DOI: 10.3303/CET2398047 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Paper Received: 20 November 2022; Revised: 23 February 2023; Accepted: 30 March 2023 
Please cite this article as: Valor D., Garcia-Casas I., Montes A., Pereyra C., Martinez De La Ossa E., 2023, Development of PLGA-PEDOT Mixed 
Polymeric Scaffolds and Their Impregnation with Natural Extracts Using Supercritical CO2, Chemical Engineering Transactions, 98, 279-284  
DOI:10.3303/CET2398047 
  

 CHEMICAL ENGINEERING TRANSACTIONS  
 

VOL.  98, 2023 

A publication of 

 

The Italian Association 

of Chemical Engineering 

Online at www.cetjournal.it 

Guest Editors: Sauro Pierucci, Carlo Pirola 

Copyright © 2023, AIDIC Servizi S.r.l. 

ISBN 978-88-95608-97-6; ISSN 2283-9216 

Development of PLGA-PEDOT Mixed Polymeric Scaffolds 

and their Impregnation with Natural Extracts using 

Supercritical CO2  

Diego Valor*, Ignacio García-Casas, Antonio Montes, Clara Pereyra, Enrique M. De 

la Ossa 

Department of Chemical Engineering and Food Technology, Faculty of Sciences, International Excellence Agrifood Campus 

(CeiA3). University of Cadiz, Campus Universitario Río San Pedro, 11510, Puerto Real (Cadiz), Spain. 

diego.valor@uca.es 

Conjugated materials are increasingly becoming a vital component in the production of therapeutic scaffolds 

through the creation of foams. This is due to the unique properties they possess that are not commonly found 

in other traditional polymers used in the industry. This preliminary study investigates the formation of conductive 

and porous scaffolds of PLGA/PEDOT:PSS and their impregnation with an ethanolic extract of mango leaves 

using supercritical CO2. The effect of various process parameters, such as pressure, temperature, contact time, 

and polymer ratio, on the formation of the scaffolds and their properties were studied. The results were analyzed 

using scanning electron microscopy, DPPH assay, and the Folin-Ciocalteu method for polyphenols. The 

produced scaffolds showed various pore sizes (10 to 100 µm), and high antioxidant capacities and impregnation 

of the phenolic compounds were achieved. The results suggest that the scaffolds and the proposed technique 

have potential for various biomedical applications due to their porous structure and antioxidant capacities. 

1. Introduction 

The current research interest on composite 3D structures has increased considerably, especially due to the 

support for cell attachment that they provide to in vitro culture cells (Drury and Mooney, 2003). The flexibility 

and porosity of these structures are their most important properties for their use in tissue engineering, and 

polymers or mixtures of these are commonly used materials, owing to their mechanical properties (Chan and 

Leong, 2008). To mimic the extracellular matrix, materials must have an internal structure with interconnected 

pores or large voids, called scaffolds (Lawrence and Madihally, 2008). 

Recent research has shed light on the advantages of utilizing biodegradable and conductive copolymers to 

facilitate cell proliferation and tissue repair (Wu et al., 2016). The presence of conductivity in organisms plays a 

crucial role in maintaining biological processes such as nerve signal transmission and tissue healing. 

Consequently, the potential of using electrical stimulation to enhance these processes is gaining recognition 

(Korupalli et al., 2021). Conductive polymers, such as polyaniline, polythiophenes, polypyrrole or poly(3,4-

ethylenedioxythiophene) (PEDOT), have a positive effect on cell proliferation and adhesion with or without 

electrical stimulation and demonstrate a high degree of biocompatibility (Yu et al., 2022). PEDOT:PSS, a 

polymer studied in this work, has been incorporated into scaffolds with different techniques, such as 

electrochemical polymerization surrounding a pre-synthesized scaffold, vapor-phase polymerization for neural 

stem cell proliferation and differentiation or freeze-drying embedding PEDOT:PSS in porous materials, 

achieving improved mechanical properties through the addition of different aggregates (Richardson-Burns et 

al., 2007).  

Conductive polymers lack important characteristics for tissue engineering such as strength, flexibility, solubility, 

or durability. To overcome these limitations, conductive polymers are often combined with non-conductive 

polymeric compounds, which provide mechanical properties required for tissue repair. Poly(lactic-co-glycolic 

acid) (PLGA), polylactide (PLA), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyvinyl acetate (PVAc) 

279



or polycaprolactone (PCL) are the most commonly used compounds (Marsudi et al., 2021). Among these, PLGA 

is the most widely used for biomedical objectives, thanks to its mechanical properties, adjustable 

biodegradability ratio, and facility of processing in different techniques (Ishaug et al., 1997; Shen et al., 2008). 

PLGA has excellent processability, which facilitates the production of scaffolds with various pore sizes, and 

adequate mechanical properties, biocompatibility, and degradability to promote the regeneration of tissues. 

However, its degradation can lead to a local acidic microenvironment (Pan and Ding, 2012). 

Additionally, studies in the literature suggest that bioactive capabilities can be added to porous scaffolds by 

impregnating them with compounds from natural compounds (Satpayeva et al., 2022). For example, a natural 

extract from mango leaves with pharmacological properties considered an excellent agent for the treatment of 

degenerative diseases, such as Alzheimer's or cancer was used in the present work (Fernández-Ponce et al., 

2015). Supercritical carbon dioxide (scCO2) foaming is employed for the formation of functional porous scaffolds. 

CO2 has a high dissolution in polymers and is a non-toxic, inexpensive, and reusable compound. Therefore, this 

technique is considered a green process (Jacobs et al., 2008). In the supercritical state, CO2 has properties of 

density, diffusivity, and low viscosity that allow it to permeate the polymeric matrix, causing plasticization of the 

matrix by decreasing its glass transition temperature. After a controlled contact time, depressurization of the 

system leads to supersaturation, with the subsequent cell nucleation phenomenon during phase separation, 

resulting in the formation of the porous scaffold structure (Kosowska et al., 2022; Valor et al., 2022). To carry 

out foaming and impregnation in a single step, the polymer mixture and the extract are introduced concurrently 

and maintained in contact with supercritical carbon dioxide (scCO2) to facilitate its permeation into the polymer 

and induce a plasticizing effect that facilitates the impregnation process. The objective of this preliminary 

research is to developed conjugated systems with PEDOT and PLGA with the incorporation of mango leaves 

extracts, to use in tissue engineering. The authors evaluated the influence of temperature, pressure or contact 

time on the morphology, total impregnated compound and its antioxidant capacities. 

2. Materials and methods 

2.1 Materials 

PLGA (lactide:glycolide 75:25) (poly(lactic-co-glycolic acid)) with Mw 76,000-115,000 and PEDOT:PSS 

(Poly(2,3-dihydrothieno-1,4-dioxin)-poly(styrenesulfonate)) 3.0-4.0% in H2O were purchased from Sigma-

Aldrich (Spain). CO2 (99.8% purity) was supplied by Linde (Spain). DPPH (2,2-diphenyl-1-picrylhydrazyl), 

ethanol (99.5%), Folin–Ciocâlteu reagent (FCR) and Na2CO3 were supplied by Sigma–Aldrich (Madrid, Spain). 

2.2 Production of ethanolic extract from mango leaves 

The mango leaf extract was obtained through maceration extraction using a sample of 40 g of dried and crushed 

mango leaves. Ethanol (99.5%) was used as a solvent for 3 hours at a mild temperature of 45°C with agitation 

to avoid degradation of the leaf compounds. Ethanol was chosen for its high solubility in the supercritical phase, 

making it easy to remove from the final product. The resulting ethanolic suspension was filtered using 

commercial filter paper and then stored in darkness at 4 °C prior to assay. A rotary evaporator was employed at 

45°C to achieve the desired total concentration. 

2.3 Supercritical foaming-impregnation process 

The experiments involving polymer foaming and impregnation were conducted using a SSI pilot plant (Thar 

technologies), as illustrated in Figure 1. The equipment is equipped with a high-pressure pump and a thermal 

bath, enabling CO2 injection in its liquid state. A temperature-controlled electric heat exchanger, located just 

before the CO2 inlet to the 257 mL foaming vessel, helps regulate the temperature. The required pressure of 

the entire system is controlled through an automatic backpressure regulator valve. Finally, the system includes 

a micrometric valve, which enables adjustment of the vessel depressurization rate and CO2 venting through a 

cyclonic separator. 

The PLGA and PEDOT:PSS polymer mixture was processed using a mechanical press to produce tablets, 

which were placed on an aluminum support. 3 mL of the ethanolic extract of mango leaves were introduced into 

the vessel without being in direct contact with the polymer mixture. Then, supercritical CO2 was injected into the 

vessel under the desired conditions for the foaming/impregnation process. The mixture was maintained under 

supercritical conditions for a determined contact time, causing the polymer to plasticize. The system was then 

depressurized at a controlled rate using the micrometric valve, resulting in the foaming and impregnation of the 

compounds present in the extract through the formation of a porous structure inside the tablet. 

 

280



 

Figure 1: Schematic diagram of SSI pilot plant. 

A total of seven experiments were conducted to investigate the impact of various parameters such as polymer 

ratio, temperature, pressure, and contact time on the scaffold formation process and their properties. The 

conditions used in each experiment are shown in Table 1. The tablet was always made using 25 mg of PLGA, 

while the amount of PEDOT was varied in some experiments. The depressurization rate was kept constant at 

0.5 MPa/min, while the concentration of the extract used was always 20 mg extract/mL ethanol. 

Table 1: Conditions used for the foaming/impregnation process. 

Run  
PEDOT 
(µL) 

Pressure 

(MPa) 

Temperature 

(K) 

Time 

(h) 

1 100 12 318 0.5 

2 50 12 318 0.5 

3 50 20 318 0.5 

4 50 20 333 0.5 

5 50 20 318 2 

6 50 20 318 6 

7 50 20 318 24 

2.4 Scanning electron microscopy 

The morphology and pore size of the foamed scaffolds were analyzed using a Scanning Electron Microscope 

(SEM). A Nova NanoSEM 450TM (Elecmi, Zaragoza, Spain) with an accelerating voltage of 15 kV was used for 

the analysis. To enhance conductivity and improve image quality in SEM, a 10 nm film of gold was coated on a 

cross-section of each sample before analysis. The SEM images were processed using Scion image software. 

2.5 Antioxidant activity of extract and scaffolds 

The antioxidant activity was calculated in order to evaluate the success of the runs carried out, for both mango 

leaves extract and the impregnated scaffolds. The methodology employed was derived from the one suggested 

by Brand-Williams and Scherer (Brand-Williams et al., 1995; Scherer and Godoy, 2009), but with necessary 

adaptations. The method was adapted to determine the antioxidant capacity of the impregnated scaffolds. A 

specific quantity of processed scaffolds was submerged in 4 mL of a solution containing 6×10-5 mol/L DPPH 

and left for 2 hours. The absorbance of the DPPH at the beginning and end of the process was measured at 

515 nm. The results were expressed as % IO per 100 mg of impregnated scaffold, where % IO represent the 

percentage of DPPH inhibition, Eq (1). The analyses were carried out in triplicate. 

 

% Inhibition Oxidation =  
(𝐴0 − 𝐴𝑖) 

𝐴0
 * 100   (1) 

2.6 Total polyphenol content impregnated 

The analysis of total phenolic content impregnated in the scaffolds was conducted using a modified version of 

the Folin-Ciocalteau method, as described by Singleton and Rossi (Singleton et al., 1999), for microplates. The 

281



total release of polyphenols presents in scaffolds was achieved via 5-hour stirring in ethanol, and a 12.5 μL 

aliquot from this release was mixed with 12.5 μL of Folin-Ciocalteu's reagent and 200 μL distilled water, then 

shaken for 5 minutes. Subsequently, 25 μL of a sodium carbonate solution (20% w/v) was added, and the 

mixture was stirred for another 5 minutes. After 60 minutes of incubation at room temperature in the absence of 

light, absorbance was measured at 725 nm by means of a Synergy HTX multi-mode reader (Aligent, Biotek, VT, 

USA). A calibration curve was established using gallic acid (20–300 μg/mL). The analyses were performed in 

triplicate to ensure reproducibility. 

3. Results and discussion 

Several successful experiments were carried out in which key variables in both the supercritical foaming process 

and the impregnation of bioactive compounds present in the extract were modified, including temperature, 

pressure, contact time, and the ratio of conjugated polymers used. Figure 2 shows scanning electron microscopy 

images of the experiments where impregnated scaffolds were obtained. Generally, the foaming/impregnation 

process was able to modify the morphology of the initial material, resulting in porous structures, moderately 

ordered, with several pore size distributions depending on the conditions used. The morphology of the original 

tablet Figure 2(a) can be observed, in which it appears that PEDOT:PSS forms a film throughout the system, in 

which no pores are apparently observed at the magnification used. The pore sizes obtained ranged from 12 µm 

in run 7 to larger pores of up to 80-100 µm, as observed in run 2. The increased use of PEDOT seems to lead 

to a smaller pore opening when comparing the results of test 1 with those of test 2. The ability to modify sample 

porosity with slight changes in operational parameters makes supercritical foaming an attractive alternative for 

scaffold formation in tissue engineering. In addition, all samples showed a conductivity in the order of 10-5 S/cm 

in the 2-point conductivity method, so that an acceptable spreading of the PEDOT:PSS over the whole surface 

was obtained. 

 

 
(a) Original tablet 

 

 
(b) Run 1 

 

 
(c) Run 2 

 

 
(d) Run 3 

 
(e) Run 4 

 
(f) Run 7 

Figure 2: SEM images of (a) original PLGA/PEDOT:PSS tablet and  formed scaffolds (1,2,3,4 and 7). 

The obtained results with respect to the antioxidant capacity exhibited by the scaffolds when subjected to the 

DPPH oxidation test as well as the amount of polyphenolic compounds impregnated are shown in Table 2. It 

can be observed that the increase in pressure has a positive effect when comparing runs 2 and 3 (12 and 20 

MPa, respectively) under isothermal conditions. In cases where the impregnation vessel contains an excessive 

amount of active compound, the saturation of the CO2-phase with the compound can be maintained. As the 

pressure (or density) of CO2 increases, the solubility of the active compound in the supercritical CO2-phase also 

increases, resulting in a higher concentration of the active compound in the CO2-phase. This generates a greater 

concentration gradient of the active compound between the CO2-phase and the polymeric matrix, which 

promotes the active compound impregnation in the material. Additionally, the swelling phenomenon of the 

polymer is enhanced with increased CO2 pressure, which facilitates the diffusion of the active compound inside 

282



the polymer. This positive effect of pressure on the active compound loading has been reported in several 

studies (De Souza et al., 2014). 

Table 2: Percentage of inhibition in the DPPH test (%IO) and total content of impregnated polyphenols (TPC). 

Run 
% Inhibition 

Oxidation 

TPC 

(mgGAE/g 

sample) 

1 41.76 209.30 

2 35.42 176.05 

3 48.11 218.48 

4 12.56 60.23 

5 49.77 209.58 

6 58.56 234.56 

7 39.21 196.16 

 

Regarding the effect of temperature, the temperature was increased up to 60ºC under isobaric conditions to test 

its effect. A lower antioxidant activity and impregnation of compounds were clearly obtained. Increasing the 

temperature under isobaric conditions may potentially hinder the impregnation of the active compound into the 

polymer matrix. This is because as the temperature increases, the solvent power of CO2 decreases, resulting 

in a decrease in the amount of active compound dissolved in the supercritical CO2 phase. Studies have reported 

a decrease in active compound incorporation in various polymeric materials when the temperature or pressure 

was increased beyond certain limits. For example, an increase in temperature from 40 to 60 °C at 15 MPa 

caused decreased incorporation of flax oil in β-glucan aerogels (Comin et al., 2012). 

Foaming/impregnation experiments were also carried out at different times while keeping pressure and 

temperature constant. Both the impregnation of phenolic compounds and the % inhibition in the oxidation of 

DPPH in the samples increase with contact time, reaching maximum performance at 6 hours (Run 6). The 

incorporation of active compounds into polymers using scCO2-assisted impregnation involves both 

thermodynamic and kinetic processes. The attainment of equilibrium is dependent on thermodynamic factors 

such as the concentration gradient of active compounds and the physical-chemical properties of the polymer 

and fluid phase, including their chemical affinity. On the other hand, the time needed to reach equilibrium is 

controlled by the kinetics of the impregnation process. It may also be due to degradation of the bioactive 

compounds over time as indicated in several papers in the literature (Almeida et al., 2013; Pantić et al., 2016). 

4. Conclusions 

In this preliminary work the formation of PLGA/PEDOT:PSS using the supercritical foaming technique and its 

impregnation with a high antioxidant extract of mango leaves was achieved. The influence of pressure, 

polymeric ratio, temperature and contact time on the final activity of samples was studied, always under 

supercritical conditions. Scaffolds with a porous structure were obtained by modifying their initial morphology, 

reaching pores ranging from 10 to 100 µm. Successful impregnation of bioactive compounds into the scaffolds 

with proven antioxidant activity was obtained. The positive effect of pressure and time was observed, while 

increases in temperature led to lower compound loadings. Therefore, supercritical CO2 foaming and the 

properties of the obtained scaffolds have high potential for use in tissue engineering. 

Acknowledgments 

We gratefully acknowledge the Spanish Ministry of Economy, Industry and Competitiveness (Project PID2020-

116229RB-I00) for financial support and Central services of Science and Technology of University of Cadiz for 

measurement and developed analyses. 

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