biomolecules

Review
Biofunctionalization of Natural Fiber-Reinforced
Biocomposites for Biomedical Applications
Tânia D. Tavares , Joana C. Antunes, Fernando Ferreira and Helena P. Felgueiras *
Centre for Textile Science and Technology (2C2T), Department of Textile Engineering, University of Minho,
Campus of Azurém, 4800-058 Guimarães, Portugal; taniatav13@hotmail.com (T.D.T.);
joana.antunes@det.uminho.pt (J.C.A.); fnunes@det.uminho.pt (F.F.)
* Correspondence: helena.felgueiras@2c2t.uminho.pt; Tel.: +351-253-510-283; Fax: +351-253-510-293




Received: 30 December 2019; Accepted: 15 January 2020; Published: 16 January 2020


Abstract: In the last ten years, environmental consciousness has increased worldwide, leading to
the development of eco-friendly materials to replace synthetic ones. Natural fibers are extracted
from renewable resources at low cost. Their combination with synthetic polymers as reinforcement
materials has been an important step forward in that direction. The sustainability and excellent
physical and biological (e.g., biocompatibility, antimicrobial activity) properties of these biocomposites
have extended their application to the biomedical field. This paper offers a detailed overview of the
extraction and separation processes applied to natural fibers and their posterior chemical and physical
modifications for biocomposite fabrication. Because of the requirements for biomedical device
production, specialized biomolecules are currently being incorporated onto these biocomposites.
From antibiotics to peptides and plant extracts, to name a few, this review explores their impact on
the final biocomposite product, in light of their individual or combined effect, and analyzes the most
recurrent strategies for biomolecule immobilization.

Keywords: natural fibers; biocomposites; surface modification; specialized biomolecules;

immobilization methods

1. Introduction
The use of eco-friendly materials has been increasing with time as a result of global environmental
awareness. The development of recyclable and environmentally sustainable materials has become an
attractive and important field of research. Natural fibers are among these materials and are gradually
replacing synthetic fibers made from non-renewable petroleum-based resources [1,2].
Composites are formed of a strong load-carrying material (reinforcement) embedded within a
“weaker” material (matrix). Because of the beneficial properties, abundance and low cost of natural
fibers, these are considered a new generation of reinforcements for polymer matrices. By themselves,
natural fibers are very unpredictable (with properties varying from batch-to-batch) and do not possess
the mechanical resilience desirable for most applications; as such, combinations with polymer matrices
have been proposed [3,4]. A biocomposite is considered a material that is composed of at least one
natural resource. The natural fiber added value endows the biocomposites with a wide range of
physical, mechanical and biological properties [5]. Manufacture of biocomposites can be accomplished
by different processing techniques, including compression molding, injection molding, resin transfer
molding, sheet molding, hand lay-up, filament winding, extrusion and pultrusion. These processes
allow the natural fibers, which are presented in the form of loose fibers, nonwoven mats, aligned yarns
and/or woven fabrics, to be placed in the desired direction to acquire specific mechanical properties
in the final product [6]. There are other factors that must be considered as well to attain desirable
properties, such as the type of natural fiber, the chemical compatibility between the fiber and matrix

Biomolecules 2020, 10, 148; doi:10.3390/biom10010148 www.mdpi.com/journal/biomolecules

Biomolecules 2020, 10, 148 2 of 44

phases, the corresponding surface energies and the quality of the interface [7]. The interfacial bonding
between both materials in a biocomposite are affected by the natural fiber’s hydrophilicity and polymer
matrix hydrophobicity. Chemical and physical methods are required to treat the surface of the fiber to
optimize this interaction [3].
The natural fibers’ abundance, availability and low-cost have made biocomposites very attractive
for several industrial applications. However, in biomedicine, specific requirements must be met
prior to their use. The most important is to be accepted by the human body without causing any
adverse response, namely inflammation, allergies and/or early rejection associated with toxicity.
Biocompatibility is, therefore, essential for the successful development of a biomedical device [8,9].
Even though biocomposites on their own have been reported in medical textiles [10], the addition of
specialized biomolecules with particular properties, such as antimicrobial, anti-inflammatory, analgesic,
sedative, anti-oxidative, UV-protection or chemical stability, to name a few, have demonstrated
improved performance on specific biomedical applications. Biomolecules such as peptides, antibiotics,
nanoparticles (NPs) or plant extracts functionalized onto biocomposites contribute significantly to
their biocompatibility towards host cells, while improving other dormant material properties [11–15].
These combinations have been desirable for prospective applications in sutures, coatings for cell
culture and drug delivery matrices, as well as for 3D scaffolds for ligaments, bone, cartilage, skin
and vasculature engineering [10]. Still, even though they have demonstrated tremendous potential,
research in this field is only now taking the first steps with the use of biocomposites for biomedicine,
requiring further study and understanding. The present work explores this subject further by
introducing some of the most recent (last ten years) biomolecule–biocomposite combinations and
their final product properties. Fiber extraction, separation and chemical and physical processing prior
to interfacial bonding with polymer matrices were also discussed. Finally, a detailed and critical
analysis of the biomolecule’s inherent characteristics and the most recurrent methods employed for
their immobilization onto natural fibers, fabrics and biocomposites was provided.

2. Natural Fibers
Natural fibers can be sourced from plants, minerals and animals [16]. The several physical and
mechanical properties that characterize these fibers, such as low cost, low density, high specific strength
and stiffness, processing flexibility, biodegradability and non-toxicity, allow an easy replacement of
synthetic fibers [17]. Nowadays, plant-based fibers are very commonly used in many industrial sectors,
such as textiles, automobiles, packaging, construction, sports equipment and medicine [3,18]. These
are also known as ligno-cellulosic fibers, which can be extracted from inexpensive and available natural
resources, and depending on the part of the plant from which they are sourced, can be classified
into bast fibers (jute, flax, hemp, kenaf and ramie), seed fibers (cotton, milkweed, coir and kapok),
leaf fibers (sisal, pineapple, agave, banana and abaca), grass fibers (sugarcane bagasse and bamboo),
straw fibers (rice, corn and wheat) or wood fibers (softwood and hardwood) [2,16,18,19]. There are
other natural fibers that are considered regenerated fibers, meaning that are produced from natural
sources with human interference. Soybean is an example of this type, which undergoes chemical
manipulation to be turned from a plant into a fiber [20]. Silk, wool, hair and feathers are examples of
animal-based fibers composed mainly of proteins and are the second most important source of natural
fibers [2,21]. However, compared to plant-based fibers they are stronger and more bioactive. Because
of their high costs and lower accessibility, their use is restricted to biomedical applications [8,22]. In this
field, natural fibers have attracted a research interest towards potential applications [23]. Medical
textiles can be used from a simple gauze for wound dressings to sutures, reconstruction and repair of
tissues and bones [24]. The materials for medical purposes require very specific characteristics, such as
biodegradability, biocompatibility, functionability, bioresorbability, sterilizability, manufacturability,
as well as mechanical properties [9]. Table 1 shows the mechanical properties of potential natural fibers
for biomedical applications compared to human tissues.
Biomolecules 2020, 10, 148 3 of 44

Table 1. Mechanical properties of natural fibers and human tissues comparatively (adapted from
[1,2,8,16,19,25]).

Tensile Strength Elongation at Break Young’s Modulus

(MPa) (%) (GPa)
Natural fibers
Jute
393.0–773.0 1.5–1.8 13.0–26.5
(Corchorus capsularis)
Flax
345.0–1100.0 1.3–10.0 27.6
(Linum usitatissimum L.)
Hemp
550.0–900.0 1.6 30.0–70.0
(Cannabis sativa)
Kenaf
295.0–1191.0 3.5 53.0
(Hibiscus cannabinus)
Ramie
348.0–938.0 1.2–8.0 44.0–128.0
(Boehmeria nivea)
Cotton
264.0–800.0 7.0–8.0 5.5–12.6
(Gossypium sp.)
Milkweed
381.0 2.1 8.2
(Calotropis gigantea)
Coir
131.0–175.0 15.0–25.0 4.0–6.0
(Cocos nucífera)
Kapok
90.0–95.0 1.8–4.2 4.0
(Ceiba pentandra)
Sisal
500.0–800.0 2.0–25.0 9.4–22.0
(Agave sisalana)
Pineapple
170.0–1627.0 2.4 60.0–82.0
(Ananas comosus)
Agave
430.0–580.0 3.0–4.7 13.2
(Agave americana L.)
Banana
529.0–914.0 3.0 27.0–32.0
(Musa sepientum)
Sugarcane bagasse
20.0–290.0 1.1 17.0
(Saccharum officinarum)
Bamboo
140.0–230.0 – 11.0–17.0
(Bambusoideae)
Rice
450.0 – 1.2
(Oryza sativa)
Corn
160.0–175.0 – 4.5–5.1
(Zea mays)
Wheat
275.0 – 4.5–6.5
(Triticum sp.)
Softwood
1050.0 – 40.0
(different species)
Hardwood
1000.0 – 38.0
(different species)
Silk
650.0–750.0 18.0–20.0 16.0
(Bombyx mori)
Wool
120.0–174.0 25.0–35.0 2.3–3.4
(Ovis aries)
Human tissues
Hard tissue (e.g., tooth, bone) 130.0–160.0 1.0–3.0 17.0–20.0
Skin 7.0–6.0 78.0 –
Tendon 53.0–150.0 9.4–12.0 1.5
Elastic cartilage 3.0 30.0 –
Heart valves 0.5–2.6 10.0–15.3 –
Aorta 0.1–1.1 77.0–81.0 –

In the last years, the use of natural fibers as reinforcement of composites has received considerable
attention as substitutes of glass, ceramic and metal-based materials in various industries [1,17,18].
Biomolecules 2020, 10, 148 4 of 44

The application of these fibers has started in the automotive and aircraft sectors. However, nowadays
they are being used in electrical and railway devices, as well as in civil engineering for structural and
infrastructure applications such as roofs and bridges [16,19,26]. Biocomposites consist of a polymer
matrix embedded with natural fibers; however, their binding is considered a challenge because of
the numerous chemical structures of both the fibers and the polymers. Their performance depends
on the properties of the individual components and their interfacial compatibility. Thus, it becomes
necessary to modify the natural fibers resorting to specific treatments. Generally, the composition of
the fiber structure is changed using reagent functional groups [1]. The reinforcement of a synthetic
polymer with treated natural fibers introduces a positive effect on their mechanical and tribological
performance. However, this performance depends of type, fraction or treatment of the fibers, type of
polymer or manufacturing process [3,22]. Commonly, increasing the natural fiber amount in a polymer
matrix leads to increased mechanical properties [8]. The matrix material is responsible for binding and
protecting the natural fiber since, due to their fibrous nature, they cannot be used by themselves to
sustain considerable loads [26].

Fiber Separation and Extraction

Fiber separation and extraction are very important as they can affect the fibers’ quality, yield,
chemical composition, structure, etc. [7,27]. Commonly, the separation of the plant-based fibers from
the fiber crops is made by the retting process. This method consists in removing non-fibrous tissues
attached to fibers through decomposition and degradation of hemicellulose and pectins, releasing
individual fibers [2,28]. Table 2 compares five types of retting processes, namely dew, water, mechanical,
enzymatic and chemical retting. Traditional methods, dew and water retting, rely on biological activity
of microorganisms from the soil and are the most commonly used [29–31]; however, these have
several disadvantages (Table 2). To overcome these limitations, improvements in fiber processing
techniques are crucial to ensure consistently high-quality fibers and reduced environmental impact
in terms of water waste and energy consumption [27,29]. Apparently, there is no single method that
can give optimum results in all aspects. Enzymatic retting has been demonstrated to be the most
promising solution due to its high enzyme specificity, better controllability, shorter duration and low
environmental impact [32–35]. Nevertheless, the high cost of the process has not yet made it feasible at
an industrial scale [7]. After the retting process, non-fibrous materials must be completely removed.
For this, the fibers are extracted (breaking, milling, scutching or decortication), cleaned, refined and
processed (spinning or weaving) to be used in a specific application [27].
Animal-based fibers come from diverse sources and, as such, the extraction occurs in different
ways. The most used, silk, is obtained from silkworm cocoons that are composed of fibroin (fiber)
and sericin (gum) proteins endowed with different biological and physicochemical properties [36,37].
Sericin is responsible for coating and protecting the fibroin, which needs to be extracted to release
the fibers. Degumming is a process during which sericin is removed by thermo-chemical treatment
of the cocoons, by boiling in a mild soap solution that dissolves the sericin gum binding the fibers
and untangles them. Lastly, it is washed in cold water to remove the remaining sericin and other
contaminations [8,38,39]. Wool fibers from sheep are, probably, the most widely used at an industrial
scale and are mainly composed of keratin. Fiber extraction is accomplished manually by shearing and
collecting “wool grease”, which has many impurities that must be washed and removed to extract clean
wool [2,21]. Chicken feathers are also composed of keratin. The extraction of these fibers, composed of
fiber (keratin) and quill, is initiated with a wash in water and ethanol to remove dirt and other particles
present on the feather surface and dried under natural light. Then, barbs are mechanically separated
from the quill, treated with NaOH, and further washed and dried [40–42].
Biomolecules 2020, 10, 148 5 of 44

Table 2. Properties and limitations of the five types of retting processes.

Duration of
Retting Type Description Advantages Disadvantages References
Retting
Plant stems are cut and Influenced by
distributed in the field uncontrollable weather
exposed to the action Low cost and conditions and
Dew Retting of pectinolytic sustainable soil-contaminated 2–10 weeks [2,7,31,43]
microorganisms that process. fibers; reduces fiber
disrupt pectins strength, consistency
surrounding the fiber. and quality.
Plant stems are Large consumption
submerged in water and contamination of
Produce
(river, ponds or tanks) water (superior
uniform and
Water Retting where anaerobic environment impact); 7–14 days [2,7,29,30]
high-quality
bacteria develop and extensive stench of
fibers.
break down the fermentation gases and
pectins. high labor costs.
Simple process
The fibers are
that produces
separated by
Mechanical huge quantities High cost and lower
mechanical means, 2–3 days [2,27,43]
Retting of fiber in a fiber quality.
such as a decorticator
short retting
or hammermill.
time.
The process is
done under
Fiber separation is controlled
made using conditions, is
Enzymatic [7,27,28,
pectin-degrading fast and clean; High cost 8–24 h
Retting 33–35]
enzymes (pectinases) produces
in a bioreactor. high-quality
and consistent
fibers.
The process is
unaffected by
Pectins are removed weather High processing cost
from the plant by conditions and and consumption of
Chemical
dissolution in water can produce water, chemicals and 75 min–1 h [2,7,44]
Retting
tanks filled with consistent and energy (superior
chemical solutions. high-quality environment impact).
fibers in short
times.

3. Treatments of Natural Fibers for Successful Biocomposite Production

As mentioned earlier, it is important to modify the natural fiber surface to achieve a good
interface bonding with the polymer matrix. Because of their low water and moisture absorption,
and wettability [45], natural fibers require further chemical and surface treatments to optimize their
performance as reinforcement agents.

3.1. Chemical Treatments in Plant-Based Fibers

Plant-based natural fibers are composed of cellulose, hemicellulose, lignin and wax [46]. Table 3
shows the percentage of chemical compounds in some of the most common natural fibers. Cellulose is
the strongest and stiffest component of the fibers, endowing the fiber surface with several hydroxyl
(-OH) groups and making them hydrophilic in nature. In addition, waxy substances cap the fiber
reactive functional groups acting as an interference to interlock with the matrix that results in poor
interfacial interaction with the hydrophobic polymer matrix. To turn the fibers less hydrophilic and,
consequently, increase their mechanical and physical properties, modifications are necessary. Generally,
the fiber structure composition is altered by chemical treatments using functional groups to react with
the surface available hydroxyl groups. This can be accomplished through [3,45–49]:
Biomolecules 2020, 10, 148 6 of 44

Table 3. Chemical composition of some of the most common natural fibers (adapted from [1,2,27,45,50–53].

Fiber Cellulose (wt %) Hemicellulose (wt %) Lignin (wt %) Wax (wt %)

Bast fibers
Jute 61.0–71.5 13.6–20.4 12.0–13.0 0.5
Flax 71.0 18.6–20.6 2.2 1.7
Hemp 70.2–74.4 17.9–22.4 3.7–5.7 0.8
Kenaf 45.0–57.0 21.5 15.0–19.0 –
Ramie 68.6–76.2 13.1–16.7 0.6–0.7 0.3
Seed fibers
Cotton 82.7–91.0 5.7 – 0.6
Milkweed 55.0 24.0 18.0 1.0–2.0
Coir 32.0–43.0 0.2–0.3 40.0–45.0 –
Kapok 13.0–35.0 23.0–32.0 13.0–21.0 –
Leaf fibers
Sisal 67.0–78.0 10.0–14.2 8.0–11.0 2
Pineapple 70.0–82.0 – 5.0–12.0 –
Agave 68.4 4.9 4.9 0.3
Banana 63.0–64.0 6.0–.0 5.0 –
Abaca 56.0–63.0 20.0–25.0 7.0–12.4 3
Grass fibers
Bagasse 55.2 16.8 25.3 –
Bamboo 26.0–43.0 30.0 21.0–31.0 –
Straw fibers
Rice 41.0–57.0 33.0 8.0–19.0 8.0–38.0
Corn 38.0–40.0 28.0 7.0–21.0 –
Wheat 38.0–45.0 15.0–31.0 12.0–20.0 –
Wood fibers
Softwood 40.0–45.0 7.0–14.0 26.0–36.0 –
Hardwood 38.0–50.0 19.0–26.0 20.0–30.0 –

Cellulose alkalization by removing the remaining fiber components (hemicellulose, lignin and
wax) with sodium hydroxide (NaOH), cleaning the surface and increasing its roughness to improve
adhesion to the polymer matrix;
Silanization treatment forming silane groups that act as a fiber-matrix coupling agent, creating a
siloxane bridge between them. Silanol (Si-OH) groups react with -OH groups of the fibers and the
matrix functional groups;
Acetylation by introducing an acetyl group on the fiber surface. Here, the -OH groups react with
the acetyl groups decreasing their hydrophilic nature;
Peroxide treatment by generating free radicals that react with the -OH groups of both fiber and
polymer. This treatment requires an alkaline pre-treatment;
Benzoylation treatment using benzoyl chloride to treat the fibers and decrease their hydrophilic
nature by replacing of -OH groups with benzoyl groups. In this method, an alkaline pre-treatment
is required;
Potassium permanganate treatment by forming highly reactive permanganate ions that react with
the -OH groups, generating cellulose-manganate to initiate graft copolymerization;
Stearic acid treatment by inducing the interaction between reactive carboxyl groups of stearic acid
with the fiber -OH groups, and thus improving water resistance properties;
Isocyanate treatment by acting as a coupling agent between the fiber and the matrix. Isocyanate
functional groups react with the cellulose and lignin -OH groups, forming a chemical linkage by means
of strong covalent bonds;
Maleated coupling treatment by means of maleic anhydride, which is used to modify the fiber
surface and the polymeric matrix, ensuring high compatibility between them. Maleic anhydride is
Biomolecules 2020, 10, 148 7 of 44

grafted onto the polymer, becoming available to react with the cellulose -OH groups by means of
hydrogen or covalent bonds.
Many other chemical treatments can be used to treat fibers in order to reduce the number of
hydroxyl groups and improve the fiber adhesion to the matrix, including acrylation, acrylonitrile
grafting, triazine, zirconate, titanate, sodium chlorite, fungal and enzyme treatment. Chemical
treatments comprehend a class of the most important approaches to improve natural fiber adhesion to
a polymeric matrix, modifying their microstructure, improving tensile strength, wettability, surface
morphology and increasing the number of available chemical groups [46].

3.2. Chemical Treatments in Animal Fibers

Animal-based fibers are mainly composed of structural proteins; hence, specific chemical
modifications must be employed to these fibers, including coupling reactions (cyanuric
chloride-activated, carbodiimide and glutaraldehyde coupling), amino acid modification (arginine
masking, sulfation of tyrosine and azo-modified tyrosine) and grafting reactions (tyrosinase-catalyzed
and poly(methacrylate) grafting). The primary structure of silk fibroin (SF), the protein from silkworm,
contains a repetitive sequence of glycine-alanine-glycine-alanine-glycine-serine amino acids, which
self-assemble into an anti-parallel β-sheet structure. The crosslinking between β-sheets along the
protein is done by means of strong hydrogen bonds and Van der Waals interactions that endows silk
with excellent mechanical properties [36,54]. SF is widely used in biomedical applications. However, it
is essential to modify the SF surface chemistry to better control the interaction between silk and the
living systems. SF possesses many reactive functional groups that facilitate crosslinking with other
polymers, thus increasing its use as a reinforcing fiber [21]. Due to the presence of several reactive amino
acids in SF, chemical modifications via coupling and grafting reactions and amino acid modifications
can be applied. Wool and chicken feathers are mainly composed of keratin, a structural protein similar
to SF. The chemical structure of keratin is predominantly an α-helix in chicken feathers [55] and a
super coiled polypeptide chain with an α-helix and β-sheet in wool [56]. These structures are tightly
packed via cross linkages, hydrogen bonds, Van der Waals and electrostatic interactions.
Chemical modifications play an important role in fiber functionalization, improving existing
physicochemical properties or incorporating new ones. The fiber protein amino acid residue side chains
may be conveniently conjugated with a variety of chemical groups [57]. These modification methods can
be classified into coupling reactions, amino acid modification and grafting reactions. Coupling reactions
are mainly used to immobilize peptides, molecules and polymers in fiber proteins. Copper-catalyzed
azide-alkyne cycloaddition reactions, cyanuric chloride, carbodiimide and glutaraldehyde are very
effective coupling agents [58,59]. The amino acid modifications are made through arginine masking,
which is used to regulate the surface charge, sulfation/oxidation of tyrosine, which causes the hydrolysis
of the fiber protein [58], and azo-modified tyrosine that can be used to install small molecules into
fiber protein, resulting in hydrophobic and hydrophilic derivatives [60]. The grafting reactions include
tyrosinase-catalyzed grafting and poly(methacrylate) grafting. Still, the chemical treatments discussed
in Section 3.1. may also be applied to these protein fibers when used as composite reinforcements due
to their several reactive functional groups [61,62].

3.3. Physical Surface Treatments

In addition to the mentioned chemical treatments, it is also very common to improve the fibers’
surface through physical surface treatments. Some of these approaches are used to functionalize the
natural fibers’ surface and consist of the use of plasma, ultrasounds and UV-light. Plasma treatment
is one of the most common surface modification methods. Cold plasma treatment is required to
remove the surface impurities which, consequently, induces modifications in the surface properties,
such as wettability, flame resistance, printability, etc., and increases surface roughness leading to
better mechanical interlocking and interfacial adhesion between the fiber and polymer [47,63]. The
hydrophilic/hydrophobic surface character can also be changed with the incorporation of free radicals
Biomolecules 2020, 10, 148 8 of 44

capable of reacting with oxygen or other gases [48]. Plasma is a partially ionized gas that reacts with
the fiber surface. Plasma is generated by applying an electrical field between two electrodes, which
transmit energy, accelerating the gas electrons that collide with neutral gas molecules or atoms under
atmospheric pressure or in a vacuum. In the case of a plasma vacuum, the gas is introduced at a low
pressure in a vacuum chamber causing ionization by means of atom removal or bond rupture, giving
rise to free radicals and crosslinking. However, this method requires an expensive closed system and
is considered a batch process [64,65]. The treatment with atmospheric plasma is more attractive for
industry, as it allows the samples to be treated in situ rather than restricted to a vacuum chamber. It is a
continuous and uniform treatment, reliable and reproducible [66]. The atmospheric plasma technique
can be divided into different types of discharge, such as corona-discharge, dielectric barrier discharge,
glow discharge and atmospheric pressure plasma jet.
Corona treatment is a process based on low-frequency discharges applied in two opposing
electrodes and grounded metal roll. These discharges induce ionization of the nearby atmosphere
generating plasma. The fiber is placed in the gap between the electrodes and is bombarded with
high-speed electrons, inducing surface oxidation and increasing the amount of high reactive free
radicals [64,67]. It is a low-cost process with low energy consumption and exhibits several advantages
compared with others plasma treatments [48]. The dielectric barrier discharge (DBD) technique is
similar to the corona treatment. However, here, there is one or more dielectric barriers in the path
between the electrodes, acting as an insulator. These accumulate the transported charge and distribute
it over the entire electrode area. The gas between the electrodes is not ionized and only serves as a
reservoir to absorb the energy dissipated. The main disadvantage of DBD is that it is not completely
uniform and has a short duration [68,69]. The atmospheric pressure glow discharge (APGD) is a
more stable, uniform and homogeneous surface treatment than DBD. This technique is generated in
helium or argon by applying low voltages through parallel conductive electrodes at higher frequencies.
The glow of the discharge refers to the characteristic luminescence resultant from excitation collisions
followed by de-excitation [63,70]. In the atmospheric pressure plasma jet (APPJ) there are two tubular
metal electrodes separated by a gap. Between the electrodes, a quartz cylindrical tube is inserted where
helium (or other gases) flows. The plasma is launched into the surrounding air in the form of a plume
or bullet, directly into the sample. This process can provide a local and very precise treatment [64].
APPJ is suitable for industrial and research applications, namely treatment of heat-sensitive materials,
biological material sterilization and several biomedical devices [71].
Ultrasound treatment, while not as common as plasma treatment, is also effective in surface
modifications. This method causes the cavitation effect, which is the formation, by ultrasonic irradiation,
of small collapsing bubbles that generate powerful shock waves. The impact of the shock waves on the
fiber surface leads to surface peeling, erosion and particle breakdown. Cavitation is responsible for
the physical and chemical effects of ultrasound in solid/liquid and liquid/liquid systems and is more
effective in heterogeneous systems than homogeneous systems. The effect of ultrasound treatment is
related to its frequency; at low frequencies, violent cavitation is produced, and the effects are highly
localized. On the other hand, with high frequency, the cavitation is less violent due to the shorter
lifetime of the bubbles [49,72,73].
Ultraviolet treatment is based on UV-light, an electromagnetic radiation with a potential energy
source capable of promoting photochemical reactions in the molecular structure of the fibers’ surface [74].
UV-treatment is a clean and cost-effective process that can be used in industrial applications [48]. In
addition to the processes described earlier, there are other physical methods of surface modifications,
such as ozone treatment, gamma-ray irradiation treatment, laser treatment and ion beam treatment [47].

4. Biomolecules and Their Immobilization Methods onto Biocomposites

Incorporation of biological cues onto the filament surface, through immobilization of bioactive
ligands, peptides, NPs, enzymes, plant extracts or essential oils (EOs), has been used to obtain
effective and specific biological functions of the composition. Immobilization of yeast invertase
Biomolecules 2020, 10, 148 9 of 44

onto polyethylenimine (PEI)-coated cotton flannel for food modifying processes is one of the earliest
cases of a biologically functional natural fiber by means of surface modification [75]. On the other
hand, cotton and wool fabrics bearing covalently attached alkylated PEI exerted high bactericidal
and antifungal activity [76] for wound dressing production, being a first example of the medical use
of textiles functionalized with bioactive compounds [77]. Since then, biomolecules of all kinds have
been immobilized on and within biocomposite materials for a variety of biomedical applications,
including therapeutics, diagnostics, wound healing, tissue engineering, etc. A list highlighting the
most recent (last ten years) formulations of biomolecule-modified biocomposites and respective “final
product” properties is provided in Table 4. For the purpose of this review, inorganic NPs were
considered biomolecules due to the biological and biomedical impact of their combination with
selected biocomposites.
In the following sub-sections, a detailed analysis of these promising bioactive molecules applied
in the production or modification of natural fiber-reinforced composites (Table 4) is provided
together with a brief introduction about the approaches or methodologies required to attain such
modified biocomposites.
Biomolecules 2020, 10, 148 10 of 44

Table 4. Application of biomolecules in the production of natural fiber-based composites for potential biomedical applications and respective properties. Most of the
selected combinations have already been established for biomedical uses. However, there are a few that, even though the publications do not state those as potential
applications, the authors feel that the combinations or the principles described may be of interest for biomedical uses and as such were included. This table compiles
examples of natural fiber-reinforced composites modified with multiple biomolecules reported in the last 10 years.

Specific Biomolecule Natural Fiber-Reinforced Biofunctionalized Fibers/Fabric/Composite Production and

Category References
Composites Properties
Name Characteristics
Penicillin-type antibiotic that works by stopping Drug-loading capacity increased with decreasing fabric
the growth of bacteria. Used to treat several Woven cotton porosity. Degradation of the fabric composites influenced drug
Amoxicillin bacterial infections like, middle ear infection, fabric/polylactic acid release rate. Water absorption decreased with increasing PLA [14]
strep throat, pneumonia, skin and urinary composite concentrations. The mechanical properties of the composites
infections, etc. were consistent with the fabric’s density and weight.
Composite fibers showed a smooth and uniform morphology
Sericin (outer layer of silk with suitable porosity, mechanical stability and water vapor
FDA approved glycylcycline antibiotic used in
Tigecycline fibers)/poly(vinyl alcohol) transmission rate. They also revealed antibacterial activity [78]
the treatment of skin tissue infections.
Drugs/ composite against Escherichia coli and Bacillus subtilis. In vivo testing
Antibiotics showed this composite to accelerate wound healing.
Keratin extracted from wool and filled with hydrotalcite NPs
intercalated with anionic diclofenac gave rise to a new
Nonsteroidal anti-inflammatory drug used to composite. These showed a less pronounced swelling, porosity
Keratin/hydrotalcite NPs
Diclofenac treat pain and inflammation associated with and degradation and a greater thermal stability compared to [79]
composite
arthritis. pure keratin films. Diclofenac release profile was more stable
on the modified composites, which were also able to support
fibroblast-like cells adhesion.
Cellulose nanofibers derived from waste sugarcane bagasse
Colorless liquid soluble in organic solvents, were mixed with starch granules to produce a low porosity
Sugarcane bagasse/starch
Dimethyl phthalate commonly used as an insect repellent and biocomposite with enhanced water uptake. The initial [80]
granules composite
ectoparasiticide. dimethyl phthalate release burst was reduced, gaining a
superior controlled release efficiency overtime.
TEMPO AgNPs, averaging 50.0 ± 2.0 nm, were formed in situ and
(2,2,6,6-tetramethylpiperidine- deposited on the surface of jute cellulose fibers by microwave
[81]
1-oxyl radical) selectively heating. The versatile jute-AgNPs nanocomposites
oxidized jute fiber demonstrated superior thermal stability and high crystallinity.
Inorganic particles endowed with superior
Regenerated silk fibers were fabricated through the dry–wet
antimicrobial activity. Their mechanism of
Silver (Ag) spinning process and modified via master batch or dipping
action is not yet completely understood but it is
process with different concentrations of PHMB and AgNPs.
clear it is significantly affected by the particles’ Silk
The bactericidal efficiency of the master batch treated fabrics
nanoscale dimensions. fibers/polyhexamethylene [82]
was dependent on the concentration of the antibacterial agent
biguanide (PHMB) fabric
as well as particle size. In the dipping process, a compromise
Nanoparticles was made between the good inhibition effect and the least
(NPs) amount of color change on the bio-fibers.
Biomolecules 2020, 10, 148 11 of 44

Table 4. Cont.

Specific Biomolecule Natural Fiber-Reinforced Biofunctionalized Fibers/Fabric/Composite Production and

Category References
Composites Properties
Name Characteristics
Sugarcane bagasse was successfully grafted with acrylamide
Sugarcane and glycidyl methacrylate and further modified in a colloidal
bagasse/acrylamide/glycidyl suspension of AgNPs, gaining superior antimicrobial action [83]
methacrylate composites against Escherichia coli, Staphylococcus aureus, Aspergillus flavus
and Candida albicans.
Linen fabrics coated with chitosan and modified with AgNPs
via in situ synthesis with tamarind seed coat extract showed
Linen (from flax efficient multifunctional properties, with bacterial reduction of
[84]
family)/chitosan composite 100%, UPF rating of 50+ and antioxidant activity of 97%.
Except for flame retardancy, all properties were retained to a
satisfactory level even after 50 washing cycles.
Cotton fabric grafted with carboxymethyl chitosan and
immobilized with AgNPs, via amidation reaction with the
L-cysteine groups available at the fabric surface, demonstrated
Cotton/carboxymethyl
enhanced antibacterial functions, sustained even after 180 [13]
chitosan/L-cysteine composite
cycles of washing. Cytotoxicity assays showed insignificant
effects on human immortalized keratinocyte cells, revealing the
safety of the material for contact with the human skin.
Polymer–AgNPs nanocomposites modified cotton fabrics
prepared by in situ chemical oxidative polymerization,
Cotton/polypyrrole-silver
displayed enhanced conductivity. AgNPs were also responsible [85]
nanocomposites
for the increased antibacterial activity of the composite against
Staphylococcus aureus and Escherichia coli.
Cotton–polyester textiles were successfully impregnated
during washing and ironing processes with five impregnation
solutions containing Ag/Cu in the form of bimetallic NPs (alloy
Silver and copper
Inorganic particles with exceptional and core-shell) as well as ionic species. The antimicrobial
(Ag/Cu bimetallic Cotton/polyester composite [86]
antimicrobial and antifungal properties. activity of the fabrics was observed and did not become
NPs)
compromised after 20 washing cycles. Surfaces treated with
solutions containing Ag+ /Cu2+ and AgNPs/Cu2+ inhibited
fungi growth significantly.
Inorganic particles with antimicrobial properties.
CuO-modified cotton fabrics revealed excellent resistance to
Copper oxide (CuO) CuO has unique optical, catalytic and chemical Polycotton-based fabric [87,88]
microorganisms (bacteria and fungi) at different concentrations.
properties at nanoscale.
Biomolecules 2020, 10, 148 12 of 44

Table 4. Cont.

Specific Biomolecule Natural Fiber-Reinforced Biofunctionalized Fibers/Fabric/Composite Production and

Category References
Composites Properties
Name Characteristics
Kenaf fiber–polyester composites produced via
vacuum-assisted resin infusion process followed by CaCO3
Kenaf fiber/polyester
NPs impregnation exhibited increased modulus of elasticity, [89]
composite
modulus of rapture, tensile modulus and tensile strength, and a
reduced swelling capacity and moisture absorption.
Inorganic particles endowed with an ultra-fine
solid structure and high economic value that CaCO3 was incorporated within the composite via the
Calcium carbonate Kenaf bast fibers-polyolefin inorganic nanoparticle impregnation method. The tensile
play an important role in reinforcing and
(CaCO3 ) matrices/polypropylene modulus and strength of the fibers increased significantly after [90]
toughening materials and enhancing
electrostatic attraction. composite NPs incorporation, as the compatibility of the modified kenaf
fibers and polypropylene was significantly improved.
Impregnation of the bamboo fibers with CaCO3 increased the
fiber density, filling the morphological voids and creases, and
Bamboo fiber/polypropylene
improving the interfacial compatibility of the composite. The [91,92]
composite
modified composites exhibited improved tensile strength,
modulus of elasticity, and elongation at break.
Composites were prepared by pad-dry-cure method which
Like AgNPs, these inorganic particles are generated a functional silica matrix that induced the in situ
Silver chloride
capable of great antimicrobial activity, by acting Wool/polyester composite synthesis of AgCl NPs. Ag-modified surfaces were successful [93]
(AgCl)
as leaching antibiotics. against bacteria and fungi at concentrations superior to 0.5 mM
AgNO3 .
Cotton fabrics were modified with a film of chitosan or by a
Zeolites are crystalline aluminosilicates that conventional pad–dry–cure process in which chitosan–zeolite
exhibit adsorption properties and ion-exchange composites were immobilized onto the fabric surface. The
Silver zeolites (SZs) capabilities. By encapsulating silver, they allow Cotton/chitosan composites altered fabrics displayed improved antibacterial properties [94]
an optimized release of the NPs and ensure against Escherichia coli, Staphylococcus aureus, Candida albicans
antimicrobial activity without adverse effects. and Trichophyton rubrum. Evidences of thermoregulating
properties were also found.
The modified cotton fabric showed superhydrophobic
properties and excellent antibacterial action against Escherichia
Zeolitic imidazolate Inorganic particles endowed with a large surface Cotton/ZIF-8-polydimethyl
coli and Staphylococcus aureus. Fabrics retained their excellent [95]
framework-8 (ZIF-8) area, and strong hydrophobicity. siloxane fabric
antibacterial property and superhydrophobicity after 300 cycles
of abrasion and 5 cycles of washing.
Kenaf fiber reinforced composites were produced via
Hydrophilic, inorganic particles, non-toxic and vacuum-assisted resin transfer molding process and
Aluminum
odorless that exhibit good dispersion and can Kenaf fibers/polyester impregnated with Al(OH)3 NPs. The NPs addition increased
hydroxide [96]
generate very easily hydrogen bonds with composite the composite modulus of elasticity, modulus of rupture, tensile
(Al(OH)3 )
cellulosic fibers. modulus and tensile strength, while the water thickness of
swelling was reduced.
Biomolecules 2020, 10, 148 13 of 44

Table 4. Cont.

Specific Biomolecule Natural Fiber-Reinforced Biofunctionalized Fibers/Fabric/Composite Production and

Category References
Composites Properties
Name Characteristics
Cotton fabrics treated with reduced graphene oxide were
Titanium dioxide Inorganic particles with photocatalytic activity, successfully decorated with two types of TiO2 NPs doped with
doped with iron and self-cleaning properties and base Cotton/reduced graphene 1% iron and nitrogen atoms and synthesized in different
[97]
nitrogen atoms substrate-dependent oxide composite hydrothermal conditions. NPs-modified fabrics were found
(TiO2 ) superhydrophilicity/superhydrophobicity. harmless for human skin cells and capable of inhibiting the
growth of Staphylococcus aureus and Enterococcus faecalis.
Sonosynthesis and sonofabrication of Fe3 O4 NPs was
accomplished on cotton/polyester composite fabrics, with
Iron oxide Inorganic particles with photocatalytic activity appropriate saturation magnetization. Composites
Cotton/polyester composite [98]
(magnetite, Fe3 O4 ) and antimicrobial properties. demonstrated a 95% antibacterial efficiency against
Staphylococcus aureus and a 99% antifungal effect against
Candida albicans, along with enhanced mechanical properties.
Linen fabric was modified with chitosan followed by in situ
Inorganic particles with outstanding catalytic, synthesis of CeO2 NPs. The modified fabric displayed effective
Cerium oxide electronic and magnetic properties. They are Chitosan/linen (from flax antibacterial activity against Staphylococcus aureus and
[99]
(CeO2 ) also highly efficient in absorbing UV radiation family) composite Escherichia coli bacteria. They were also endowed with
and protecting against corrosion. properties like wrinkle resistance, UV-protection and flame
retardancy, which were maintained after 5 washing cycles.
PtNPs were synthesized in situ on silk-based fabrics through
Inorganic particles very stable and effective for
heat treatment. Color strength increased with the concentration
antimicrobial applications. PtNPs have high
of the Pt ions. The modified fabrics exhibited good washing
Platinum (Pt) activity and selectivity for catalytic reaction, Silk-based fabrics [100]
fastness and excellent rubbing color fastness. They also
good recyclability, and can enhance the
demonstrated significant catalytic functions and a significant
cleansing function of the skin surface.
antibacterial effect against Escherichia coli.
Due to the high surface area of the bamboo NPs, incorporation
Biocompatible, organic particles endowed with Woven-nonwoven kenaf
allowed for a strong bond between kenaf and polyester to be
Bamboo superior mechanical properties, namely ultimate fiber/unsaturated polyester [101]
generated with improved wettability and excellent mechanical
tensile, toughness and Young’s modulus. composite
and thermal properties.
Lignocellulosic jute fabrics were treated with laccase and then
used as reinforcement materials to prepare
polypropylene-based composites. Laccase-treated
Laccases are multi-copper glycoproteins that jute/polypropylene composites exhibited high breaking [102]
catalyze the mono-electronic oxidation of Lignocellulosic
Enzymes Laccase strength, storage modulus, and melting temperature. Data
phenols and aromatic or aliphatic amines to jute/polypropylene composite
suggests a good interfacial adhesion between the jute and the
reactive radicals and reduce molecular oxygen polypropylene.
to water in a redox reaction.
Biomolecules 2020, 10, 148 14 of 44

Table 4. Cont.

Specific Biomolecule Natural Fiber-Reinforced Biofunctionalized Fibers/Fabric/Composite Production and

Category References
Composites Properties
Name Characteristics
Grafting of dodecyl gallate onto jute fibers via laccase was
investigated as a reinforcement of polypropylene-based
composites. The composite hydrophobicity and breaking [103]
strength increased after grafting, and the composite fracture
section became neat and regular.
Alkyl gallates with different aliphatic chain lengths, such as
propyl gallate, octal gallate and dodecyl gallate, were
enzymatically grafted onto jute by laccase and then
incorporated onto polypropylene matrices. After modification, [104]
the tensile and dynamic mechanical properties of the
composites improved, while water absorption and swelling
decreased.
A composite of milkweed, polyethylene and polypropylene
Arginyl-glycyl-aspartic acid (RGD) is the most was made by carding and further treated with atmospheric
common and well documented peptide motif Milkweed/polyethylene/poly pressure plasma to functionalize the surface with carboxylic
Peptides RGD-peptide [11]
responsible for cell recruitment and attachment propylene composite acid groups for RGD-peptide binding. Plasma treatment
to the extracellular matrix. accelerated the degradation of milkweed. The composite was
seen to promote MC3T3 osteoblast-like cells recruitment.
Cecropin-B is an antibacterial peptide found in
the hemolymph of the pupae of H. cecropia. It is
composed of 35–39 a.a. in length and assumes
AMPs immobilization was accomplished via exhaustion
an amphipathic α-helice structure that facilitates
method. The functionalized AMPs reduced significantly the
Antimicrobial microbial penetration.
Cecropin-B/[Ala5] bacterial growth, with Cecropin-B resulting in 71.67% reduction
Peptides [Ala5]-Tritrp7 is a synthetic peptide that results Wool-based materials [12]
-Tritrp7 against Staphylococcus aureus and 85.95% against Klebsiella
(AMPs) from the replacement of the first Pro at position
pneumoniae, while [Ala5]-Tritrp7 promoted a 66.74% and 88.65%
5 in tritrpticin by Ala (Tritrp7). The substitution
reduction, respectively.
of Pro-5 to Ala in Tritrp7 leads to the formation
of amphipathic α-helices, which stimulates an
effective cell leaching and thus bacteria death.
Baicalin bonded with the silk fabric via electrostatic interactions
Major component of the root of Scutellaria
between the ionized carboxyl groups in the extract and the
Baicalin baicalensis Georgi. It possesses multiple
positively charged amino groups in the fabric. The treated
(5,6,7-trihydroxyfla bioactivities including antibacterial, antioxidant, Silk-based fabrics [105]
Plant fabric exhibited excellent antioxidant activity, high antibacterial
vone-7-O-glucuronid) anticancer, anti-inflammatory, and antiviral
Extracts performance against Escherichia coli and Staphylococcus aureus,
activities.
and very good UV-protection.
Biomolecules 2020, 10, 148 15 of 44

Table 4. Cont.

Specific Biomolecule Natural Fiber-Reinforced Biofunctionalized Fibers/Fabric/Composite Production and

Category References
Composites Properties
Name Characteristics
Propolis is a gum gathered by honeybees from
Cotton fabrics were treated with propolis at different
various plants. It is not toxic to humans or
concentrations using the pad–dry–cure technique. Surfaces
Propolis mammals. Propolis has been reported as Cotton-based fabrics [106]
were found highly antibacterial, water repellent and capable of
anticancer, antioxidant, anti-inflammatory,
protecting against UV-radiation.
antibacterial, antifungal and antiviral.
Microcapsules containing Psidium guajava Linn. leaf extract
Psidium guajava Linn., from the Myrtacae family, were prepared by in situ polymerization using urea and
Psidium guajava Linn. also known as guava, is characterized by its formaldehyde for encapsulation and applied by direct printing
Cotton-based fabrics [107]
leaf extract exceptional antidiabetic, anticough, antioxidant, onto cotton fabrics. The extract modified fabrics showed
antibacterial and antispasmotic properties. antibacterial activity against Staphylococcus aureus but were not
effective against Escherichia coli bacteria.
Aloe Vera is a highly abundant, natural plant Bleached cotton fabrics were immersed in the extracted
that has antimicrobial activity against various solution for specific periods of time, padded, dried and cured.
pathogens. External application of Aloe Vera gel Modified fabrics became very effective against pathogens,
Aloe Vera gel Cotton-based fabrics [108]
penetrates the skin directly and produces a namely Bacillus subtillis, Pseudomonas aeruginosa, Bacillus pumalis
soothing, pain-relieving and anti-inflammatory and Escherichia coli. The antimicrobial finishing did not affect
effect on arthritic joints and tendonitis. the physical properties of the fabric.
Jatropha curca is a plant indigenous of India
composed of phenolic, terpenoids, flavonoids, An ecofriendly natural antibacterial finish was applied to
Jatropha curcas leaf alkaloids, glycosides, steroids, tannin, etc., cotton-based fabrics via dip coating. Modified fabrics were
Cotton-based fabrics [109]
extract which endows the extract with antibacterial characterized as bactericides and bacteriostatic against
properties (bactericide and bacteriostatic). It is Staphylococcus aureus bacteria.
also known for its anti-cancerous properties.
Cotton and non-woven fabrics were decorated via
sonochemical process with FF loaded with curcumin. A
Cotton and non-woven
sustainable, controlled release of curcumin was attained using
fabrics/diphenylalanine (FF) [110]
Bright yellow compound produced by Curcuma this functionalization process, which was modulated by the
peptide nanotubes
longa plants. It is endowed with many functions, sonication time, conferring potential antimicrobial and
Curcumin including anti-inflammatory, anticancer, anti-inflammatory properties to the fabric.
antiviral, antiarthritic and antioxidant Composite microspheres loaded with curcumin and made of
properties. poly(methyl methacrylate) stabilized with cellulose
Sisal fibers/poly(methyl
nanocrystals prepared from sisal fibers were produced. Results [111]
methacrylate) composites
showed curcumin loaded composites to display long-term
photostability and good encapsulating ability.
Biomolecules 2020, 10, 148 16 of 44

Table 4. Cont.

Specific Biomolecule Natural Fiber-Reinforced Biofunctionalized Fibers/Fabric/Composite Production and

Category References
Composites Properties
Name Characteristics
Ocimum sanctum plant is found in India and has
The composite fabric was treated with the herbal extract at
antibacterial, antioxidant, antibiotic,
different concentrations, using glutaraldehyde as cross-linking
antiatherogenic, immunomodulatory,
agent and sodium hypophosphite as catalyst by the exhaustion
Ocimum sanctum leaf anti-inflammatory, analgesic, antiulcer,
Cotton/polyester composite method. Modified fabrics inhibited Gram-positive bacteria [112]
extract chemo-preventive and antipyretic properties.
growth in more than 92%. Although, the treated fabrics
Besides it is very abundant and easily accessible,
showed enhanced crease recovery property, there was a
economically feasible, and possesses minimal
marginal reduction in tensile properties.
side effects.
Cotton fabrics were modified with monochlorotriazinyl
β-cyclodextrin, as an eco-friendly encapsulating/hosting
compound, to create core-shaped hydrophobic cavities for
Rosemary, lavender, Bioactive oils endowed with antimicrobial Cotton/monochlorotriazinylβ-
individual loading of EOs. The modified fabrics revealed [113]
clove and cinnamon properties. cyclodextin fabric
improved antibacterial activity and durability. The antibacterial
activity of the treated knitted cotton fabrics was superior to that
of woven fabrics.
Microencapsulation of citronella oil was done by complex
coacervation onto wool fabrics. The multi-core structure of the
Biopesticide with a non-toxic mode of action
Wool/gelatin and gum Arabic microcapsules allowed the oil diffusion by a Fickian
Essential Citronella that works as a mosquito repellent due to its [114]
biopolymers mechanism in the first release stage and by non-Fickian kinetics
Oils (EOs) eco-friendly and biodegradable nature.
on the second stage. The textile structure influenced the release
model due to the interaction between the fabric and water.
Sugarcane bagasse fiber-reinforced starch foam composites
were prepared with different oregano essential oil contents.
The addition of oregano oil increased the composite
Oregano oil comes from the leaves and shoots of Sugarcane bagasse/starch
antimicrobial properties, particularly against Gram-positive [115]
Oregano foam composite
the oregano plant and is botanically known as bacteria, but decreases its water absorption capacity and
Origanum vulgare. It is a natural antibiotic and hygroscopicity. The biodegradation rate and flexural strength
antimicrobial agent with antioxidant, of the composite slightly decreased with increasing oil content.
anti-inflammatory and anti-cancerous properties.
Green composites were obtained by twin-screw extrusion
It may also be involved in lowering cholesterol.
Coconut followed by compression molding. Coconut fibers were
fibers/poly(3-hydroxybutyrate- impregnated with oregano essential oil by spray coating and
[116]
co-3-hydroxyvalerate) (PHBV) then incorporated into PHBV. The green composites displayed
composite enhanced physical performance and superior bacteriostatic
effect against Staphylococcus aureus bacteria.
Biomolecules 2020, 10, 148 17 of 44

Table 4. Cont.

Specific Biomolecule Natural Fiber-Reinforced Biofunctionalized Fibers/Fabric/Composite Production and

Category References
Composites Properties
Name Characteristics
Transparent composites were produced via solvent casting and
further modified by the incorporation of cinnamon oil.
Cinnamon oil is derived from the bark or leaves
Scanning calorimetry analysis showed that the oil-modified
of several trees, including the Cinnamomum
Durian skin fiber/polylactic composites were less crystalline than the controls, suggesting
Cinnamon verum tree and the Cinnamomum cassia tree. [15]
acid composite their structure was less rigid and flexible. The oils decreased
Possesses antibacterial, antifungal, antidiabetic
the water vapor permeability and improved the composite
and antioxidant properties.
antimicrobial activity against Gram-positive and
Gram-negative bacteria.
Biomolecules 2020, 10, 148 18 of 44
Biomolecules 2020, 10, x FOR PEER REVIEW 16 of 40

4.1. Bioactive
4.1. Bioactive Biomolecules
Biomolecules

4.1.1. Antibiotics
The discovery of penicillin and streptomycin in 1929 and 1943, respectively, respectively, foreshadowed
foreshadowed the
age of antibiotics [117]. In fact, only two years later, the first definition for antibiotics was proposed:
“chemical substance
“chemical substance ofof microbial
microbial origin
origin that
that possesses
possesses antibiotic
antibiotic powers”
powers” [118].
[118]. This definition only
included those
included those antibiotics
antibiotics produced
produced byby microorganisms
microorganisms but but did
did not
not consider
consider those
those of synthetic origin
produced by other biological products
or produced products of non-microbial
non-microbial origin
origin (but still endowed with antagonistic
effects on
effects on the
the growth
growth ofof microorganisms)
microorganisms) [119].
[119]. As such, acceptable variations of this definition have
proposed over
been proposed over the
the years.
years.
Currently,the
Currently, theantibiotics
antibioticsavailable
availableininthe
themarker
markerare are either
either produced
produced byby microbial
microbial fermentation
fermentation or
or are
are synthetically
synthetically preparedfollowing
prepared followingthe
the backbone
backbone structure of ofexisting
existingantibiotics.
antibiotics.They target
They the
target
physiology
the physiologyand and
biochemistry
biochemistryof bacteria (Figure
of bacteria 1) by
(Figure 1) affecting the the
by affecting membrane
membrane structure, the
structure,
peptidoglycans
the peptidoglycans or the
or cell
the wall biosynthesis;
cell wall by interfering
biosynthesis; withwith
by interfering protein synthesis
protein via interaction
synthesis with
via interaction
ribosomal
with subunits;
ribosomal by meddling
subunits; with the
by meddling withDNAthe and
DNA RNA
andreplication and transcription
RNA replication of nucleic
and transcription of
acid synthesis
nucleic and metabolism;
acid synthesis and/or
and metabolism; by interfering
and/or by interferingwith metabolic
with metabolicpathways
pathwaysand,and,this
this way,
way,
inhibiting DNA
inhibiting DNAsynthesis.
synthesis.Ultimately,
Ultimately,thethe effective
effective actionaction against
against these targets
these targets inhibitsinhibits
bacteria bacteria
growth,
growth, compromises
compromises the cell and,
the cell integrity integrity and,
finally, finally,
leads leads
to cell deathto cell death The
[119,120]. [119,120]. The and
structural structural and
metabolic
metabolic differences
differences betweenand
between bacteria bacteria and mammalian
mammalian cells enablescellsantibiotics
enables antibiotics
to inducetoselective
induce selective
toxicity
toxicity pathogens
against against pathogens
without without
harmingharming
the host thecellshost cells [121].
[121].

Figure 1. Antibiotic modes of action on bacteria (used with permission from [121]).
[121]).

For
For their
their efficiency
efficiency andand effectiveness,
effectiveness, antibiotics
antibiotics represent
represent aa primary
primary treatment
treatment method
method for for
infections
infections and chronic diseases. However, the increasing and indiscriminate use of antibiotics has
and chronic diseases. However, the increasing and indiscriminate use of antibiotics has led
led
to
to the
the development
development of of tolerance
tolerance and
and the
the emergence
emergence of of antibiotic-resistant
antibiotic-resistant pathogens.
pathogens. In In fact,
fact, this
this has
has
become a serious global issue with devastating consequences for patient care [122]. The
become a serious global issue with devastating consequences for patient care [122]. The recognition recognition of
the correlation between antibiotic use and resistance development has catapulted research
of the correlation between antibiotic use and resistance development has catapulted research devoted devoted
to
to the
the discovery
discovery andand design
design ofof new
new compounds
compounds effective
effective against
against multi-drug-resistant
multi-drug-resistant pathogens
pathogens and and
multi-organism
multi-organism biofilms [117,120,123]. In this context, many efforts have been made towards
biofilms [117,120,123]. In this context, many efforts have been made towards the the
design
design of
of new
new drugs,
drugs, and
and the
the development
development of of nanostructured
nanostructured platforms
platforms forfor the
the local
local and
and controlled
controlled
delivery
delivery ofofantibiotics.
antibiotics.One of the
One of most common
the most strategies
common consistsconsists
strategies in the immobilization of antibiotics
in the immobilization of
at the surface of inorganic NPs or encapsulated within nano-sized shells [124].
antibiotics at the surface of inorganic NPs or encapsulated within nano-sized shells [124]. Functionalization
Functionalization or modification of polymer-based composites has also been one of the most
recurrent strategies in biomedicine [125].
Biomolecules 2020, 10, 148 19 of 44

or modification of polymer-based composites has also been one of the most recurrent strategies in
biomedicine [125].
With the concomitant rising interest in the use of renewable feedstocks, there has been great
opportunities for the use of natural-origin materials in medical applications. Cellulose, for instance,
is one of the most abundant polymers on Earth that can be harvested from natural fibers (Table 3).
Butylparaben and triclosan antibiotics have been incorporated within the cationic β-cyclodextrin
cellulose complexes cavities to improve the antibiotic’s solubility and, consequently, release kinetics.
The antibiotic-loaded complexes were found to inhibited bacteria action by affecting the bacteria
metabolism instead of damaging the cell membrane [126]. The incorporation of the ciprofloxacin
hydrochloride antibiotic has also been attempted on a similar cellulose-based fibrous structure.
β-cyclodextrin were covalently bonded to the cellulose fibers via citric acid, which prolonged the
antibiotic release process and improved its antibacterial activity, particularly against Escherichia coli
bacteria [127]. Research on the use of biocomposites as platforms for antibiotic delivery is fairly recent.
Feather keratin/polyvinyl alcohol biocomposites have been produced by crosslink with dialdehyde
starch for an improved compatibility. Dialdehyde starch was employed with the goal of decreasing the
relative crystallinity and enthalpy of the composite, while increasing the water stability. Rhodamine
B dye was used as a substitute of a model drug to explore the ability of this composite to sustain
prolonged and stable drug release. Data confirmed this premise [128]. Research has continued on this
subject and there are now woven cotton/polylactic acid composite systems loaded with amoxicillin [14],
sericin (outer layer of silk fibers)/poly(vinyl alcohol) composites modified with tigecycline [78] and
even keratin/hydrotalcite nanoparticle composites functionalized with diclofenac [79]. Acquired data
shows the promising future of these new formulations and their ability to overcome the limitations of
the use of free antibiotics, and their overall potential in biomedicine.

4.1.2. Nanoparticles (NPs)

NPs are defined as solid colloidal particles of 1 to 100 nm in size and have been used in the
biomedical field for a variety of purposes, including drug design and delivery, diagnostics and
therapeutics. They can be engineered in the form of nanospheres, nanocapsules, liposomes, dendrimers
and micelles from a variety of materials, including those from organic and inorganic origins [129,130].
The influence of NP parameters, such as size, shape, charge, colloidal stability, corrosion, stiffness and
so forth, on interactions with molecules, living cells and animal models has been researched. However,
interfacing inorganic NPs with biological settings have led to the most influential and outstanding
discoveries [130–132]. For that reason, even though inorganic NPs are not considered biomolecules,
their multiple biomedical applications and the various advantages offered when combined with
biocomposites has led the authors to open an exception and include them in this section.
NPs are characterized by a large surface area-to-volume ratio. In the case of inorganic NPs, they
can be subdivided into magnetic, metallic, bimetallic or alloy and metal oxide [133]. Much literature
has focused on iron oxide NPs because of their superior chemical, biological and magnetic properties,
including chemical stability, non-toxicity, biocompatibility, high saturation magnetization and high
magnetic susceptibility. Maghemite (γ-Fe2 O3 ) and magnetite (Fe3 O4 ) are the most biocompatible
oxidation states of iron. However, these forms tend to oxidize, requiring an additional coating made
of other biocompatible materials, e.g., polymers [134]. Gold, silver and their respective compounds
are the most widely employed metal NPs in biomedicine. Gold’s unique electronic and optical
properties have resulted in important biosensor and bioimaging applications. Further, its easy
functionalization with organic molecules allows for active or passive drug delivery systems to be
engineered. Silver NPs are endowed with unique physicochemical properties that include high
electrical and thermal conductivity, chemical stability, catalytic activity, enhanced optical properties
and exceptional antibacterial performance. The antimicrobial activity of NPs, like silver, has been
confirmed against a variety of microorganisms, including Gram-positive and Gram-negative bacteria
and fungi. Because of their large surface area and reduced sizes, NPs can disrupt the cell wall
Biomolecules 2020, 10, x FOR PEER REVIEW 18 of 40

the most explored,

Biomolecules with exceptional bactericidal properties being identified in cotton-, linen-, sugarcane
2020, 10, 148 20 of 44
bagasse-, silk- and jute-reinforced composites [13,81–85]. The bimetallic NPs comprehend those NPs
that combine more than one metal or are produced from metallic alloys. Silver/copper NPs are a
and provoke membrane damage; penetrate intracellularly and cause protein denaturation, enzyme
frequent example in this class. They have been used in the modification of cotton–polyester composites
inactivation, DNA rupture or ribosome disassembly; and even induce oxidative stress (Figure 2) [135].
at different ratios and oxidation states with excellent antimicrobial properties against bacteria and fungi
Silver NPs have contributed significantly to advances in medical textiles that include the production
[86]. Metal oxide NPs are characterized by their unique physical and chemical properties and superior
of wound dressings and protective coatings for medic devices [133]. In fact, the combination of
density. Size-related alterations in response to an increasing number of surface and interface atoms have
these NPs with biocomposites is the most explored, with exceptional bactericidal properties being
been observed in NPs made of CuO, ZnO, SnO2, Al2O3, MgO, ZrO2, AgO, TiO2, CeO2, etc. Conjugation
identified in cotton-, linen-, sugarcane bagasse-, silk- and jute-reinforced composites [13,81–85].
with biomaterial substrates has proven very effective in stabilizing these NPs and improving their
The bimetallic NPs comprehend those NPs that combine more than one metal or are produced from
performance. In fact, combinations with biocomposites have shown their harmless activity on human
metallic alloys. Silver/copper NPs are a frequent example in this class. They have been used in the
cells and improved antimicrobial action and UV-protection [97–99].
modification of cotton–polyester composites at different ratios and oxidation states with excellent
Derived from plant- and animal-based sources, organic NPs are highly biocompatible, nontoxic
antimicrobial properties against bacteria and fungi [86]. Metal oxide NPs are characterized by their
at various concentrations and often inexpensive. Most organic NPs are produced from natural-origin
unique physical and chemical properties and superior density. Size-related alterations in response to
polymers, such as polysaccharides (e.g., chitosan, hyaluronic acid and cellulose) and proteins (e.g.,
an increasing number of surface and interface atoms have been observed in NPs made of CuO, ZnO,
albumin, elastin, collagen and silk). However, contrary to inorganic NPs, whose reproducibility is
SnO2 , Al2 O3 , MgO, ZrO2 , AgO, TiO2 , CeO2 , etc. Conjugation with biomaterial substrates has proven
maintained with production, organic NPs have a significant batch-to-batch variability, displaying a
very effective in stabilizing these NPs and improving their performance. In fact, combinations with
range of physical and chemical properties that result from the poor control over the synthesis and
biocomposites have shown their harmless activity on human cells and improved antimicrobial action
fabrication processes. Because of that, very little reports have been published on the combination of
and UV-protection [97–99].
these NPs with biocomposites [99,101,136].

Figure 2.
Figure 2. Overview
Overviewofofthe antimicrobial
the action
antimicrobial mechanisms
action of silver
mechanisms NPsNPs
of silver (used with with
(used permission from
permission
[135]).
from [135]).

4.1.3.Derived
Enzymes:from plant- and animal-based sources, organic NPs are highly biocompatible, nontoxic at
Laccase
various concentrations and often inexpensive. Most organic NPs are produced from natural-origin
Laccases, EC 1.10.3.2, p-diphenol:dioxygen oxidoreductase (60–100 kDa), are part of a larger
polymers, such as polysaccharides (e.g., chitosan, hyaluronic acid and cellulose) and proteins (e.g.,
group of enzymes termed multicopper enzymes that catalyze the oxidation of organic and inorganic
albumin, elastin, collagen and silk). However, contrary to inorganic NPs, whose reproducibility is
substrates. Laccase is a glycosylated monomer or homodimer protein composed of carbohydrates
maintained with production, organic NPs have a significant batch-to-batch variability, displaying a
like hexoamines, glucose, mannose, galactose, fucose and arabinose. To function, laccase depends on
range of physical and chemical properties that result from the poor control over the synthesis and
Cu atoms distributed among its three different binding sites.
fabrication processes. Because of that, very little reports have been published on the combination of
Laccase was first described by Yoshida in 1883 and was then characterized as a metal containing
these NPs with biocomposites [99,101,136].
oxidase by Bertrand in 1985, making it one of the oldest enzymes ever studied [137]. Laccases are
widely
4.1.3. distributed
Enzymes: among plants, e.g., trees, cabbages, turnips, beets, apples, asparagus, potatoes,
Laccase
pears and other vegetables; insects of genera Bombyx, Calliphora, Diploptera, Drosophilia, Lucilia,
Laccases,
Manduca, ECOryctes,
Musca, 1.10.3.2,Papilio,
p-diphenol:dioxygen oxidoreductase
Phormia, Rhodnius, (60–100 kDa),
Sarcophaga, Schistocerca are part of
and Tenebrio; anda larger
fungi,
group of enzymes termed multicopper enzymes that catalyze the oxidation of organic and inorganic
such as Monocillium indicum, Cerena maxima, Coriolposis polyzona, Lentinus tigrinus, Pleurotus eryngii
substrates.
and othersLaccase is a Trametes
from the glycosylated monomer
species. or homodimer
Laccase activity hasprotein composed
also been of carbohydrates
reported like
in few bacteria,
Biomolecules 2020, 10, 148 21 of 44

hexoamines, glucose, mannose, galactose, fucose and arabinose. To function, laccase depends on Cu
atoms distributed among its three different binding sites.
Laccase was first described by Yoshida in 1883 and was then characterized as a metal containing
oxidase by Bertrand in 1985, making it one of the oldest enzymes ever studied [137]. Laccases are widely
distributed among plants, e.g., trees, cabbages, turnips, beets, apples, asparagus, potatoes, pears and
other vegetables; insects of genera Bombyx, Calliphora, Diploptera, Drosophilia, Lucilia, Manduca, Musca,
Oryctes, Papilio, Phormia, Rhodnius, Sarcophaga, Schistocerca and Tenebrio; and fungi, such as Monocillium
indicum, Cerena maxima, Coriolposis polyzona, Lentinus tigrinus, Pleurotus eryngii and others from the
Trametes species. Laccase activity has also been reported in few bacteria, including Bacillus subtilis [138].
Fungal laccase is perhaps the most widely researched, as its presence has been documented in virtually
every fungus examined for it. Most fungi produce both intra- and extracellular enzymes, being the
phenols, amines and benzoic acid, responsible for inducing the synthesis of laccase. Laccase can
oxidize any substrate with characteristics similar to p-diphenol. Some fungal laccases are also capable
of oxidizing monophenols and ascorbic acid. However, the primarily role of fungal laccase is to
decompose lignin and/or to influence the polymerization of its oxidation by-products [137,139].
The activity of laccase-mediated systems is dependent on the redox potential of the enzyme and
the stability and reactivity of the radical groups. Laccases are capable of catalyzing the mono-electronic
oxidation of phenols and aromatic/aliphatic amines to reactive radicals and, simultaneously, reduce
molecular oxygen to water in a redox reaction. Studies have shown that the phenolic sites of lignin
macromolecules can be oxidized to phenoxyl radicals by laccase, and then undergo covalent coupling
to initiate the polymerization of lignins. Laccase-oxidized phenols or non-oxidized amines can also be
grafted to the radicalized lignins or lignocellulosic surfaces to produce engineered materials with novel
functions [102,140,141]. As natural fibers, namely jute, are rich in lignin, the use of laccase to generate
novel functions or induce stronger interfacial adhesion between non-polar resins in fiber-reinforced
polymer biocomposites has been highly desirable [102–104].

4.1.4. Peptides: RGD Motif

Peptides are versatile building blocks that adopt specific secondary structures, providing a
unique platform for the design of self-assembling biomaterials with hierarchical 3D macromolecular
architectures, nanoscale features and tunable physical properties. Various peptide motifs have
been identified and used in biomedical applications [142]. However, the widely occurring
arginine-glycine-aspartate amino acid sequence, also known as the RGD motif, is the most investigated.
This simple tripeptide (75 kDa) endowed with cell adhesion properties (adhesion peptide) and located
in the III10 module of the fibronectin protein is very complex and depends on flanking residues,
the protein 3D structure and the individual features of the integrin-binding pockets [143]. For instance,
by bonding with integrins, the RGD sequence allows fibronectin to assemble into fibrils and forming
the primitive structure of the extracellular matrix. However, this motif is not restricted to fibronectin;
indeed, it occurs within more than 100 proteins with either a cell adhesive activity or being functionally
silent [143–146].
As pointed earlier, surface modification of biomaterials is of prime importance for biomedical
applications, with biocompatibility being one of the major requirements (the material must be non-toxic
to the relevant cells). Introduction of chemical stimuli, in the form of an RGD motif, along the
biomaterial surface can facilitate its recognition and reception by the host cells. For that reason,
functionalization by either inserting peptides coupled with binding agents or by embedding them into
a polymeric matrix has been extensively researched and new bioactive biomaterials developed [147,148].
For instance, a composite of milkweed, polyethylene and polypropylene has been engineered and
modified with the RGD peptide for bone replacement. The altered biocomposite was seen to promote
MC3T3 osteoblast-like cells recruitment and, thus, to facilitate osteointegration [11]. Because of the
particular functions and loads bone substitutes must endure, there is still much to be researched
Biomolecules 2020, 10, 148 22 of 44

about the synergistic effect of natural fibers and the RGD motif. At this moment, most research on
RGD-functionalized surfaces focus on metal-based biomaterials or polymer composites.

4.1.5. Antimicrobial Peptides (AMPs)

AMPs are an integral part of the innate immune system, working as the first line of defense in a
variety of organisms. They can be of natural or synthetic origin, are typically very short (5–100 amino
acid residues), of low molecular weight (less than 10 kDa), positively charged (cationic with a net
charge of +2 to +9) and amphiphilic. Most AMPs reported to date can be characterized as one of
the following four types, based on their secondary structures: β-sheet, α-helix, extended and loop.
Even though the β-sheet structure is the most common, it is only formed when the peptide comes in
contact with
Biomolecules a membrane
2020, [149–153].
10, x FOR PEER REVIEW In the case of natural origin AMPs, they can be isolated from 20 both
of 40
prokaryotes and eukaryotes. Most AMPs are produced by specific cells, at all times; however, there are
those
are whose
those production
whose is inducible.
production Still,Still,
is inducible. theythey
are quickly mobilized
are quickly afterafter
mobilized microbial infection
microbial and
infection
act rapidly to neutralize a broad range of microbes (Figure 3) [149,150,153–156].
and act rapidly to neutralize a broad range of microbes (Figure 3) [149,150,153–156]. To date,To date, hundreds of
AMPs have
hundreds ofbeen
AMPs identified
have beenand their importance
identified and their in the innatein
importance immune system
the innate explored.
immune system explored.

Biological functions
Figure 3. Biological functions of
of AMPs. AMPs
AMPs bind
bind to
to bacterial
bacterial membranes
membranes through
through electrostatic
interactions either
interactions either to
to disrupt the membrane or to inhibit
inhibit intracellular
intracellular functions. Some AMPs
functions. Some AMPs also
modulate host
modulate host immunity
immunity by by recruiting/activating
recruiting/activating immunocytes
immunocytes or or by
by controlling
controlling the
the inflammatory
inflammatory
response (used
response (used with
with permission
permission from
from [157]).
[157]).

Unlike antibiotics,
Unlike antibiotics, which
which target
target specific
specific bacteria
bacteria cell
cell functions
functions (Figure
(Figure 1),
1), most
most AMPs
AMPs target
target the
the
microorganism’s lipopolysaccharide layer, which is exclusive to them. As eukaryotic
microorganism’s lipopolysaccharide layer, which is exclusive to them. As eukaryotic cells are rich in cells are rich
in cholesterol
cholesterol andand possess
possess a lowa low anionic
anionic charge,
charge, theyout
they are areofout
theof the of
focus focus
many of AMPs
many [151,154].
AMPs [151,154].
AMPs
AMPs can be classified based on their target microorganism as antibacterial,
can be classified based on their target microorganism as antibacterial, which target bacterial which target bacterial cell
cell
membranes, compromising the lipid bilayer structure; antiviral, which neutralize
membranes, compromising the lipid bilayer structure; antiviral, which neutralize viruses by viruses by integrating
the viral envelope
integrating the viralor envelope
the host cell
or membrane;
the host cellantifungal,
membrane; which kill by targeting
antifungal, which killeither the cell wall
by targeting or
either
the intracellular components of fungi; and antiparasitic, which kill through direct
the cell wall or the intracellular components of fungi; and antiparasitic, which kill through direct interaction with the
parasite cellwith
interaction membrane [149,157].
the parasite cell membrane [149,157].
Functionalization of biomaterialswith
Functionalization of biomaterials withAMPs
AMPsisisa arecent
recentpractice
practice that
that is is gaining
gaining much
much interest
interest in
in the biomedical field. However, guaranteeing the antimicrobial performance
the biomedical field. However, guaranteeing the antimicrobial performance of these peptides while of these peptides
while immobilized
immobilized remains remains a challenge,
a challenge, as it isasdependent
it is dependent not on
not only only onbase
the the base substrate’s
substrate’s physical
physical and
and chemical properties but also on the selected immobilization process.
chemical properties but also on the selected immobilization process. If blended with a polymer If blended with a polymer
solution, for
solution, for instance,
instance, thethe AMPs
AMPs solubility
solubility can
can bebe compromised
compromised using using organic
organic solvents
solvents asas they
they may
may
deteriorate the
deteriorate thebiomolecules
biomoleculesoror induce
induce aggregation,
aggregation, hindering
hindering theirtheir ability
ability to penetrate
to penetrate or bind ortobind
the
cell membrane. Cellulose acetate/poly(vinyl alcohol) composite films have been produced by solvent-
casting followed by phase inversion for prospective applications in wound healing. The produced
films were functionalized with LL37 by two methods, blending and surface binding via dopamine.
Data reported a significant reduction of the LL37 antimicrobial action when immobilized by
blending, proving the immobilization via binding agent more effective [158]. Physical binding
Biomolecules 2020, 10, 148 23 of 44

to the cell membrane. Cellulose acetate/poly(vinyl alcohol) composite films have been produced
by solvent-casting followed by phase inversion for prospective applications in wound healing.
The produced films were functionalized with LL37 by two methods, blending and surface binding via
dopamine. Data reported a significant reduction of the LL37 antimicrobial action when immobilized by
blending, proving the immobilization via binding agent more effective [158]. Physical binding methods,
which include adsorption and layer-by-layer approaches, require the biomolecules dissolution prior to
the physical adsorption by means of non-covalent or multidentate interactions [149]. Yet, this is not
always feasible. A synthetic hybrid of cecropin and melittin has shown the tendency to form dimmers
when in solution, augmenting its hemolytic activity and, thus, reducing its ability to penetrate the
microbial membranes [159]. Still, when immobilized by covalent bonding on polyurethane-based
substrates its action was significantly enhanced against Gram-positive bacteria [160]. Compared to
physical binding methods, covalent immobilization offers many advantages, including minimizing
AMPs leaching, providing long-term stability and lowering toxicity. Here, AMPs can be coupled
to the surface via grafting, which requires covalent bonding of intact AMPs to the material surface,
or via “surface initiated” methods, in which the synthesis of the AMPs is made through initiators
of reactive groups covalently immobilized onto the biomaterials’ surface [149,155]. Because of their
expensive and delicate nature, very little reports have been published on the functionalization of
biocomposites with AMPs. One of the few works reports the modification of wool-based fibers
with the Cecropin-B/[Ala5]-Tritrp7 hybrid AMP via the exhaustion method [12]. This modification
improved the natural fibers’ antimicrobial action, both against Gram-positive and Gram-negative
bacteria, and revealed the potential of these surfaces for biomedical uses.

4.1.6. Plant Extracts

Plants are the most important source of natural drugs used in conventional medicine. Recent
findings have demonstrated that near 72,000 (≈17%) of the 422,000 identified flowering species present
a therapeutic potential. These values are continuously increasing since the bioactive molecules present
in a plant species have also been identified in other plant species that are related with the former, thus
increasing rapidly the diversity of plants that can be used in herbal medicine [161].
Plants produce proteins, lipids, carbohydrates and chlorophyll as the primary metabolic products
after photosynthesis. These are easily found in nature, particularly in the seeds and vegetative
tissues of tall plants. The secondary metabolites, however, are more difficult to identify and extract,
being until recently discarded and their therapeutic potential ignored. These secondary biochemical
pathways are capable of synthesizing raft chemicals in response to specific environmental stimuli,
such as pathogen attacks [162]. Their roles comprehend the protection of the host by acting as
antioxidant, free radical-scavenging, UV-light absorbing and antiproliferative agents, or by defending
the plant against microorganisms such as bacteria, fungi and viruses [162,163]. The major classes of
antimicrobial compounds extracted from plants are the phenolics, which antimicrobial action includes
enzyme inhibition by the oxidized compounds (e.g., reaction with sulfhydryl groups) or through
non-specific interactions with the proteins; the terpenoids (EOs), which give the plants their odors
and are suggested to disrupt the membrane of bacteria, fungi, viruses and protozoa via lipophilic
compounds; the alkaloids, which are heterocyclic nitrogen compounds capable of interfering with
the DNA of pathogens by intercalating it; and the lectins and polypeptides, which are often cationic,
thus allowing the formation of ion channels in the microbial membrane or inhibiting the adhesion
of microbial proteins to host polysaccharide receptors by competing with those [164]. Mainstream
medicine is increasingly receptive to the use of antimicrobial agents derived from plants as traditional
antibiotics become ineffective [165]. As such, the incorporation of plant extracts onto polymeric-based
substrates, natural fibers or even biocomposites are already widely investigated. Some of the most
promising examples of the incorporation of plant extracts from different origins are highlighted in
Table 4. Special consideration was given to natural fiber fabrics endowed with antimicrobial properties
and to biocomposites with potential as regenerative medicine.
Biomolecules 2020, 10, 148 24 of 44

4.1.7. Essential Oils (EOs)

Aromatic plants are very common in traditional medicine as antimicrobial agents. EOs are volatile,
natural, complex compounds characterized by a strong odor that can be harvested from the essence of
these aromatic plants [166]. In nature, EOs play antibacterial, antiviral and antifungal roles, as well as
being insecticides, protecting plants against insects and herbivores or by reducing their appetite. They
are also responsible for the attraction of specific insects that will then disperse pollens and seeds and
promote the plant’s propagation [167,168]. EOs are produced by more than 17,500 species of plants from
many angiosperm families, e.g., Lamiaceae, Rutaceae, Myrtaceae, Zingiberaceae and Asteraceae [169].
They are synthesized in the cytoplasm and plastids of plant cells, stored in complex secretory structures
like glands or resin conduits, and only then presented as drops in the leaves, stems, flowers, fruits, bark
and roots of the plants [170]. EOs are mainly composed of terpenes, terpenoids and phenylpropanoids
at the levels of 20%–70% but may also contain fatty acids, oxides and sulfur derivatives [171]. They are
generally obtained by steam- or hydro-distillation of plants. Their properties were first investigated by
De la Croix in 1881 but since then many other researchers have analyzed the chemical composition and
inherent properties of these volatile compounds [167,172]. In fact, the use of EOs in biomedicine has
grown over the past four decades, being nowadays considered a potential alternative to antibiotics in
the treatment of various infectious diseases. EOs are known for their antiseptic (bactericidal, viricidal
and fungicidal), fragrance and medicinal properties, and have been employed in embalmment and
preservation of foods and as antimicrobial, analgesic, sedative, anti-inflammatory, spasmolytic and
local anesthetic remedies [173].
The EOs antimicrobial properties are frequently evaluated in light of their inhibitory or
bacteriostatic effect against the replication of microbial cells or by their lethal or bactericidal activity.
Their physiological role against microorganisms in not yet entirely understood; however, it is generally
accepted that the spreading of EOs along the bacteria cell membrane enhances membrane permeability,
which then leads to the subsequent loss of cell components. The acidification inside the cell blocks the
production of ATP and leads to the coagulation of the cytoplasm and destruction of genetic materials
(lipids, proteins, etc.) that, ultimately, lead to the cell death [167,173,174].
One of the major drawbacks associated with the use of EOs in biomedicine is their toxicity. It is
well known that at high concentrations EOs may induce allergic reactions, thus being strictly regulated
by the scientific committee on consumer products (SCCP). However, in the blood stream or in contact
with eukaryotic cells the tolerance is even lower. As such alternatives for the controlled release of
these volatile oils have been proposed. Recent studies have demonstrated that NPs functionalized
with EOs have significant antimicrobial potential against multi-drug-resistant pathogens [175]. Other
studies suggest the encapsulation of EOs onto chitosan to improve the antibacterial effect of the
oils and their controlled release, without a toxic initial burst [176]. In this context, biocomposites of
cotton modified with monochlorotriazinyl β-cyclodextrin, as an eco-friendly encapsulating/hosting
compound, have been proposed for the formation of core-shaped hydrophobic cavities for individual
loading of EOs [113]. The fibers contained in the peals of fruits like durian and coconuts have
also been combined with synthetic polymers for the encapsulation of cinnamon [15] and oregano
oils [116], respectively. Aside from enhancing the physical properties of the biocomposite, these EOs
demonstrated improved antimicrobial action against Gram-positive and Gram-negative bacteria; this
way attesting to the exceptional performance of EO-modified biocomposites.

4.2. Immobilization Methods

There are three major methods to immobilize biomolecules onto natural fibers: physical
adsorption, physical entrapment and covalent attachment [77,177]. Physical adsorption includes:
(1) van der Waals interactions, (2) electrostatic interactions, (3) hydrophobic effects and (4) affinity
recognition [177–179]—all methods that imply self-organization (the molecules or ions adjust their
own positions to reach a thermodynamic equilibrium) [180]. However, once adsorbed, the molecules
may be further crosslinked to each other [177–179]. Van der Waals forces (including hydrogen
Biomolecules 2020, 10, 148 25 of 44

bonding) are the most ubiquitous form of interaction between two material bodies, being caused by
the electromagnetic fluctuations derived from the continuous movements of positive and negative
charges within all types of atoms, molecules and bulk materials. They bring the bodies together.
Through the use of stabilizing ligands or appropriate solvents, these interactions can be controlled to
provide a useful tool with which to guide self-assembly [181]. Electrostatic forces hold ions together
in an ionic compound [182]. They can be either attractive (between oppositely charged ions) or
repulsive (between like-charged ions) and even directional, as in the case of structures with asymmetric
surface-charge distributions or permanent electric polarization [181]. Electrostatic forces offer a type
of bond that is low demanding in terms of the directionality and the distance between oppositely
charged functional groups, having the least steric demand of all chemical bonds [183], in addition
to the possibility of forming multi-center bonds [184]. Furthermore, the magnitude and length scale
of these interactions can be regulated, namely by choosing the solvent (e.g., dielectric constant)
and/or the concentration and chemical nature (e.g., size and valence) of the surrounding charged
counterparts [181]. The use of these forces are a non-specific approach to immobilize biomolecules when
the biomolecule has an isoelectric point higher or lower than seven and the surface a positive or negative
charge [178]. Hydrophobic interactions involve separation of hydrophobic parts of amphiphilic objects
from water molecules [180,181,185–189]. Hydrophobic interactions have been used to functionalize
hydrophobic surfaces, using biomolecules like ligands attached to hydrophobic sequences. Surfaces
with hydrophobic gradients have also been prepared [177]. But non-specific adsorption tend to
provide little control in biomolecule orientation or activity, having low durability [178]. Finally, affinity
interactions relate to the principle of complementary biomolecules interactions, by exploiting the
selectivity of specific interactions (antibodies and antigens or haptens, lectins and free saccharidic
chains or glycosylated macromolecules, nucleic acids and nucleic acid-binding proteins, hormones
and their receptors, avidin and biotin, polyhistidine tag and metal ions). A marked advantage is their
high selectivity, along with the possibility to control the orientation of immobilized biomolecules, high
retention of the bioactive compound activity, mild reaction conditions and relative simplicity of the
immobilization processes [178,181].
On the other hand, physical “entrapment” systems comprehend imprisonment of the bioactive
compound within (1) microcapsules, (2) hydrogels, and (3) physical mixtures, such as matrix drug
delivery systems [177]. Main advantages include simplicity, ability to use similar protocols for different
biomolecules and simultaneous immobilization, stability and protection of the bioactive agent against
degradation; while limitations comprise diffusion constraints (particularly with larger molecules) and
the possibility of biomolecule leakage (if the entrapped molecule is small) [190,191]. The process of
physical entrapment itself may also be harmful to the bioactive molecule [190].
Finally, covalent attachment comprises short-range intermolecular attractive forces at the molecular
scale. Two electrons are shared by two atoms [181,182]. Covalent attachment may occur within a
polymeric chain (water-soluble polymer conjugates), onto a solid surface or within hydrogels [177].
Chemical coupling reactions should achieve very high yields under mild conditions with few side
reactions and little denaturation of the bioactive compounds [190]. Numerous covalent bonding
chemistries exist. Regardless, a main advantage of a covalent bond is that the molecule is tethered
at a site on its surface rather than in contact over a significant part of its surface as in the case of
physical adsorption. The molecule is therefore generally more remote from the binding surface.
Notwithstanding, covalent binding may excessively constrain the biomolecule or at least increase
the probability of involving the bioactive site in the interaction with the surface. The proximity of
the surface may also hinder the interaction between the bound molecule and other molecules in
the solution [192]. For this reason, the inclusion of a spacer group (also called the linker, arm or
tether) is often recommended to allow the tethered molecule to be located further from the tethering
surface [177,192]. One of the most popular tethers is a poly (ethylene glycol) (PEG) molecule that can
be derivatized with different reactive end groups [177,193]. Such spacers can provide greater steric
freedom, and thus greater specific activity for the immobilized biomolecule. The spacer arm may
Biomolecules 2020, 10, 148 26 of 44

also be either hydrolytically or enzymatically degradable, and therefore will release the immobilized
biomolecule as it degrades [177]. However, the use of a linker does not always implies higher
biomolecule activity, as the linker may adopt a conformation that interferes with the function of
the compound [192]. Coatings with PEG, PEG derivatives like PEG-containing surfactants, other
hydrogels, saccharides, proteins, choline headgroups and hydrogen bond receptors have also been
useful to confer new functionalities to a surface, stabilize and protect the load and provide stealth
effect at the host environment [178,194]. Of particular interest is the metal–ligand binding between a
soluble metal acceptor center and organic ligand donors: Attractive coordination of covalent bonds
that give rise to infinite metallo–organic architectures [195]. Both the metal and the ligand are
typically chemically modified during bond activation, which depends on the nature of the metal and
ligand structures. A metal–nitrogen bond is the most well-studied cooperation interaction, although
metal–oxygen, metal–sulfur and metal–carbon also occur frequently [196]. Indeed, recently, a wide
variety of metal−ligand bonds have been formed and used to functionalize metal NPs, beyond the
conventional metal–thiolate (M-S) linkages. NP-mediated intraparticle charge delocalization is a
unique advantage. In addition, chemical events that occur at a specific site on the NPs surface may
be propagated and even amplified to all NPs, resulting in a clear variation of the NPs spectroscopic
and electrochemical properties [197]. Metal-centered compounds with endless complex structures
and shapes enable new chemistries, like novel mechanisms of action not accessible by organic small
molecules, towards the discovery of new drugs. The metal and/or ligands can interact with nucleic acids
or amino acid residues, inhibiting the function of a targeted biomolecule. Consequently, metal–ligand
interactions are being increasingly studied for therapeutic applications [185,198]. A variety of physical
properties (redox, optical and magnetic) are also presented by the metallic donors and allow suitable
spatial and electronic arrangement for mild and selective bond activation processes, resembling highly
selective bond activation reactions that occur in enzymes under mild conditions [185,196]. Figure 4
represents each of the latest referred intermolecular forces.
 different methods, plus more than one biomolecule may be immobilized to the same support. Major
immobilization method trends comprise the exhaustion method, dip–pad–dry–cure method,
covalent chemistry and in situ inorganic NP synthesis through the hydrothermal sol-gel method. Of
interest is a successful biomolecule immobilization in a sufficient amount, along with retention of an
acceptable
Biomolecules level
2020, of bioactivity over an appropriate time period [177]. Table 5 summarizes27recent
10, 148 of 44
examples of bioactive molecule immobilization strategies onto clean and/or pre-treated natural fibers.

Figure4.4.Forces
Figure Forcesinvolved
involvedininbiomolecules
biomoleculesimmobilization
immobilizationonto ontonatural
naturalfibers.
fibers.(a)(a)van
vander
derWaals
Waalsforces;
forces;
(b) hydrogen bonds between a H-bond donor and a H-bond acceptor; (c) electrostatic
(b) hydrogen bonds between a H-bond donor and a H-bond acceptor; (c) electrostatic interactions interactions
between
betweenoppositely,
oppositely,ororlikely,
likely,charged
chargedspecies;
species;(d)
(d)hydrophobic
hydrophobiceffects
effects(here
(hererepresented
representedininthe
theform
form
ofofmicelles or bilayers); (e) example of affinity recognitions, such as an antigen–antibody
micelles or bilayers); (e) example of affinity recognitions, such as an antigen–antibody interaction; interaction;
(f)(f)covalent
covalentbond
bondbetween
betweendonors
donorsXXandandYYwithout
withouta aspacer
spacerarm
arm(left),
(left),via
viaa aspacer
spacerarmarm(middle)
(middle)and
and
metal–ligand binding between a soluble metal acceptor center (M) and organic
metal–ligand binding between a soluble metal acceptor center (M) and organic ligand donors ligand donors (X and Y)
(X and
(right); and (g)
Y) (right); andlength scales ofscales
(g) length the forces
of theinvolved,
forces taking intotaking
involved, accountinto
thataccount
hydrophobicthat interactions
hydrophobic
occur upon contact, and that antigens are bound to antibodies through electrostatic
interactions occur upon contact, and that antigens are bound to antibodies through electrostatic interactions,
hydrogen bonds,
interactions, van der bonds,
hydrogen Waals forces and Waals
van der hydrophobic
forces interactions [180,181,185–189,199,200].
and hydrophobic interactions [180,181,185–
189,199,200].
But irrespective of the method used, the same biomolecule may be immobilized by many
different methods, plus more than one biomolecule may be immobilized to the same support. Major
immobilization method trends comprise the exhaustion method, dip–pad–dry–cure method, covalent
chemistry and in situ inorganic NP synthesis through the hydrothermal sol-gel method. Of interest is a
successful biomolecule immobilization in a sufficient amount, along with retention of an acceptable
level of bioactivity over an appropriate time period [177]. Table 5 summarizes recent examples of
bioactive molecule immobilization strategies onto clean and/or pre-treated natural fibers.
Biomolecules 2020, 10, 148 28 of 44

Table 5. Recent trends on biomolecule immobilization strategies onto natural fibers.

Natural Functional
Cleaning Pre-treatment Immobilization Strategies Biomolecule Main chemical Reactions References
Fiber Groups
Dip–pad–dry method to deposit pegylated silver
Non-ionic NPs, drying 100 ◦ C 20 min, water, drying 100 ◦ C 6
Silver NPs and Zinc
detergent 80 ◦ C 30 min Metal–ligand binding with Ag+
Flax - -OH oxide NPs (inorganic [201]
min, DW 70 ◦ C 30 In situ NP synthesis by sol-gel method: immersion and Zn2+ ions from NPs
NPs)
min, 100 ◦ C 10 min in Zn(CH3 COO)2 .2H2 O 50 ◦ C 1 h stirring, NaOH,
drying 100 ◦ C 6 h
Dip–pad–dry–cure method: immersion in CA,
Esterification of linen with
NaPO2 H2 and chitosan, padding, drying 100 ◦ C 3 Chitosan
-COOH of CA; electrostatic
min, curing 140 ◦ C 5 min (Polysaccharide) and
- - -OH interaction of CA with -NH2 of [99]
In situ NP synthesis by sol-gel method: immersion Cerium oxide NPs
chitosan; Metal-ligand binding
in Ce(SO4 )2 solution 45 min, NaOH 50 ◦ C 30 min inorganic NPs)
with Ce3+ ions
under ultrasound irradiation, cold water, drying
Linen (flax Esterification with -COOH of
family) Dip–pad–dry–cure method with chitosan, BTCA Silver NPs (Inorganic BTCA; electrostatic interaction
and NaPO2 H2 , dried 80 ◦ C 4 min and cured 140 ◦ C NPs), Chitosan of BTCA with -NH2 of chitosan,
4 min (Polysaccharide), and of -COOH, NH2 and -OH
- - -OH [84]
In situ NP synthesis by sol-gel method: immersion Tamarindus indica L. groups with silver nitrate;
in AgNO3 20 min, then in mordant TSCE 60 min seed coat extract Metal–ligand binding between
under ultrasound irradiation, cold water, drying (TSCE, plant extract) phenol groups of tannings of
TSCE and Ag+ ions
Casting of a resin mixture (polyester resin with NP
filler loadings and MEKP as catalyst) onto the fibers Bamboo NPs (organic Hydrogen bonding between
Kenaf - - -OH [101]
using hand layup process, cure cold press 24 h, NPs) NPs, fiber and matrix
polymerization 105 ◦ C
In situ NP synthesis by sol-gel process: immersion
Metal–organic
in Zn(NO3 )2 .6H2 O and CH3 C3 H3 N2 H solutions in
framework (zeolitic
CH3 OH 24h, DIW with ultrasound irradiation 10 Metal–ligand binding with
- -OH imidazolate [95]
min, drying 80 ◦ C 2 h Zn2+ ions
framework-8, ZIF-8)
Immersion in THF solution with PDMS and curing
(inorganic NPs)
agent stir 5 min, drying 80 ◦ C 2 h
Esterification through the
dip–pad–cure–dry method:
Cotton immersion in CMCS In situ NP synthesis by sol-gel process: immersion
Silver NPs (inorganic Metal–ligand binding with Ag+
- solution 15 min, pad-roll, in AgNO3 10 min, drying 100 ◦ C 1 h, immersion in -SH [13]
NPs) ions
cure 180 ◦ C 5 min, DW, NaBH4 10 min, DW, drying 100 ◦ C 1 h
drying 100 ◦ C 1 h. Same for
Cys adsorption
Biomolecules 2020, 10, 148 29 of 44

Table 5. Cont.

Natural Functional
Cleaning Pre-treatment Immobilization Strategies Biomolecule Main chemical Reactions References
Fiber Groups
Silanization: drying 55 ◦ C
Ultrasound
24 h, immersion in OTS and Silver NPs (inorganic Metal–ligand binding with Ag+
treatment in DIW, Immersion in silver NP dispersion for 10 min -OH [202]
MTS in C7 H8 sealed 10 min, NPs) ions
drying
drying
Physical adsorption after
sonication process: based on
the point melting of the
substrate and carbonization of
Ultrasound treatment: immersion into a hot
Curcumin (plant the fibers at the points of their
- - dispersion of loaded FF peptide nanotubes in an Unspecific [110]
extract) contact with the silver nuclei
ice bath, DW, freeze-drying
due to the high rate and
temperature of the nanotubes
thrown to the solid surface by
sonochemical microjets
Thiol-maleimide click chemistry: immersion in
NaOH and Silanization: immersion in
CH3 C(O)CH2 CH3 with N-phenyl-male-imide N-phenyl-male-imide Thiol-maleimide click
C58 H118 O24 at 70 KH-580 solution 2 min, cure -SH [203]
◦ C 20 min and C6 H15 N 60 ◦ C 30 min while stirring, drying (organic compound) chemistry
120 ◦ C 5 min
70 ◦ C 10 min
Immersion in amoxicillin solutions 10 min,
Hydrogen bonding and
drying 72 h fume hood
NaOCl, DW, Amoxicillin electrostatic interaction with
- Solvent casting technique: pouring of PLA -OH [14]
drying 60 ◦ C 48 h (antibiotic) cationic groups of amoxicillin
solution in CHCl3 until submersion, solvent
like -NH2
evaporation 72 h vacuum
Poly(propylenimine)
dendrimers from first
and third generations
modified with
- - Deposition by extraction method [204]
1,8-naphthalimide
units and their Zn(II)
complexes
(dendrimers)
UV-photo-grafting method of Covalent bond with radical
- - alginate-Ca2+/PNIPAA hydrogel: PAAm, SA -OH MB as model drug initiators that subtracted H [205]
and other additives, UV 30 min, CaCl2 24 h, DW atoms to cotton
Biomolecules 2020, 10, 148 30 of 44

Table 5. Cont.

Natural Functional
Cleaning Pre-treatment Immobilization Strategies Biomolecule Main chemical Reactions References
Fiber Groups
In situ NP synthesis by sol-gel process:
Functionalization by
immersion in Zn(CH3 COO)2 into CH3 OH and
immersion in dopamine Zinc oxide NPs Metal-ligand binding with Zn2+
Acetone, DIW NaOH 20 min, pad-rolled, dried in vacuum. Cathecol [206]
solution at pH 8.5, DW, (inorganic NPs) ions
Then, immersion into Zn(NO3 )2 .6H2 O) and
drying vacuum
HMTA solutions 90 ◦ C 5 h, DW, drying
Ultrasound
Dip–pad–dry–cure method:
treatment:
immersion in Cys30 min, In situ NP synthesis by sol-gel method:
C12 H25 NaO3 S 30 Copper NPs Metal–ligand binding between
pad, drying 3 min 80 ◦ C, immersion in CuSO4 and CA 50 ◦ C 30 min, -SH [207]
min, ethanol 2 h, (inorganic NPs) Cys on cotton and Cu2+ ions
cure 180 ◦ C 3 min, DW (3 NaBH4 40 ◦ C 1h, DW twice, drying 4 h
DIW 30 min 3
times), drying 100 ◦ C 1 h
times
Pad–dry–cure process: immersion in
chitosan-silver zeolite composites (previously Esterification with -COOH of
- - obtained by ionic gelation method with TPP) at -OH Silver zeolites CA that also lead to chemical [94]
pH 5.5, drying 90 ◦ C 3 min, crosslinked with CA reaction with -NH2 of chitosan
140 ◦ C 2 min, water, drying
Pad–dry–cure technique: immersion in aqueous
Covalent bond of -COH of
solution of ethanol extract liquid of propolis
glyoxal with -OH of propolis
- - with glyoxal and Al2(SO4)3, padding, drying 80 -OH Propolis (plant extract) [106]
◦ C 3 min, cure 140 ◦ C 5 min, warm water 15 min, and fabric, hydrogen bonding,
physical entrapment
drying
Turbo Break
detergent (NaOH),
Silex Emulsion
detergent (fatty
alcohol
ethoxylates, Immersion in Ag3 C6 H5 O7 , C4 H6 O4 Cu as
NaOH), and precursors in water Ag+/Cu2+ and Silver
Metal–ligand binding with
Ozonit - Immersion in mixed solution with C4 H6 O4 Cu -OH NPs/Cu2+ (inorganic [86]
Ag+ /Cu2+ ions
Performance and Ag3 C6 H5 O7 , reduction with NaBH4 , ions, inorganic NPs)
detergent stabilizer PVP
(CH3 COOH,
H2 O2 , CH3 CO3 H),
Finale Liquid
detergent
(HCOOH)
Biomolecules 2020, 10, 148 31 of 44

Table 5. Cont.

Natural Functional
Cleaning Pre-treatment Immobilization Strategies Biomolecule Main chemical Reactions References
Fiber Groups
Carding together with
core-shell PE-coated PP Immersion under stirring in EDC solution in
Soxhlet extraction
fibers 80–120 ◦ C MES buffer 30 min, MES buffer twice, Peptide covalent bond with
Milkweed in acetone 24 h, -COOH RGD (peptide) [11]
Dielectric Barrier Discharge RGD-TAMRA HEPES solution pH 7.4) 3 h, NH2 with RGD peptide
vacuum-drying
plasma treatment at TWEEN-20 five times, DIW three times
atmospheric pressure
Functionalization by In situ NP synthesis by sol-gel method:
Filter, wash, Silver NPs (inorganic Metal–ligand binding with Ag+
Kapok immersion in dopamine immersion in AgNO3 UV irradiation under Catechol [208]
drying NPs) ions
solution at pH 8 24 h stirring 30 min, DW, drying vacuum
Washing, Solvent casting method: Hydrogen and covalent
chopping, drying PLA and durian skin Cinnamon oil addition to the previously formed Cinnamon (essential bonding between the
Durian skin -OH [15]
grinding, drying fiber, dissolution in ChCl3 composite oil) PLA/durian skin fiber and
and sieving while stirring, EPO, 24 h aldehydes in cinnamon oil
Ultrasound
Functionalization by In situ NP synthesis by sol-gel method:
treatment: acetone, Silver NPs (inorganic Metal–ligand binding with Ag+
immersion in dopamine immersion in Ag3 C6 H5 O7 , microwave Catechol [209]
ethanol and DW, NPs) ions
solution at pH 8.5 irradiation, rinse in DW, drying
15 min
Ultrasound Exhaustion bath with loaded microcapsules,
-COOH, Covalent bonding between
treatment: water, Mikracat B crosslinking agent and Sapamine Lavender oil (essential
Air plasma treatment -OH, loaded microcapsules and [210]
detergent and softener 1 h pH 7, padding, crosslinking 1 h 130 oil)
Bamboo -COH fabric
Na2 CO3 , 1 h 60 ◦ C ◦ C, drying

In situ NP synthesis by sol-gel method:

Immersion in HAuCl4 , 15 min RT, 80 ◦ C 60 min
Water 70 ◦ C 3 min, in oscillating water bath, DW, drying; or Gold and silver NPs Metal–ligand binding with
- -OH [211]
DW Immersion in AgNO3 , 15 min RT, 80 ◦ C 60 min (inorganic NPs) Au3+ /Ag+ ions
in oscillating water bath, NaOH for pH 10, 80 ◦ C
60 min, DW, drying
In situ NP synthesis by sol-gel method:
◦ C, Immersion in H2 PtCL6 at pH 5 10 min, 90 ◦ C 60 Platinum NPs Metal–ligand binding between
Water 50 DW - -SH [100]
min in shaking water bath, DW, drying. NaOH (inorganic NPs) Cys on silk and Pt+ ions
or CH3 COOH to adjust pH to 6
Silk In situ NP synthesis by sol-gel method:
Immersion in HAuCl4 pH 3 20 min, 90 ◦ C 60 min
Warm water 5 min, Gold and silver NPs Metal–ligand binding with
- in shaking water bath, DIW, drying 70 ◦ C; or -SH [212]
DIW (inorganic NPs) Au3+ /Ag+ ions
Immersion in AgNO3 pH 10 20 min, 90 ◦ C 60
min in shaking water bath, DIW, drying 70 ◦ C
Biomolecules 2020, 10, 148 32 of 44

Table 5. Cont.

Natural Functional
Cleaning Pre-treatment Immobilization Strategies Biomolecule Main chemical Reactions References
Fiber Groups
Dip dyeing process: immersion dye solution pH
Electrostatic interaction with
3 90 ◦ C 60 min Tea stem extract (plant
- - -SH polyphenol groups of the [213]
Mordant treatment with FeSO4 , Fe2 (SO4 )3 and extract)
extract
TiOSO4 60 ◦ C 30 min, tap water, drying
3 times Na2 CO3 Exhaustion method: immersion in silver NP
Silver NPs (inorganic Electrostatic interaction with
boiling point 30 dispersion (previously reduced by SA) in - NH2 [214]
NPs) -COOH from SA
min, DW, drying shaking bath pH 4 40 ◦ C 40 min, drying
In situ NP synthesis by sol-gel method:
Immersion in Silver NPs (inorganic Meta–ligand binding with Ag+
– -SH [215]
AgNO3 90 ◦ C 3 ◦ C /min from 30 ◦ C, CfA, 90 ◦ C NPs) ions
30 min with agitation, DIW, drying
Layer-by-layer
self-assembly: alternate
Three times immersion in PAH and PAA
Immersion in heparin 4 ◦ C 24h, PBS and DW Heparin Electrostatic interaction with
Na2 CO3 98 ◦ C 30 3 ◦ C 100 rpm 30 min -NH2 [216]
under ultrasonic irradiation 10 min (polysaccharide) sulfate groups of heparin
min, DW, drying followed by rinsing DW 1
min 3 times (outermost
layer: PAH), drying 24 h
Kapok flower extract Bonding with phenol groups of
Exhaustion method: in rota dyer, mordant
(plant extract) and tannings of TSCE and amide
Non-ionic soap at treatment with TSCE 90 ◦ C 60 min, squeeze, -CONH
- Tamarind seed coat -CONH groups of wool; [217]
80 ◦ C 20 min dyed with natural dye KFE 90 ◦ C 60 min, cold -OH
extract (TSCE, plant hydrogen bonding between
water, dried
extract) mordanted wool and KFE
Metal–organic
Hydrogen bonding and
Immersion in Cu(NO3 )2 and C6 H3 (COOH)3 -SH framework-199
- - Metal-ligand binding with Cu2+ [218]
solution 85 ◦ C, wash with DMF, drying -OH (HKUST-1, inorganic
ions
NPs)
Exhaustion method: immersion in LRM extract,
Ultrasound Lycium ruthenicum Hydrogen bonding and van der
warm water, cold rinse, drying 60 ◦ C 15 min.
Wool treatment: acetone - -OH Murray extract (LRM, Waals forces with anthocyanin [219]
Mordant treatment with FeSO4 and Fe2 (SO4 )3 60
3 h, drying 50 ◦ C ◦ C 30 min, rinse, drying plant extract) of the extract

Mordanting with
Immersion in natural dye solution 91–93 ◦ C 1h
KAl(SO4 )2 , FeSO4 and Pomegranate peel Electrostatic interaction with
Soaking in water manual agitation, non-ionic detergent Safewash, -CONH [220]
SnCl2 91–93 ◦ C 1 h under extract (plant extract) phenolic compounds of dye
tap water, drying
stirring, tap water
Biomolecules 2020, 10, 148 33 of 44

Table 5. Cont.

Natural Functional
Cleaning Pre-treatment Immobilization Strategies Biomolecule Main chemical Reactions References
Fiber Groups
Na2 CO3 bath pH
8.5 60 ◦ C 30 min Exhaustion method: immersion in AMP solution
Cecropin-B and Electrostatic interaction with
and non-ionic - 40 ◦ C 1–3 h while stirring, 5-cycle washing with -COOH [12]
[Ala5]-Tritrp7 (AMPs) terminal -NH2 of peptides
detergent Nekanil WOB detergent 40 ◦ C 60 min, drying 37 ◦ C 4 h
907, DW, drying
Exhaustion-dyeing process: immersion in
Non-ionic Poly(amidoamine) Electrostatic interaction with
dendrimer derivative dye 30 ◦ C pH 5-5.5, 100 ◦ C
detergent Lotensol - -NH2 dendrimer terminal -COOH of dye [221]
within 25 min + 60 min, non-ionic detergent 50
60 ◦ C 20 min ◦ C 20 min (dendrimers) molecules

Abbreviations: Ag3 C6 H5 O7 : silver citrate; AgNO3 : silver nitrate; Al2 (SO4 )3 : aluminum sulfate; AMP: antimicrobial peptide; BTCA: 1,2,3,4-Butanetetracarboxylic acid; CHCl3 :
chloroform; CH3 C(O)CH2 CH3 : butanone; CH3 CO3 H: peracetic acid; C4 H6 O4 Cu: copper (II) acetate; C6 H3 (COOH)3 : trimesic acid; C6 H15 N: triethylamine; C7 H8 : toluene;
C12 H25 NaO3 S: sodium 1-dodecanesulfonate; C58 H118 O24 : polyoxyethylene lauryl ether; CA: citric acid; Ce(SO4 )2 ; CfA: caffeic acid; CH3 C3 H3 N2 H: 2-methylimidazole; CH3 OH:
methanol; CMCS: carboxymethyl-chitosan; Cu(NO3 )2 : copper nitrate; CuSO4 : copper sulfate; Cys: L-cysteine; DIW: dionized water; DMF: dimethylformamide; DW: distilled water; EDC:
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; EPO: epoxidized palm oil; FF: diphenylalanine; Fe2 (SO4 )2 : ferric sulfate; FeSO4 : ferrous sulfate; HAuCl4 : tetrachloroauric
acid; HCOOH: formic acid; H2 PtCl6 : chloroplatinic acid; HMTA: hexamethylenetetramine; ITX: 2-isopropylthioxanthone; KAl(SO4 )2 : potash alum; KH-580: silane coupling agent; MB:
methylene blue; MEKP: methyl ethyl ketone peroxide; MES: 2-(N-Morpholino)ethanesulfonic acid; NIPAAm: N-isopropylacrylamide; NP: nanoparticle; Na2 CO3 : sodium carbonate; NaBH4 :
sodium borohydride; NaPO2 H2 : sodium hydrophosphite; NaOCl: sodium hypochlorite; NaOH: sodium hydroxide; OTS and MTS: long and short silanes; PDMS: polydimethylsiloxane;
PE: polyethylene; PLA: polylactic acid; PP: polypropylene; PVP: polyvinylpyrrolidone; RGD: arginylglycylaspartic acid; SA: sodium alginic acid; SnCl2 : stannous chloride; TAMRA:
carboxylic acid of tetramethylrhodamine THF: tetrahydrofuran; TiOSO4 : titanium sulfate; TPP: sodium tripolyphosphate; Zn(CH3 COO)2 .2H2 O: zinc acetate dihydrate; Zn(NO3 )2 .6H2 O:
zinc nitrate hexahydrate.
Biomolecules 2020, 10, 148 34 of 44

5. Conclusions and Future Perspectives

Biocomposite materials are a relatively recent addition to the composites class, with desirable
properties for biomedical applications. Along this review, the advances on this front were
highlighted, and the improvements made by the introduction of attractive biomolecules and respective
immobilization processes and the selection of specific fiber and/or surface treatments were analyzed
in detail. It is now clear that the success of a biocomposite relies greatly on the compatibility of the
individual materials and the interactions formed. Here, the pre-treatment of the natural fibers and
the surface modifications are essential. Immobilization of biomolecules onto these biocomposites
represents a step forward to their use in specific biomedical applications. Addition of drugs, NPs,
peptides or even plant extracts were found to improve the biocompatibility and antimicrobial resistance
of the biocomposites, qualities that are fundamental to a successful implantation. Still, challenges
remain and should be properly addressed in future works. One of the major challenges lies with the
understanding of the material’s individual properties and the proper selection of the processing tools.
It is well known that the manufacturing process, and all the inherent stages of production, have an
important influence on the final product. Because of their biological origin, the extraction of the natural
fibers and their consequent properties are more difficult to predict. Hence, variations between batches
are encountered, which may also explain why biocomposites developed in the laboratory have limited
success during clinical trials.
Production of biocomposites for biomedical uses relies on a different set of rules than other
composites for other applications. They need to be tailored and optimized to fit the desired local
and global arrangement of the reinforcement phase so that the implantable biocomposite can become
structurally compatible with the host tissues. Efforts should be made to harness the potential of textile
biocomposites for the design of implants with improved performance. The modification of materials
with a biomolecule of interest has been very important to reach this goal, particularly for external-use
medical textiles. Yet, the long-term durability and reliability of internal-use biomaterials made from
these biocomposites require further research efforts. In view of their clear potential, which is intimately
related to their flexibility in introducing surface modifications via biomolecules, it is expected that
biocomposite materials will find increasing uses in biomedicine.

Author Contributions: Writing-original draft preparation, T.D.T., J.C.A. and H.P.F.; writing-review and editing,
T.D.T., J.C.A., F.F. and H.P.F.; supervision, F.F. and H.P.F.; funding acquisition, F.F. and H.P.F. All authors have read
and agreed to the published version of the manuscript.
Funding: This work was funded by the Portuguese Foundation for Science and Technology (FCT), FEDER funds
by means of Portugal 2020 Competitive Factors Operational Program (POCI) and the Portuguese Government
(OE) by means of projects POCI-01-0145-FEDER-028074 and UID/CTM/00264/2020.
Conflicts of Interest: The authors declare no conflict of interest.

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