Development and characterization of electrospun curcumin-loaded antimicrobial nanofibrous membranes

Abstract

Curcumin is a naturally occurring hydrophobic polyphenol compound. It exhibits a wide range of biological activities such as antibacterial, anti-inflammatory, anti-carcinogenic, antifungal, anti-HIV, and antimicrobial activity. In this research work, antimicrobial curcumin nanofibrous membranes are produce by an electrospinning technique using the Eudragit RS 100 (C₁₉H₃₄ClNO₆) polymer solution enriched with curcumin. The morphology and chemistry of the membrane are analyzed using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. Kirby Bauer disk diffusion tests are carried out to examine the antibacterial effectiveness of the membrane. Experimental results show that the nanofibers produced are of uniform thickness morphology and curcumin is successfully incorporated into the nanofibrous mat, while no chemical bonding was observed between curcumin and the polymer. The antimicrobial curcumin nanofibrous membranes can be effectively applied as antimicrobial barrier in a wide variety of medical applications such as wound healing, scaffolds, and tissue engineering.

Keywords

antimicrobial nanofibrous membrane curcumin electrospinning SEM FTIR Kirby Bauer disk diffusion test

Nanofibers have emerged as exciting new class of materials, useful for many applications owing to their thickness (≤100 nm) and large surface-to-volume ratio.¹ Depending on polymer type, attainable fiber assembly, production rate, and cost, several techniques have been developed to produce nanofibers. Among these are electrospinning,² melt-blowing,³ template synthesis,⁴ self-assembly,⁵ multi-component spinning,⁶ flash spinning,⁷ and nanolithography.⁸

It is recognized^2–8 that electrospinning is the most versatile fabrication process that can produce nanofibers from a wide range of polymeric materials at high production rate and low cost. Electrospinning is a process of applying high voltage between a polymer solution contained in a syringe with a capillary tip and a collector. Electrospun fibers are collected at the collector during the fabrication process in the form of membranes. The properties of nanofibers can be customized by regulating the voltage, concentration and viscosity of the polymer solution, and solvent composition.⁹ Till the recent past, electrospinning of more than 100 varieties of natural and synthetic polymers, composites and ceramics into ultra-fine nanofibers have been demonstrated. Nanofibers can also be functionalized by mixing with functional additives or surface modification.^10,11 Electrospun nanofibrous membranes have found a wide range of applications in health care, energy storage, environment engineering and biotechnology due to their relatively light weight, high surface area, and interconnected porous structure.¹⁰

Recent developments in the field of nanostructures have opened up exciting opportunities for new materials design, such as nanofibrous membranes with antimicrobial properties for medical applications such as wound dressings.^12,13 Conventional wound dressings provide basic environments for wounds with regards to barrier to evaporation and protection from outside bodies. However, the biologically functional electrospun antimicrobial nanofibrous membranes encourage healing, and also compensate the functions of conventional wound dressing.¹⁴ Antimicrobial agents inhibit the growth of microorganisms by bacteriostatic and bactericidal responses (they can, however, also be harmful in the case of specific allergies¹⁵). Compared with synthetic alternatives, natural antimicrobial agents are less harmful, biocompatible, economically viable and readily available. Natural antimicrobial agents are the major chemical constituents of some common plants such as turmeric, aloe vera, basil and neem, etc. Turmeric was used in ancient times to promote wound healing because of one of its major chemical components, curcumin. Figure 1 shows the structure of the curcumin molecule. Curcumin is an anti-oxidant, anti-inflammatory, anti-cancer molecule used to reduce pain and accelerate the process of wound healing.^16,17 Curcumin dissolves in organic solvents such as ethanol, acetone and dimethyl formamide (DMF), and its anticoagulant property makes it suitable for drug-eluting stents.¹⁸ Several researchers have successfully incorporated curcumin into biomaterials and observed its medical efficacy. Nguyen et al.¹⁶ produced curcumin-loaded poly-lactic acid (PLA) nanofibers and reported its potential antimicrobial effectiveness. Xie et al.¹⁹ fabricated curcumin-loaded silk fibroin nanofibrous membranes and suggested that the drug-loaded nanofibrous membranes can be useful as biomaterials with antimicrobial and antitumor function.

Figure 1.

Curcumin (C₂₁H₂₀O₆) structure.

A wide range of natural (collagen, gelatin, chitosan, elastin, silk fibroin and alginate) and synthetic (PGA, PLGA, PCL, PLLA-CL, PEO and PVA) polymers are used to produce nanofibrous membranes for biomedical applications.^20–22 Natural polysaccharide polymers are biocompatible and biodegradable compared with synthetic polymers, and they display improved structural properties. In return, they have inferior mechanical properties. Consequently, copolymers of natural and synthetic polymers are sometimes mixed to enhance the functional properties of membrane.²³ Among the synthetic copolymers, Eudragit RS 100 (C₁₉H₃₄ClNO₆) is greatly appreciated for biomedical applications because of its exceptional permeability and swelling properties.²⁴ In this research work, for the first time, an economical and effective antimicrobial nanofibrous membrane is fabricated by applying an electrospinning technique on the Eudragit RS 100 polymer solution enriched with curcumin (a natural antimicrobial agent). Surface morphology and chemical composition of the nanofibrous membrane are studied using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), respectively. To evaluate application in the medical field, Kirby Bauer disk diffusion tests are carried out to examine the antibacterial effectiveness of the antimicrobial curcumin-loaded nanofibrous membranes. Based on the performance properties of the fabricated antimicrobial scaffold, its use is recommended in industrial applications such as tissue engineering, medical devices and wound care dressings.

Experimental work

Materials

A copolymer of acrylic and methacrylic acid esters with quaternary ammonium groups [ethyl prop-2-enoate; methyl 2-methylprop-2-enoate; trimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium; chloride], also called Eudragit RS 100 (C₁₉H₃₄ClNO₆) and supplied by Sigma-Aldrich Germany was used as electrospinning polymer. Ethanol and DMF, used as solvents, were purchased from Duksan Chemicals (Korea). Analytical grade curcumin was recovered from turmeric powder, obtained from a local market.

The conductivity, viscosity and surface tension of the solutions were measured using conductivity meter, Ostwald viscometer and stalagmometer, respectively. The nanofibrous membranes were successfully developed using sprocket disk needleless electrospinning. Figure 2 represents the schematic diagram of needleless electrospinning setup. The setup is composed of a high-voltage supply, a solution bath, an intermediate disk equipped with spinneret for fabrication of nanofibrous membrane and a collector to receive nanofibers and produce membranes.

Figure 2.

Schematic diagram of needleless electrospinning setup.

Finite element method was used to analyze the mechanism, profile and intensity of electric area across the spinneret of sprocket disk in needleless electrospinning setup. The geometry of the bullet spinneret was produced using SolidWorks software (2014 version) and COMSOL Multiphysics 5 software was used for modeling.

Surface morphology and chemical composition of electrospun nanofibers were analyzed by scanning electron microscopy (SEM, Inspect S50) and Fourier transform infrared spectrometer (FTIR, Nicolet iS5), respectively. Mueller–Hinton agar plates were used to evaluate the response of curcumin-loaded nanofibrous membrane toward bacteria growth.

Methods

Curcumin extraction

To extract curcumin, turmeric powder was mixed with ethanol (95%) in a ratio 1:5 for 15 days at room temperature with the help of a magnetic stirrer. By doing so, the main ingredients of turmeric diffused into the solvent and the active substance (curcumin) could be recovered by filtration and evaporation.

Polymer solution preparation

The polymer solution for electrospinning of nanofibrous membrane without antimicrobial activity was prepared by adding 3.2 mg of Eudragit RS 100 polymer to an 8% solution of HCON (CH₃)₂ containing 20 mL of ethyl alcohol and 20 mL of DMF. The polymer solution was stirred for 36 h continuously to reach uniformity.

To produce antimicrobial nanofibrous membrane, the polymer solution was enriched with curcumin before electrospinning in order to obtain three mixtures of different concentrations (1 wt%, 5 wt% and 9 wt%). Each of them was stirred for 36 h continuously at room temperature to obtain a homogeneous solution. The viscosity, conductivity and surface tension of the prepared polymer solutions were measured and the respective values are reported in a following section.

Nanofibrous membranes preparation

Intermediate disk needleless electrospinning setup was used to produce the nanofibrous membrane specimens. The electrospinning polymer solution was added to the solution bath and spinneret teeth were dipped into the polymer solution. A 12V DC potential was supplied between the wheel and a twine electrode submerged in the polymer solution bathtub.

The optimized value for spinning distance was set to 12 cm and potential at 35 kV. Such values were calculated by using finite element method and modeling software. Figure 3 shows the distribution of electric field around the intermediate disk equipped with spinneret. It was observed that electrical intensity is denser (red color) on the upper sphere of the spinneret in comparison with other parts.

Figure 3.

Electric field profile around the intermediate disk equipped with spinneret.

Electrical intensity is the opposite of the gradient of the implemented capacity difference (E = −∇ V, where, V = Electrical potential and E = Electrical intensity). Consequently, a geometric form that has large curvature should be able to produce a strong electrical discharge under the same applied electrospinning conditions, that is under the same electrical potential and electrical intensity. This is why the electrical intensity is denser on the upper sphere in comparison with the other parts of the spinneret.

With the increase in electric potential, up to the optimized value, the sprocket disk starts rotating and cohesive forces between the spinneret and polymeric solution are established. During the rotation, the spinneret transfers the polymer solution from the bathtub to the collector in the form of nanofibers. The layers of nanofibers accumulate on the collector and develop a fibrous membrane. It is important to note that the amount of the electrospun nanofibers increases with time.

The nanofibrous mat accumulated on the collector surface was recovered. Figure 4 shows the macro images of the membranes recovered during the electrospinning experiments conducted in this study.

Figure 4.

Macro images of curcumin-loaded nanofibrous membrane. (a) Image of a 2 cm × 3.5 cm piece of nanofibrous membrane, (b) 3 × magnified image.

Kirby Bauer disk diffusion method

The antibacterial activity of the nanofibrous membranes against Staphylococcus aureus (ATCC 25923) was conducted following the standard test method Kirby Bauer disk diffusion. To do so, a standard bacterial culture of Staphylococcus aureus was used and a liquid culture at 150 million/mL was spread across the (Mueller–Hinton) agar plate using a moist swab. Then the circular pieces (disks) of antimicrobial nanofibrous membrane were placed carefully on the floor of the agar plate using forceps. The infected forceps were cleaned each time by putting it in a bacteria incinerator for 10 s. The agar plates containing the antimicrobial nanofibrous membrane samples were incubated at 37℃ for 24 h. The developed inhibition zones were measured using metric ruler to analyze the sensitivity of the antimicrobial nanofibrous membrane to bacteria.

Results and discussion

Properties of polymer solution

The viscosity, surface tension and conductivity of the prepared polymer solutions for electrospinning of nanofibrous membranes were measured at 0 wt%, 1 wt%, 5 wt% and 9 wt% curcumin concentrations. The results, reported in Table 1, show a decreasing trend in viscosity and surface tension of the polymer solution with the increase in curcumin concentration. In order to achieve uniform and beadless nanofibers, the concentration of the electrospinning polymer solution should be above entanglement concentration (Ce), which is the minimum concentration required to develop nanofiber.²⁵ Lower viscosity of the polymer solution may cause electrospraying instead of electrospinning, while lower surface tension of the polymer solution leads to bead-free nanofibers production in electrospinning.²⁶ The addition of curcumin to the polymer solution was kept to the level to keep the viscosity above the entanglement concentration (Ce) threshold; therefore, at the attained viscosity, the workable range of polymer solution is expanded. A higher conductivity was observed when the concentration of curcumin was increased in the polymer solution. Wang et al.¹⁷ also reported the increase in conductivity with the addition of curcumin to the polymer solution and observed that the enhanced electrical conductivity favors the electrical drawing effect on the jet fluid and decreases the fiber fineness.

Table 1.

Properties of the prepared polymer solutions enriched with curcumin

Solution number	Curcumin concentration	Viscosity [cP]	Conductivity [mS]	Surface tension [dyne/cm]
1	Polymer solution without curcumin	1415 ± 9	0.75 ± 0.03	87 ± 1
2	Polymer solution with 1% curcumin	1165 ± 5	0.96 ± 0.06	81 ± 2
3	Polymer solution with 5% curcumin	769 ± 7	1.50 ± 0.06	78 ± 1
4	Polymer solution with 9% curcumin	687 ± 4	1.80 ± 0.01	74 ± 1

Morphology

Figure 5 shows the SEM images showing the morphology of the electrospun nanofibrous membranes. The results evidence that the nanofibers are apparently produced with uniform thickness, and no accumulation of polymer mass in the form of beads was observed across the fibrous membrane. Researchers in the recent past reported that there are several electrospinning process parameters such as polymer concentration, applied voltage, type of solvent system, solution fed rate, type of collector and rotation speed that can affect the morphology of the nanofibers.^16,27,28 In this study, for all the specimens prepared, the electrospinning process parameters were first optimized and then kept identical during the series of experiments. As a direct consequence, diameter, layer thickness and distribution were found to be uniform in all nanofiber sheets.

Figure 5.

SEM images of membrane having (a) 1%, (b) 5% and (c) 9% curcumin content.

The absence of bead formation and apparently even distribution of nanofibers in the nanofibers cluster shows the uniform dispersion of curcumin in the polymer and in the membrane. A relative discontinuity of nanofibers was noticed in Figure 5(a) and (b). However, uniform and continuous nanofibers were observed when the curcumin content in the polymer solution was increased to 9 wt%, as highlighted by Figure 5(c). The relative discontinuity noticed in Figure 5(a) and (b), can be ascribed to the lower capacity of these two polymer solutions to carry charges in comparison with the 9 wt% polymer solution. The latter seems to be the best doping concentration to obtain continuous and uniform thickness electrospun nanofibers at the designated optimized electrospinning conditions.

FTIR analysis

FTIR spectra of the polymer (Eudragit RS 100), antimicrobial agent (Curcumin) and the electrospun curcumin-loaded (9 wt%) nanofibrous membrane are reported in Figure 6. The FTIR spectrum of the polymer (Eudragit RS 100), Figure 6(a), confirms the presence of methyl band at 2930 cm⁻¹, ester C=O band at 1724 cm⁻¹ and C–O band at 1160 cm⁻¹. Similar observations are reported in the literature by other researchers.^29,30

Figure 6.

FTIR spectra of (a) polymer (Eudragit RS 100), (b) antimicrobial agent (curcumin) and (c) antimicrobial electrospun nanofibrous membrane.

The FTIR spectrum of the antibacterial agent used in this study, Figure 6(b), evidences the presence of hydroxyl, methyl, carbonyl and C=C bands at 3307 cm⁻¹, 2950 cm⁻¹, 1640 cm⁻¹ and 1560 cm⁻¹ bands, respectively. The observations are supported by the findings reported by Rohman et al.³¹ and Siregar et al.³² while working on curcumin-based drugs for medicinal applications.

Curcumin-loaded electrospun nanofibrous membrane FTIR spectrum, Figure 6(c), indicates the peaks for the constituents of polymer and curcumin such as at 3310 cm⁻¹ (phenolic OH), at 1740 cm⁻¹ (C=O ketone), at 2970 cm⁻¹ (alkane CH₃ stretching), at 1550 cm⁻¹ (C=C) and at 1160 cm⁻¹ (C–O). These results confirm the incorporation of antimicrobial agent (curcumin) into the nanofibrous membrane, while no apparent chemical linkages between the antimicrobial agent and polymer are observed.

Kirby Bauer disk diffusion test results

Inhibition zones were measured around the nanofibrous membrane without curcumin and 9 wt% curcumin-loaded nanofibrous membrane disks in the agar plate. For the ease of comparison, the black arrows, visible in the pictures, identify the specimens loaded with antimicrobial agents. From results presented in Figure 7(b), it is evident that the curcumin spreads into the agar and stops the growth of bacteria in the indicated zones compared with the zones having disks of nanofibrous membrane without curcumin. It is thus possible to conclude that the bacteria were totally killed after the incubation process and antibacterial activity of the nanofibrous membranes lasts even after 7 days. In order to avoid the spread of bacteria, different antimicrobial liquids were used on hand gloves during the material handling in the antimicrobial experiments. The lids look differently because the writing was erased during the experiment and it was replaced.

Figure 7.

Kirby Bauer disk diffusion, agar plate loaded with disks (a) before and (b) after incubation at 37℃ for 24 h.

The inhibition of bacterial growth is confirmed by several researchers. Xie et al.¹⁹ exhibited the drug-release properties of SF/PEG nanofibrous membranes loaded with curcumin. Nguyen et al. ¹⁶ reported higher closure rates of wounds treated with curcumin-loaded PLA nanofibers. Wang et al. proposed that PVP nanofibers loaded with curcumin display enhanced bioavailability and effective anti-cancer effect.¹⁷ Hussain et al. proposed that alginate containing natural polyphenol-based antimicrobial agents can be a potential candidate for wound care.³³ The current results show promising properties for the development of curcumin-based antimicrobial membranes for medical applications.

Conclusion

In this research work, an economical and readily available natural antimicrobial agent, curcumin, extracted from a turmeric plant is successfully incorporated into a nanofibrous membrane using the electrospinning technique for medical applications. The nanofibrous membranes were characterized for morphology, chemistry and antimicrobial effectiveness. The SEM results show the formation of smooth, uniform and beadless nanofibers, clustered in a fibrous mat/membrane when the concentration of curcumin was 9 wt%. The FTIR analysis confirmed the incorporation of antimicrobial agent curcumin into the nanofibrous membrane without chemical linkages between the antimicrobial agent and the polymer. The Kirby Bauer disk diffusion test results verified the antimicrobial effectiveness of the fabricated curcumin-loaded nanofibrous membranes.

Future work

Future work in progress is in the direction of incorporating several other naturally occurring antimicrobial agents such as aloe vera, neem and basil, and their blends, into the nanofibrous membranes for high-end medical applications at lower cost.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The experimental work was performed in Nano spinning lab college of Textile Engineering, Bahauddin Zakariya University, Multan 60800, Pakistan where needleless electrospinning setup is established by financial support from the Bahauddin Zakariya Multan, Pakistan under research support Grant (No.DR&EL/D-234).

Footnotes

ORCID iD

Jean-Noël Jaubert

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