean equations reported in equation 3 and 4 where γ i is the surface energy of component i, γ12 is the interfacial energy of components 1 and 2, and d and p superscripts stand for dispersive and polar parts of surface free energy of the components, respectively (Davoodi et al., 2016). (1) (
)
√
√
(2)
Harmonic Mean:
(
Geometric Mean:
(√
) √
(3) )
(4)
Finally, the wetting coefficient (ωa) has been used to predict the thermodynamic equilibrium distribution of nano-fillers in the polymer blend as shown in equation 5. (5)
12
For in-vitro hydrolytic degradation studies, the sheet specimens with the 1×1×0.15 cm3 surface area and weight of 0.15 gr were hot pressed and incubated in 50 ml phosphate buffer with pH 7.4 at 37oC while being stirred. The specimens were periodically removed, dried up, weighed and the weight loss percentage was calculated (S. M. Davachi et al., 2012; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016). The SEM micrographs were obtained by Vega Tescan, Czech, on the fracture surface of samples in Liquid N2 to observe the dispersion of TC and nHA. 3. Results and discussion 3.1. ATR-FTIR Spectra Figure 1 depicts the FTIR-ATR spectra of PLASCL20X nanocomposites. The C=O stretching vibrations and vibrations of C-O bonds in ester groups (PLA and PCL) display peaks at 1751, 1181 cm-1. The peaks at 1362 and 1453 cm-1 are indicative of the CH3 and CH2 groups of PLLA and PCL, respectively, which appeared due to the physical dispersion of PCL in PLA matrix and also partial miscibility of these polyesters (Davoodi et al., 2016). The C-C peaks can be visible at 870 cm-1. In step with our previous studies, the C-Cl stretching bond shows a peak at 754 cm-1, while the CH stretching and C=C ring-related vibrations multiple bonds occur at ~2940 cm-1 both of which demonstrate the existence of TC (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016).
13
Figure 1. FTIR-ATR spectra of PLASCL20X nanocomposites.
The nHA functional groups such as PO43- showed a series of peaks at 560, 594, 1008 and 1220 cm−1. The OH- absorption peaks at 634 and 3027 cm-1 of nHA also appeared with very low intensity. Subsequently, a broad peak observed in the range of 3000-3600 cm-1 indicates the presence of H2O in the nHA powder; however, because of the low intensity of the aforementioned peaks, the amount of H2O is rather insignificant (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016; Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). The intensity of the peaks was increased upon addition of nHA. As a result of nHA incorporation, the PO43- and C-O peaks emerged at 1042~1129 and 1453 cm-1, respectively. The increase in the intensity of carbonyl groups at 1751 cm-1 was also observed due to the existence of this functional group in both polymer and nHA. The peaks of the hydroxyl group cannot be observed because of their low severity in nHA, or perhaps, hydrogen bonding with polymer carbonyl groups. It has been suggested that the hydroxyl groups might actively support the cellular attachment and increase the hydrolytic degradation rate in an in-vitro investigation (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). Based on our previous studies, the main structure of PLA and PCL has not been modified during the melt blending, and the results clearly state that 14
since PCL had a chemical reaction with starch and TC due to indexes complexes of starch (Davoodi et al., 2016), nHA could be homogeneously and physically dispersed throughout the blend. 3.2. Thermal Properties Thermal behavior of PLASCL20X nanocomposite blends was studied by DSC technique, as depicted in Figure 2. The important thermal properties of these materials are also reported in Table 2. As is observed, upon addition of nHA, melting temperature (Tm) of PCL phase in nanocomposites was increased comparing to PLASCL20. A slight growth in Tm was also noted, with growth in nHA content. In fact, such rise is quite negligible which suggests that nHA might exist in Starch-PCL phase (SCL) or near the mentioned phase, however, no sign of agglomeration was observed in this phase (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016). In contrast with the PLASCL blends which showed a peak with a small shoulder belonging to the Tg of PLA, such shoulder was not detected in these nanocomposites since they have utterly overlapped with Tm of PCL. In PLASCL samples, both Tm and Xc of PCL were decreased because the thermal histories were supposed to be removed via heating the samples, and due to slow rate of PCL crystallization, the chains had not enough time to form ordered structures (Jenkins & Harrison, 2006); however, upon addition of nHA, both of the mentioned parameters are increased due to the fact that nHA could act as nucleating agent. It was found that upon further enhancement in nHA content, the crystallinity of PCL shows a growing trend. The increased crystallinity has been reported for those systems containing PCL as the dispersed phase within the PLA phase at the matrix (Chavalitpanya & Phattanarudee, 2013; Davoodi et al., 2016; Noroozi, Schafer, & Hatzikiriakos, 2012). The reason for this phenomenon has been ascribed to the fact that the interface of such system can serve as a nucleating agent in the course of crystallization stage of PLA (Kusanagi, 15
Chatani, & Tadokoro, 1997). It was observed that as the nHA content increased, the PLA’s crystallinity was increased comparing to PLASCL20. Another possible reason for the observed growth in Xc is that SCL may disrupt the regularity of the chain structures, and the free space between chains was increased (Davoodi et al., 2016); however, upon addition of nHA, the free space decreased, and as a result, Xc increased. The exothermal peaks right before Tm of PLA phase belongs to the recrystallization of those imperfect crystals of PLA into α crystals of higher perfection, and upon addition of nHA, a slight growth was observed. The Tm shows nearly no change, and the results are in agreement with the previous reports (Seyed Mohammad Davachi, Kaffashi, Torabinejad, Zamanian, et al., 2016).
Table 2. Thermal and surface behavior of PLASCL20X samples PLASCL20 (Davoodi et al., PLASCL201 PLASCL203 PLASCL205 2016)
Contact Angle
Thermal Properties
Tm (oC) (PCL) Tg (oC) (PLLA) Tm (oC) (PLLA) ΔHm (J/g) (PCL) Xc (%)a (PCL) ΔHm (J/g) (PLLA) Xc (%)b (PLLA)
a,b
θm
53.7
53.63
54.17
50.1
54.33
PLASCL207 54.67
Overlapped with PCL Tm
163.0
163.03
163.04
163.06
163.34
14.8
14.93
15.12
15.48
16.13
53.1
53.59
54.27
55.56
57.89
21.5
22.08
24.13
24.58
24.91
46.2
47.48
51.89
52.86
53.57
68.37
67.11
65.42
62.89
60.18
By considering the melting enthalpy of 100% crystalline PCL as 139.3 J/g (Davoodi et al., 2016) and PLLA as 93 J/g (Seyed Mohammad Davachi & Kaffashi, 2015b)
Finally, it is also evident from Figure 2 that upon addition of nHA, the crystallization peaks become narrower, or the widths of the peaks are reduced, comparing to PLASCL20. Furthermore, the rate of crystallization was enhanced with an increase in nHA content. 16
Figure 2. DSC thermograms of PLASCL20X nanocomposites
3.3. Antibacterial activity, cell viability and in-vitro degradation of PLASCL20X nanocomposite blends To investigate the antibacterial activity of TC in the presence of nHA in PLASCL20X nanocomposite blends, PLASCL203 sample, as a representative, was placed on the seeded agar plates of E. coli and S. aureus to measure the effectiveness of TC. According to Figure 3a and 3b, the inhibition zone around the sample were 4 and 7 mm, respectively. Similar to PLASCL20 and our other studies, the inhibition zone against S. aureus was more pronounced rather than E. coli., which confirms antibacterial activity against both Gram-negative and Gram-positive bacteria, while it has greater activity against Gram-positive species (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016; Davoodi et al., 2016). According to Petersen, TC is able to decrease the bacterial growth and polymer adherences directly from the polymer surface with the
minimum
antimicrobial
release.
Moreover,
secondary bonding
between
the
microorganism and the polymer is considered to be interrupted by TC vibrational fluctuating molecular bond rotations as a possible mechanism to prevent the microbial surface
17
attachments (Petersen, 2016). The MTT assay within three and seven days after seeding was examined for investigation of in-vitro biocompatibility, the effect of nHA content variation on the discharge of TC, and the overall effect on fibroblast cell growth. As depicted in Figure 3c, with an increase in nHA content, the cell numbers are increased due to the better cell attachment on the surface of the nanocomposites (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016; Torabinejad et al., 2014). It was previously reported that upon addition of PCL, the number of attached cells were decreased due to the weaker attachment of the cells on the surface of the blends (Davoodi et al., 2016). The addition of TC facilitated the attachment of starch and PCL while the blend showed antibacterial activity, the destructive impact of TC decreased, despite the fact that, a slight reduction in MTT assay trend was observed.
18
Figure 3. PLASCL20X nanocomposite blends (a) Antibacterial activity against E. coli (b) Antibacterial activity against S. aureus (c) MTT assay after 3 and 7 days (d) Hydrolytic Degradation at 37 oC (e) Accumulative release profile in 6 months
The addition of TC can kill the cells alongside the microbes and bacteria, which is a negative effect of using TC, however by encapsulation the negative effect reduced (Seyed Mohammad 19
Davachi & Kaffashi, 2015b; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016; Davoodi et al., 2016). It was seen that with the addition of nHA and increase in its content, the antimicrobial release was enhanced due to the increased degradation. However, an interesting result in PLASCL20X nanocomposite blends is that with an increase in nHA content, the adverse effect of TC on the cells is disappeared. In fact, the MTT assay results clearly show the cell growth in the presence of antibacterial agent alongside with nHA, which has been previously observed for nHA in PLA matrix (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). For in-vitro degradation investigation, the PLASCL20X samples were immersed in phosphate buffer at pH of 7.4 and temperature of 37oC. PLASCL20 showed a 15-month degradation time, which was faster than PLA and PCL due to the addition of hydrophilic starch (Davoodi et al., 2016). As can be seen in Figure 3d, upon addition of nHA, the blends exhibit faster degradation times comparing to PLASCL20, due to the hydrophilic nature of nHA. With the increase in nHA content, the degradation happens faster, wherein PLASCL201 and PLASCL207 were degraded after 13 and 10 months, respectively. 3.4. Drug release rate studies and kinetics According to the TC calibration curve in our previous work (Seyed Mohammad Davachi & Kaffashi, 2015b), the drug release of the PLASCL20X nanocomposite blends at 37 oC in buffer solution are shown in Figure 3e. The blends exhibit a burst release at the first 24 h which for PLASCL201 and PLASCL203 overall is less than 1 μg/ml, and is in agreement with the previous studies that used LATC30 (Seyed Mohammad Davachi & Kaffashi, 2015b; Seyed Mohammad Davachi, Kaffashi, Zamanian, et al., 2016; Davoodi et al., 2016). The amount of release for the samples with different nHA contents is 0.12, 0.14, 0.16 and 0.18 μg/ml every day, which shows a growing trend with an increase in the nHA content. The minimum inhibitory concentration (MIC) of TC is between 0.025- 1 μg/ml for medical and 20
packaging applications, and the obtained results seem suitable (Davoodi et al., 2016; Suller & Russell, 2000). The drug release was monitored up to six months to examine the drug release mechanism for the long-term treatments. The degradation and release time need to show similar profiles since a continuous drug release during the application is desirable. Based on previous results, PLASCL20 degraded and released the drug in 15 months (Davoodi et al., 2016). PLASCL201 released the drug in 13.5 months; PLASCL203 released it in 12 months, which was near their degradation times while the other samples released the drug earlier than their degradation which is not desirable. Based on these results, only PLASCL203 releases the drug precisely within its degradation period. For medical applications, a slower release profile is preferable. The accumulative release profiles for PLASCL blends were analyzed by using the Peppas-Korsmeyer, zero-order, first-order, Higuchi, and Hixon–Crowell models, to investigate the TC release mechanism. Table 3 reports the regression coefficients (R2) as well as their equation constants calculated and fitted to the obtained data. According to the Peppas-Korsmeyer model, the drug delivery mechanism with the increase in nHA content shows different behavior, as it first changes from non-Fickian diffusion (anomalous transport) to Fickian diffusion up to 3% nHA, and with an increase in nHA amount, it again shows nonFickian behavior. The ‘n’ values around 0.5 have to be utilized for the Fickian diffusion of non-well-known pharmaceutical polymeric dosage, and as all the samples show the same range, the ideal result should be obtained at the lower amounts and PLASCL203 has the lowest ‘n’ among the other samples and with round approximation, it can be well fitted to Peppas-Korsmeyer equation (Seyed Mohammad Davachi, Kaffashi, Torabinejad, & Zamanian, 2016). Table 3. Regression coefficients and constants of models fitted to the release of triclosan in PLASCL20X blends Peppas-Korsmeyer Zero Order First Order Higuchi Hixon-Crowell Samples R2 n K0 R2 K1 R2 KH R2 Ks R2 PLASCL20 0.9589 0.56 0.0045 0.9986 0.001 0.7331 0.2905 0.9424 -0.0005 0.9063 PLASCL201 0.9595 0.58 0.005 0.9974 0.001 0.7311 0.3052 0.9294 -0.0005 0.9103 PLASCL203 0.9616 0.54 0.0055 0.9986 0.001 0.7051 0.3403 0.9451 -0.0005 0.9239 PLASCL205 0.9561 0.60 0.0064 0.9987 0.001 0.6835 0.3922 0.9443 -0.0005 0.9293
21
PLASCL207
0.9454
0.62
0.0073
0.9986
0.001
0.7285
0.4497
0.9395
-0.0006
0.9335
The release of a low-soluble drug in the release environment is verified as the regression coefficients for different blends showed an excellent fitting with the zero-order model. All the samples show relatively high regression coefficient with Higuchi model due to the absorption of TC in phosphate buffer solution. As the drug diffusion is faster than the matrix degradation, the drug release mainly happens due to the diffusion (Davoodi et al., 2016). Because of rather a long-time degradation of PLASCL blends, the release mechanism could not fit well in the first order and Hixon–Crowell models. However, with the increase in nHA content and faster degradation of the matrix, the regression coefficient of Hixon–Crowell is enhanced and displays reasonable regression coefficients due to the consistent amount of LATC30 in the PLASCL20X samples in comparison with the PLASCL20. According to the release studies and the regression coefficients of different models, the zero order, PeppasKorsmeyer and Higuchi models are the best models to describe the PLASCL20X nanocomposite blends release behavior. Overall, based on the release profiles and matrix hydrolytic degradation, PLASCL203 is recommended as the best candidate with the optimum results. 3.5. Morphological Studies and Surface Behavior The change in the hydrophilicity of nanocomposites surfaces was measured using the contact angle of distilled water and the polymer. The results of a water droplet sitting on the surface of PLASCL20 and nanocomposites are reported in Table 2. As expected, because of nHA hydrophilic nature, with increasing its content, the angles were reduced. As mentioned earlier, PLASCL20 consists of discrete spherical domains of starch–PCL, uniformly dispersed in the PLA matrix. Deionized water and diiodomethane were selected as the test liquids for measuring contact angles. The calculation of wetting coefficient in the previous work (ωa > 1) has shown TC nanoparticles were much more localized in the PLA phase 22
(Davoodi et al., 2016). Based on the equation 1-5 and knowing the deionized water and diiodomethane contact angles of PLA, nHA, and SCL20 tablets, the wetting coefficient for nHA was calculated as well.
Figure 4. (a) SEM image of PLASCL203 fracture surface (b) EDX mapping of Cl and Ca elements in PLASCL203 (c) SEM image of electrospun PLASCL203 fibers (d) Cell attachment on PLASCL203 fibers after 12 h.
Deionized water contact angles of PLA, nHA, and SCL20 were measured as 72.5, 60.5, and 54o and their diiodomethane contact angles were measured as 55.5, 12.3, and 37, respectively. Hence, the wetting coefficient for nHA was calculated to be -0.541 and -0.538 based on the harmonic mean and geometric mean equations, respectively. This result implies that nHA is more preferably located at the interface of PLA and SCL20 phases. Therefore, one could infer that nHA was thermodynamically driven into the interface of phases during
23
the melt mixing process. Since SCL20 is finely dispersed in the PLA matrix, it indicates that nHA has been dispersed throughout the whole blend. The attained results were further approved by SEM images in Figure 4. To observe the dispersion of LATC30 and nHA in the ternary blends and prove the previous claims, SEM imaging with EDX (energy dispersive Xray) were taken from PLASCL203 as a representative and depicted in Figure 4a,4b. TC contains “Cl” groups (Green dots) and nHA contains ‘‘Ca’’ groups (Red dots) as was detected. As TC modified the interface of starch and PCL phase, the good distribution of Cl groups is a sign of good dispersion of SCL phase, while, the homogenous dispersion of Ca groups in the PLA matrix and alongside the Cl groups confirms that nHA has been dispersed in the whole blend. The SEM image of melt electrospun PLASCL203 fibers is also shown in Figure 4c. The diameter of the prepared samples ranges from 45 to 65 µm as they need to host the fibroblast (L929) cells with the size of 5-10 µm (Higuchi & Tsukamoto, 2004) (Figure 4c). The good cell attachment on PLASCL203 melt electrospun fibers after 12h can be seen in Figure 4d, and due to the presence of nHA, they may exhibit a higher extent of surface attachment comparing to PLA and PCL.
4. Conclusion An optimized interface-modified antibacterial ternary blend based on PLA/PCL/starch was melt-mixed with nHA at different concentrations. According to FTIR results, no chemical reaction occurred, and the main structure of PLA and PCL was not modified during the melt blending. Moreover, it was found that nHA has been physically dispersed throughout the whole blend. Unlike the PLASCL blends, both Tm and Xc of PCL were increased in the case of nanocomposites. The Xc showed the same trend for PLA phase, while the Tm has not changed, and Tg of PLA shows a complete overlapping with Tm of PCL. Moreover, with an increase in nHA content, the crystallization peaks became narrower confirming an increased
24
rate of crystallization. The antibacterial properties of the prepared blends against Grampositive and Gram-negative bacteria was demonstrated as well. The MTT assay showed an increase in the number of cells in the presence of antibacterial agent accompanied with nHA making the nanocomposite blend a more suitable candidate for medical applications. It was also observed that by increasing nHA content, the hydrolytic degradation was increased comparing to PLASCL20. According to the contact angle results, it was proved that nHA was thermodynamically driven into the interface of PLA and SCL20 phases, and since SCL20 is homogenously dispersed throughout the PLA matrix, nHA was dispersed in the whole blend. Finally, microfibers of PLASCL203 were prepared via melt electrospinning process, and the attachment of fibroblast (L929) cells on these fibers were observed. It was revealed that the PLASCL203 shows a desirable drug release, excellent thermal and antibacterial properties, and the combined use of nHA alongside the encapsulated TC (only 1.5% triclosan) eliminates the adverse effects of the antibacterial drug. Therefore, PLASCL203 could be a suitable alternative for a wide range of applications especially in medical applications, and it also can be used in both hard and soft tissue engineering applications as well.
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