Materials Science and Engineering C 61 (2016) 744–752

Contents lists available at ScienceDirect

Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Pore formation mechanism of porous poly(DL-lactic acid)

matrix membrane
Thawatchai Phaechamud ⁎, Sasiprapa Chitrattha
Department of Pharmaceutical Technology, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Porous PLA structure has been widely used in cell transplantation, drug carrier and wound dressing. The porous
Received 2 November 2015 structure can be controlled depending on the choice of the polymer, solvent, nonsolvent and preparation param-
Received in revised form 4 December 2015 eters. In this study, the porous PLA matrix membranes were prepared by adding PEG 400 in PLA solution using
Accepted 6 January 2016
dichloromethane (DCM) as solvent prior to casting. The influence of other liquids as co-solvent on pore formation
Available online 9 January 2016
and the structural change during membrane formation were evaluated. The co-solvents affected both porous to-
Keywords:
pography and mechanical properties of PLA membrane. The porous matrix were produced when the non-solvent
Pore formation of PLA was used as co-solvent. Cryo-SEM micrographs revealed that PEG 400 still remained in the PLA porous ma-
Mechanism trix membrane. From the tracking of the structural change during film formation, the PLA–PEG solution changed
Porous membrane into porous structure by liquid liquid phase separation and solidification processes, respectively. Thermogravi-
Poly(DL-lactic acid) metric analysis revealed that PLA–PEG in DCM solution exhibited the two-step of weight loss, the first step oc-
curred from DCM evaporation and the second step occurred from the degradation of PLA–PEG matrix. The
liquid–liquid phase separation and solidification started when the amount of DCM was higher than PEG 400
for 2.67 folds and DCM amount was equal to that of PEG 400, respectively. These results could clarify the pore
formation mechanism of porous PLA membrane and will be useful for the further investigation and application.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction membrane by casting of polymer/salt composite membranes followed

by the dissolution of the salt [11]. The PLA/montmorillonite nanocom-
Poly(lactic acid) (PLA) is the polymeric material resulting from posite scaffold was prepared using a gas foaming/salt leaching method
polymerization of lactic acid. PLA can exist in three stereochemical using NH4HCO3/NaCl salt particles [12]. Porous PLA spheres were pre-
forms: poly (L -lactide) (PLLA), poly (D -lactide) (PDLA), and poly pared by a freeze-drying method were used as drug-carrier material.
(DL-lactide) (PDLLA). The stereochemical composition of PLA had a During the freezing process, the phase separation of the solutes from
significant effect on melting point; however, it had no effect on the solvent occurred as the solvent crystallizes and the pores were
glass transition temperature. PLA is a thermoplastic, high-strength, created by the sublimation of solvent crystals [13]. PLA scaffolds were
high-modulus polymer that can be made from annually renewable prepared via thermally induced phase separation (TIPS) from ternary
resources to yield the articles for use in either the industrial packaging systems using dioxane as the solvent and water as the non-solvent
field or the biocompatible/bioabsorbable medical device [1,2]. PLA has [14]. The phase separation was induced through the addition of water,
been used widely as a porous structure for cell transplantation [3,4], then a polymer-rich and a polymer-poor phases were formed. Follow-
drug carrier for controlled release [5] and wound dressing [6–8]. ing cooling below the solvent melting point and evaporation of the sol-
There are several techniques to prepare the biomaterials into various vent, a porous scaffold was obtained. PLA nanofiber scaffolds prepared
porous structures such as impregnation and sintering ceramic scaffolds by electrospinning have been reported [15]. Nano-fibrous PLA scaffolds
processing, solvent casting and particulate leaching, gas forming, non- were produced by reverse solid freeform (SFF) fabrication. A negative
woven fiber, fiber knitting, phase separation and emulsion freeze drying template for the scaffold was created using a computer-aided design
[9,10]. The PLA porous structure for medical/pharmaceutical applica- (CAD) program and the three-dimensional mold was created on a 3-D
tions has been developed with many methods. A solvent casting/salt printer using wax as template material [16]. Each method presents dis-
leaching method has been developed to prepare a highly porous PLA tinct the advantages and disadvantages which the appropriate tech-
nique should be selected to meet the requirements for the specific
type of tissue. However, the porous structure, especially pore size and
⁎ Corresponding author at: Department of Pharmaceutical Technology, Faculty of
Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand.
its distribution, could be modulated for each specific application
E-mail addresses: [email protected], [email protected] depending on the choice of the polymer, solvent, non-solvent and
(T. Phaechamud), [email protected] (S. Chitrattha). preparation parameters [17].

http://dx.doi.org/10.1016/j.msec.2016.01.014
0928-4931/© 2016 Elsevier B.V. All rights reserved.
 T. Phaechamud, S. Chitrattha / Materials Science and Engineering C 61 (2016) 744–752 745

Polyethylene glycol (PEG) has been widely used as a plasticizer, co- Thailand. Polyethylene glycol (PEG) 400 (Fluka, Sigma-Aldrich Co.,
polymer and blends for PLA. The mechanical, physical and barrier prop- Belgium), dichloromethane (DCM) (VWR International Ltd., Poole,
erties of smooth PLA film were improved when PEG 10–20%w/w was England), acetone (Qrec, New Zealand), ethanol (EtOH) (VWR interna-
incorporated [18–20]. The hydrophilicity, degradation rate and drug tional S.A.S., France), glycerin (SR Lab Co., Bangkok, Thailand), isopropyl
release rate of PEG/PLA blends and PLA–PEG copolymer film were en- alcohol (IPA) (Qrec, New Zealand), tetraethylene glycol (TEG) (Sigma-
hanced by PEG [21,22]. In our previous works, the porous PLA matrix Aldrich, Co., Missouri, USA), tetrahydrofuran (THF) (Fisher Scientific
film was produced by adding PEG 400 using solvent casting method UK, UK) and erythosine dye (food color, Best odor color CO., LTD.,
[23]. Although the porous PLA film could be formed at room tempera- Bangkok, Thailand) were used as received.
ture, the appropriated mechanical properties and porous topography
of porous PLA film were obtained by using PEG 400 at the concentration 2.2. Study of effect of solvent on the pore formation
of 150% of polymer prepared at −20 °C, 24 h and dried at 60 °C, 24 h.
According to other researches, PEG improved the wettability and degra- 2.2.1. Preparation of samples
dation rate of the PLA films. This porous topography of PLA matrix film The 10%w/w PLA solution was prepared in DCM while mixing vigor-
could be applied further as material in tissue engineering and drug de- ously at room temperature. Various liquids including acetone, ethanol
livery systems. In addition, the effect of temperature change on fabrica- (EtOH), glycerin, isopropyl alcohol (IPA), PEG 400, tetraethylene glycol
tion processes of PLA porous matrix films was also determined [24]. The (TEG), tetrahydrofuran (THF) and distilled water at the concentration
porous films were prepared at −80 °C, −20 °C or 4 °C for 24 h prior to of 150%w/w of polymer was mixed individually with the previously pre-
drying at room temperature for 24 h and −80 °C, −20 °C or 4 °C for 24 h pared PLA solution until they became homogeneous. These 10%w/w PLA
prior to drying at 60 °C for 24 h. Although they displayed the different mixtures were poured onto the glass plate, spread evenly and then allowed
porous structure, all of samples showed quite soft behavior. The porous to dry for 24 h at room temperature. The PLA structure prepared by
PLA membrane prepared at 4 °C, 24 h and dried at 60 °C exhibited the adding acetone, EtOH, glycerin, IPA, PEG 400, TEG, THF and water
highest strength, toughest and displayed the smallest porous structure. were coded as PA, PE, PG, PI, PP, PT, PF and PW, respectively.
However, the pore formation mechanism of the obtained porous PLA
membrane by adding PEG 400 has not been evaluated. 2.2.2. Morphology characterization
In this research, the pore formation mechanism in porous PLA ma- Morphology of the samples was observed using scanning electron
trix membrane produced by solvent casting method was investigated. microscope (SEM) (Maxim 200 Camscan, Cambridge, England) with
Various liquids were used for determining the effect of solvent on the 15 KeV of an accelerating voltage. The samples were strewed onto the
pore formation. The properties of liquids, morphology and mechanical carbon double adhesive tape that adhered on metal stub and then
properties of porous PLA membranes prepared from various co- sputter-coated with gold.
solvent systems were evaluated. The cryo-scanning electron microsco-
py (cryo-SEM) was applied to characterize the topography of the ob- 2.2.3. Mechanical properties
tained structure. The tracking of structural change of porous PLA film Mechanical properties such as tensile strength and elongation at
during membrane formation was conducted by stereomicroscope and break of prepared PLA films were measured using a tensile mode by
thermogravimetric analysis (TGA) was used to explain the pore forma- TA.XT2i texture analyzer (Charpa Techcenter, Godalming, Stable micro
tion mechanism. Finally, the continuous determination for percentage Systems Ltd., UK) equipped with a 5 kg load cell. Specimen samples,
weight ratio of each substance during PLA film formation was verified 3 × 0.6 cm, were cut from films (n = 6).
for the liquid–liquid phase separation and solidification processes.
2.2.4. Solvent properties study
2. Materials and methods From the literature review, the various porous structures were
handled by the various types of the polymer, solvent, non-solvent and
2.1. Materials preparation parameters. Hence, the solvent property should be one of
the important factors affecting the porous topography; therefore, the
Poly (DL-lactic acid) (Grade 2002D, 4% D-lactide, 96% L-lactide viscosity, surface tension, miscibility with DCM and polymer solubility
content, MW = 121,400 g/mol) was kindly provided by SCG, Rayong, of the used liquids were evaluated.

Fig. 1. SEM micrographs of cross section at magnification of 100× of a) PP, b) PW, c) PG, d) PT, e) PE, f) PI, g) PA and h) PF porous films.
746 T. Phaechamud, S. Chitrattha / Materials Science and Engineering C 61 (2016) 744–752

2.2.4.1. Viscosity measurement. Viscosity of the acetone, DCM, EtOH, Table 1

glycerin, IPA, PEG 400, TEG, THF and distilled water were determined Viscosity (n = 3) and surface tension (n = 6) of solvents.

using Brookfield DV-III Ultra programmable viscometer (Brookfield Solvent Viscosity (cps) (±SD) Surface tension (mN/m) (±SD)
Engineering Laboratories.Inc., USA) with a conical probe (CPE 40) in Acetone 0.46 ± 0.02 31.54 ± 0.35
the shear rate range of 10–250 rpm (n = 3). THF 0.65 ± 0.03 32.87 ± 0.64
DCM 1.02 ± 0.05 20.20 ± 0.34
EtOH 1.31 ± 0.10 29.12 ± 0.24
2.2.4.2. Surface tension measurement. Surface tension of the acetone, Water 1.34 ± 0.32 74.52 ± 0.21
DCM, EtOH, glycerin, IPA, PEG 400, TEG, THF and distilled water were IPA 2.40 ± 0.04 27.43 ± 0.29
determined using the drop shape analysis method by goniometer TEG 35.32 ± 0.66 58.28 ± 0.12
PEG 400 74.77 ± 1.36 38.20 ± 0.25
(FTA 1000, First Ten Angstroms, USA). Surface tension was obtained
Glycerin 688.90 ± 15.07 46.88 ± 2.84
by analyzing the change in the shape of a pendant drop of a
liquid suspended in air using 20-gauge needle with pump out rate
2.5757 ul/s (n = 6).
2.4.2. Morphology characterization by stereomicroscope
Sample solution was poured onto the glass plate and spread evenly.
2.2.4.3. Miscibility study. The miscibility of acetone, EtOH, glycerin, IPA, Additionally, the morphological change of sample during drying into
PEG 400, TEG, THF and distilled water with DCM were determined at membrane was investigated continuously under stereomicroscope
room temperature. The amount of each substance in the film prepara- (Motic®, SMZ-171 series, Rushmore Precision Co., Ltd. Bangkok,
tion (polymer, co-solvent and DCM at concentration of 10%, 15% and Thailand) at room temperature (25 °C, 60%RH). The images were deter-
75% w/w, respectively) was used, and the ratio of various solvent to mined at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 120 and 180 min at magni-
DCM was 1:5. Erythosine dye was dissolved/dispersed into 2 mL of var- fication of 50 ×. The digital camera was also used for observing the
ious solvents in the test tubes except DCM for noticeable evaluation of appearance of the samples at the same time interval.
solvent miscibility. The 10 mL DCM was added into each previously pre-
pared test tube and mixed for 5 min using vortex mixture. The miscibil- 2.4.3. Thermogravimetric analysis (TGA)
ity of these solvents was determined at 24 h after mixing. TGA data were obtained on a thermogravimetric analyzer (Pyris/
TGA, PerkinElmer, USA). Samples of approximately 10 mg were heated
2.2.4.4. Solubility study. The solubility of PLA in various solvents was in- at 5 °C/min from room temperature to 500 °C, under a dry nitrogen gas
vestigated at room temperature. The 0.1 g PLA was weighed into each flow rate of 40 mL/min.
test tube. Thereafter, the 10 mL of each acetone, EtOH, glycerin, IPA,
PEG 400, TEG, THF and distilled water was added into PLA. Subsequent- 2.4.4. Determination of phase separation and solidification
ly, the closed test tubes containing the mixed sample were placed into The sample solution was poured onto the glass plate, spread evenly
the sonicator bath at room temperature for 6 h. The solubility or change and weighed at room temperature (25 °C, 60%RH). Thereafter, the sam-
of PLA in these solvents was visually observed. ple plate was still placed on analytical balance (CP2245 Satorious,
Oharus Corporation, USA) for measuring the weight change. When the
2.3. The determination of PEG 400 in PLA porous matrix membrane clear solution started to be turbid (phase separation occurred), the
weight of sample was recorded. Afterward, when the sample changed
2.3.1. Preparation of sample from turbid solution to opaque membrane (solidification occurred),
PLA porous matrix membrane was prepared using a solvent casting the weight of sample was recorded. Finally, when the weight of sample
method. Briefly, 10%w/w of PLA in dichloromethane (DCM) was pre- was constant at 24 h, it was recorded. The percentage of substances at
pared. Then, PEG 400 at concentration of 150%w/w of polymer was phase separation, solidification and constant points were calculated
mixed in the previous solution. The mixtures were poured onto the from the recorded weight (n = 3), because PLA and PEG 400 weight
glass plate, spread evenly and suddenly placed at − 20 °C for 24 h and were constant but that of DCM was altered from its volatilization.
then allowed to dry at 60 °C for 24 h. The porous film on the glass
plate was obtained and coded as PPEG. 3. Results and discussion

3.1. The effect of solvent on the pore formation

2.3.2. The determination of PEG 400 in PLA porous matrix film by cryo-
scanning electron microscopy (cryo-SEM)
3.1.1. Morphology, viscosity, surface tension, miscibility and solubility
PEG 400 in PLA porous matrix membrane was characterized by
measurement
cryo-SEM (JOEL, JSM-6010lv, Japan). The samples were loaded on the
The morphology of films is shown in Fig. 1. The porous structures
cryo specimen holder and cryo-fixed in slush nitrogen (− 200 °C)
were found in the films containing PEG 400 (PP), distilled water (PW),
then quickly transferred to the cryo-unit in the frozen state. The sample
glycerin (PG), TEG (PT), EtOH (PE) and IPA (PI) as co-solvent (Fig. 1a,
was transferred in vacuum to the cold-stage of the SEM cryo-
b, c, d, e and f, respectively). Each film revealed a different topography.
preparation chamber where fracturing could be performed. After sput-
ter coating with gold, the sample was transferred into the SEM chamber,
where it remained frozen during imaging on another cold-stage and Table 2
Miscibility and solubility of solvents.
cooled by liquid nitrogen.
Solvent Miscibility in DCM (solvent:DCM, 1:5) Solubility of PLA (10 mg/mL)

2.4. Study of structural change during film formation Acetone Immiscible Slightly soluble
THF Miscible Slightly soluble
EtOH Miscible Insoluble
2.4.1. Preparation of samples Water Immiscible Insoluble
The 10%w/w PLA was dissolved in DCM while mixing vigorously at IPA Miscible Insoluble
room temperature. Erythosine dye was dissolved into PEG 400 then TEG Miscible Insoluble
mixed with the previously prepared PLA solution until it became homo- PEG 400 Miscible Insoluble
Glycerin Immiscible Insoluble
geneous as previously mentioned.
 T. Phaechamud, S. Chitrattha / Materials Science and Engineering C 61 (2016) 744–752 747

Fig. 2. a) Tensile strength and b) Elongation at break of PP, PT, PW, PI, PG and PE porous films (n = 6).

PP, PT, PE and PI exhibited the uniformity of small porous structures. PW at break of PP, PT and PW were similar and less than that of PI which
and PG showed the non-uniformity mixing between large and small showed the same trend with that of PG and PE films (Fig. 2b). The
pore sizes. The pore size of PI was less than PE, PT, PP, PG and PW, different mechanical properties of those films depended on the type of
respectively. On the other hand, there was no pore in the films using the non-solvents. These results indicated that several non-solvent
acetone (PA) and THF (PF) as co-solvent (Fig. 1g and h, respectively). systems affected the morphology and mechanical properties of PLA
This result related with the PLA solubility study (Table 2), PLA was porous film. Correspondingly, the type of solvent and its evaporation
slightly soluble in acetone and THF but insoluble in PEG 400, distilled rate influenced the mechanical properties of electrospun PLA nanofibers
water, glycerin, TEG, EtOH and IPA. PLA is soluble in most organic obtained by using different solvents (chloroform, chloroform/acetone,
solvents such as THF, chlorinated solvents, benzene, acetonitrile, and chloroform/THF and chloroform/dimethylformamide) [27]. The various
dioxane [1]. This result indicated that the pore formation mechanism solvent systems of PLA nanofibers also affected the morphology,
of PLA porous membrane related with non-solvent inducing a phase porosity and crystallinity of the nanofibers [28–30]. In addition, the
separation. The different pore size of those films related with the pore structure of a PLA scaffold was affected by type of solvent or
viscosity of non-solvents (Table 1) which the higher viscous liquid solvent/non-solvent ratios [25,26]. The non-solvent induced phase
promoted the higher pore size. In general, the viscosity of the system separation has been used for fabricating the porous PLA foam using
was increased therefore resulted in an increased coarsening time and DCM as a solvent and hexane as a non-solvent [29]. Considering the
that was favorable for the formation of large pores [25]. However, the good mechanical properties, high porosity and combined meso/
porous film using distilled water as non-solvent contained both large macroporous morphology of the foams as well as the simplicity and
and small pore sizes (Fig. 1b) due to the immiscibility of distilled versatility of the process, non-solvent induced phase separation was a
water and DCM which was difficult for maintaining of their homogene- very promising technique for the fabrication of polymer foams with
ity during drying. The non-uniformity of contents in the mixing of large unique properties.
and small pore sizes of films related with the miscibility behavior as
presented in Table 2. Distilled water and glycerin were immiscible 3.2. Cryo-SEM of PLA porous matrix film
with DCM thus their porous films exhibited the non-uniform porous
structure (Fig. 1b and c, respectively). Hence, the immiscibility of Cryo-SEM micrographs of membrane are exhibited in Fig. 3. Plain
non-solvent with DCM affected the porous structure of the PLA films. PLA film showed a dense film structure (Fig. 3a). While, the PLA porous
The surface tension of solvents is shown in Table 1 although they matrix film (PPEG) exhibited an occluded porous structure that seem to
were not affiliated with the morphology of those films. However, the have approximately spherical particles embedded within the pore
relative impact of surface tension on ice-templated ceramics (Fig. 3b and c). This was different from the SEM micrograph of PPEG
suggested that the variation of the surface tension partially influenced film in previous study that showed an opened porous structure [23].
the local particles packing and therefore the balance between Accordingly, cryo-SEM images of a novel octavinyl silsesquioxane
macroporosity and microporosity could explain the magnitude of (POSS)-PEG–PLA hydrogels revealed the relatively porous networks
porosity variations [26]. and occluded spherical particles that were produced after pore forma-
tion. These observations suggested that the formation of the occluded
3.1.2. Mechanical properties particles and the network porosity closely associated with the presence
The mechanical properties of porous PLA films prepared from of the acrylate-terminated octa-functionalized POSS-PEG (POSSPEGA)
various non-solvents were determined. The tensile strength values of macromonomer [31]. Therefore, the occluded porous structure in this
films are shown in Fig. 2a. PP was softer than PT, PW, PI, PG and PE, study occurred from PEG 400 in PPEG membrane that PEG 400 inserted
respectively. Whilst, the ductility of films determined from elongation into the PLA porous structure and was frozen by cryo-SEM conditions

Fig. 3. Cryo-SEM micrographs of cross section of a) plain PLA film (1000×), b) and c) PPEG porous matrix film at magnification of 200× and 2000×, respectively.
748 T. Phaechamud, S. Chitrattha / Materials Science and Engineering C 61 (2016) 744–752

(melting point of PEG 400 is 4–8 °C [32]). This result indicated that 3.3. Structural change during membrane formation
PEG 400 still remained in the PLA porous matrix film. This remained
PEG 400 could enhance the water penetration and promote the drug 3.3.1. Morphology characterization
diffusion for the drug-incorporated functional materials. According- The morphology of PLA and PLA–PEG solutions are presented in
ly, PEG 400 added into paclitaxel-loaded poly(D,L-lactic-co-glycolic Figs. 4 and 5, respectively. PLA solution exhibited a transparent pale
acid) (PLGA) films was exploited to enhance the release of hydro- red color from added erythosine dye that was used for tracking of
phobic paclitaxel from PLGA films [33]. The release of ibuprofen structural change. At 20 min, the solution started to form the solidifica-
from PLLA-PEG diblock copolymers film was enhanced with higher tion. The stereomicroscope image (Fig. 4aIII) showed the transparent
PEG compositions [22]. and little roughness surface and the digital camera image (Fig. 4bIII)

Fig. 4. Morphology characterization of PLA solution, a) stereomicroscope images at magnification of 50× and b) digital camera images at I) 0, II) 10, III) 20, IV) 30, V) 40, VI) 50, VII) 60, VIII)
70, IX) 80, X) 90, XI) 120 and XII) 180 min.
 T. Phaechamud, S. Chitrattha / Materials Science and Engineering C 61 (2016) 744–752 749

showed a rift of solution indicating solid–liquid phase separation. This simply appeared cloudy even though the two distinct phases coexisted
solid–liquid phase separation was owing to the polymer precipitation. [27]. In the early stage of liquid–liquid phase separation, a co-
Over time, the solution dried continuously which the more surface continuous structure developed (Fig. 6b). The late stage of liquid–liquid
roughness was evident in Fig. 4aIV to aXII and the solution became phase separation would occur due to coarsening effect, yielding the
dried-membrane in Fig. 4bIV to bXII. This indicated that PLA in DCM fragmented particles (Fig. 6c) and then the spherical particles (Fig. 6d)
changed from solution into a dense film only by the solidification [38]. According to the mechanism of liquid–liquid phase separation,
(solid–liquid phase separation) process. Accordingly, the PLA-dioxane the size and morphologies of macro- or micro-pores in the scaffold
binary system prepared by thermally induced phase separation were affected by the extent of coarsening [39,40]. However, the coars-
technique also exhibited only a solid–liquid phase separation [25,34]. ening process concomitantly tended to generate more closed pores
On the other hand, PLA–PEG solution revealed a transparent red color be- and gave rise to poor interconnection [41]. The phase separation contin-
cause erythosine was soluble in PEG 400. At the same point; 20 min, PLA– ued until the combination of the two phases reached a stable condition
PEG solution started a phase separation process. Fig. 5a III shows the ag- with the lowest free energy. Depending on the composition and tem-
gregated dye that was soluble in PEG 400. The digital camera (Fig. 5bIII) perature, this condition could be a liquid–liquid phase separation or
showed a rift of solution (liquid–liquid phase separation). Subsequently, solid–liquid phase separation (solidification). The solidification system
at 40 min, the solution started a solidification process (solid–liquid phase included the polymer precipitate and a polymer-lean liquid phase
separation). The stereomicroscope image revealed a little opaque spots with a clear boundary in between the two phases. The polymer-rich
mixed with the aggregation of dyes (Fig. 5aV) and the digital camera re- and polymer-lean phases formed into the porous structure respectively
vealed the incipient of opaque spots on the cloudy solution (Fig. 5bV). after aging and drying (Fig. 6e) as previous mentioned [36–38]. Thus,
The more opaque spots appeared when the time passed and completely the porous structure of PLA matrix film was created after the evapora-
formed into the opaque films at 180 min (Fig. 5aXII and bXII). This result tion of DCM and inserted by PEG 400 that remained in the film.
indicated that PLA–PEG in DCM changed from solution into porous film
by two steps, (liquid–liquid) phase separation and solidification, respec- 3.3.2. Thermogravimetric analysis (TGA)
tively. The porous structures produced by non-solvent induced phase The thermal degradation of PLA powder, PEG 400, PLA and PPEG
separation of PLA-DCM-hexane [29], PLLA-1,4-dioxane-water [35], membranes, PLA and PPEG solutions are illustrated in Fig. 7. PLA pow-
polycarbonate-chloroform-cyclohexane [36] and polysulfone-N,N- der, PEG 400, PLA and PPEG membranes displayed the single-step of
dimethylacetamide-water [37] as ternary systems have been reported. degradation. The initial decomposition temperature of PLA material
These porous structures were also achieved by liquid–liquid phase sepa- and PLA film were similar at 250–260 °C and the temperature ascribed
ration followed by solidification. These porous structures have been used to the maximum rate of thermal degradation were about 340 °C. Their
as membrane, bone scaffold and controlled drug release because of their rate of decomposition that were determined by calculation of slope of
suitable properties. However, the porous films obtained from PLA-DCM- weight (%) and temperature (°C) graph were also similar at 2.16–
PEG 400 ternary system exhibited a biodegradable, soft behavior and uni- 2.40%/°C. While, the initial decomposition temperature of PPEG mem-
formity porous structure [23]. Therefore, they could offer various poten- brane was at 150 °C, the temperature ascribed to the maximum rate
tial applications of functional materials including drug delivery system, of thermal degradation was at 286 °C and rate of decomposition was
tissue engineering and wound dressing applications. 0.72%/°C that was different from PEG 400. This indicated that PPEG
The schematic illustration of pore formation by phase separation membrane exhibited less resistance to the thermal degradation than
mechanism of this study is shown in Fig. 6. Addition of non-solvent PLA powder and PLA film because of their unique morphology. PLA
(PEG 400) to a polymer solution could increase the Gibbs free energy film exhibited a dense topography whereas PPEG membrane showed
of system [36] and some amount of solvent (DCM) was evaporated a porous structure (Fig. 3). However, the addition of PEG 400 could de-
and the polymer concentration gradient was formed (Fig. 6a). This con- crease the rate of decomposition of PLA films. The addition of PEG 400
sequence resulted in a phase separation (Fig. 6b, c and d). Liquid–liquid increased the mass ratio of membrane, the heat flux to the membranes
phase separation led to the formation of an interpenetrating network decreased and then decomposition rate was reduced [42]. The incorpo-
structure of a polymer-rich phase consisting of the polymer and a ration of the PEG has been reported to decrease the thermal decompo-
portion of the solvent, and a polymer-poor phase which contained the sition temperature of PLA in PCL/PLA and PLA/PEG blends [43–45]. On
non-solvent and the remaining solvent. At this stage, the mixture the other hand, the PLA and PLA–PEG solutions exhibited the two-step

Fig. 5. Morphology characterization of PLA–PEG solution, a) stereomicroscope images at magnification of 50× and b) digital camera images at I) 0, II) 10, III) 20, IV) 30, V) 40, VI) 50, VII) 60,
VIII) 70, IX) 80, X) 90, XI) 120 and XII) 180 min.
750 T. Phaechamud, S. Chitrattha / Materials Science and Engineering C 61 (2016) 744–752

Fig. 6. Schematic illustration of pore formation by phase separation mechanism.

degradation processes. The first step, an initial degradation started at a showed that increasing PLA concentration significantly speeds up the
beginning point of process, occurred from the volatilization of DCM. phase separation kinetics. Consistent with our study, liquid–liquid
The rate of decomposition of PLA and PLA–PEG solutions were 11.45 phase separation occurred when PEG 400/DCM volume ratio decreased
and 1.59%/°C, respectively. The end of decomposition temperature of but increasing the %weigh of PLA and solidification occurred when PEG
first step for PLA solution was 110 °C, whilst PLA–PEG solution was 400 ratio increased more than DCM ratio. Therefore, the phase separa-
90 °C indicated that DCM completely evaporated from the samples tion kinetics of PLA, DCM and PEG 400 ternary system might be ex-
due to its high evaporation rate (the vapor pressure of DCM is plained similar to PLA, DCM and hexane ternary system. Hexane in
400 mmHg at 24.1 °C [46]). In the second step, the rate of decomposition PLA-DCM-hexane ternary system could evaporate together with DCM
of PLA and PLA–PEG solutions were 0.96 and 0.31%/°C, respectively. after porous structure formation whereas PEG 400 in PLA-DCM-PEG ter-
However, the initial decomposition temperature and the temperature nary system still remained in the porous structure as mentioned above.
ascribed to the maximum rate of thermal degradation of PLA and This result might be influenced to their different morphology, porosity
PLA–PEG solutions were similar with the PLA and PPEG films, respec- and then applications. The average pore size of the our prepared porous
tively. This indicated that PLA and PLA–PEG solutions were changed membrane containing PEG was ~20 μm while the porosity was 55 ± 2%
into films prior to the beginning of the second step. This result as previously reported from our research group [47]. Similarly, the po-
corresponded with the morphology study of PLA and PLA–PEG solutions rosity of PLA–PEG copolymer biocomposite fabricated by solvent
that DCM thoroughly evaporated during film formation processes. casting-particulate leaching method was in the range of 50–70% [48].
Antimicrobial agent-loaded porous PLA films prepared with this
3.3.3. Determination of phase separation and solidification technique have been reported recently for their efficient inhibition of
The above investigation informed that the plain PLA film was obtain- the bacteria growth [47]. Gentamicin sulfate-loaded film inhibited
ed from the PLA solution only through solidification process. Neverthe- Staphylococcus aureus, Proteus mirabilis and Pseudomonas aeruginosa,
less, the porous PLA film was obtained from the PLA–PEG solution whereas metronidazole-loaded PLA film inhibited B. fragilis and the
through liquid–liquid phase separation and solidification processes. sustainable antibacterial activity was attained for 7 days.
The percentage of each substance in the samples at each step was deter-
mined referable from the morphology change during film formation. 4. Conclusions
This measurement was based on the constant weight of PLA and PEG
400 while DCM weight was minimized from its volatilization. The per- The PLA porous matrix film was produced by adding PEG 400 using
centage of weight change for substances of PLA solution is showed in the simple solvent casting method. The occluded porous structure of
Fig. 8a. The %weigh of PLA and DCM at the initial point were 10 and PLA porous matrix film characterized from cryo-SEM indicated that
90%, respectively. At solidification point which a rift of solution was ev- PEG 400 still remained in the PLA porous structure. The different porous
ident (Fig. 4bIII), DCM remained 79.4% whilst PLA was 20.6%. Finally, the structure of PLA films was obtained when various non-solvents of PLA
constant weight of film was reached at 24 h which DCM remained 10.6% were used as co-solvent. However, the dense films were obtained
and PLA was 89.4%. On the other hand, initially the %weight of sub- when the solvents that could partially solubilize PLA were used.
stances of PLA–PEG solution for DCM, PLA and PEG 400 was 75, 10 Therefore, the pore formation mechanism occurred from non-solvent
and 15%, respectively (Fig. 8b). At (liquid–liquid) phase separation induced phase separation. The higher viscosity of non-solvent
point that showed a rift of solution (Fig. 5b III), DCM remained 61.5%
while PLA and PEG 400 were 15.4 and 23.1%, respectively. At solidifica-
tion point that showed the incipient opaque spot on the cloudy solution
(Fig. 5bV), DCM and PEG 400 were similar as 37 and 37.8%, respectively,
while PLA was 25.2%. At last, DCM remained 9.1% whilst PLA and PEG
400 were 36.4 and 54.6% at the constant weight of membrane at 24 h.
These results indicated that the DCM remained in the prepared mem-
brane; however, it was a tiny amount and could be completely evapo-
rated by hot air oven because of its high vapor pressure. The ternary
phase diagram of PLA, DCM (solvent) and PEG 400 (non-solvent) has
not been conducted. Nevertheless, this diagram of system comprising
PLA, DCM (solvent) and hexane (non-solvent) has been determined
[36]. The boundaries between single phase, liquid–liquid phase separat-
ed and solid–liquid phase separated regions were identified. The
binodal that was the boundary separating the single phase and liquid–
liquid phase separated regions occurred at a higher PLA amount, at
low hexane/DCM volume ratios. Precipitation became more likely to- Fig. 7. TGA thermograms of PLA material, PEG 400, PLA and PPEG films and PLA and PLA–
wards the hexane-rich portion of the phase diagram. Kinetics studies PEG solutions.
 T. Phaechamud, S. Chitrattha / Materials Science and Engineering C 61 (2016) 744–752 751

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