Re:후코이단 보충제의 비교 - 후코이단 항암용량 Review 논문

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beyond reason

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 Review

Possibilities of Fucoidan Utilization in theDevelopment of Pharmaceutical Dosage Forms

Aleksandra Citkowska, Marta Szekalska and KatarzynaWinnicka *

Department of Pharmaceutical Technology, Medical University of Białystok, Mickiewicza 2c,

15-222 Białystok, Poland

* Correspondence: kwin@umb.edu.pl; Tel.: +48-85-748-5616

Received: 11 July 2019; Accepted: 2 August 2019; Published: 5 August 2019

Abstract: 

Fucoidan is a polysaccharide built from L-fucose molecules. The main source of this

polysaccharide is the extracellular matrix of brownseaweed ( Phaeophyta), but it can be also isolated from invertebrates such as sea urchins ( Echinoidea) and sea cucumbers ( Holothuroidea). Interest in fucoidan is related to its broad biological activity, including possible antioxidant, anti-inflammatory, anti fungal, antiviral or antithrombotic ects. The potential application of fucoidan in the pharmaceutical technology is also due to its ionic nature. The negative charge of the molecule results from the presence of sulfate residues in the C-2 and C-4 positions, occasionally in C-3, allowing the formation of complexes with other oppositely charged molecules. 

Fucoidan is non-toxic, biodegradable and biocompatible compound approved by Food and Drug Administration (FDA) as Generally Recognized As Safe (GRAS) category as food ingredient. Fucoidan plays an important role in the pharmaceutical technology, so in this work aspects concerning its pharmaceutical characteristics and designing of various dosage forms (nanoparticles, liposomes, microparticles, and semisolid formulations) with fucoidan itself and with its combinations with other polymers or components that give a positive charge were reviewed. Advantages and limitations of fucoidan utilization in the pharmaceutical technology were also discussed.

Keywords: polysaccharide; marine-derived; fucoidan; pharmaceutical formulations; multifunctional

polymer; fucospheres

1. Introduction

Fucoidan belongs to the large group of sulfated, rich in l-fucose polysaccharides, which was

first found by Kylin in 1913 [1]. The main source of fucoidan are marine brown algae ( Phaeophyta:

Laminariaceae, Fucaceae, Chordariaceae, Alariaceae), but it has been also isolated from marine invertebrates such as sea cucumber ( Holothuroidea: Stichopodidae, Holothuriidae), from sea urchins eggs ( Echinoidea: Strongylocentrotidae, Arbaciidae) [2], and from seagrasses ( Cymodoceaceae) [3]. The chemical structure and molecular weight of fucoidan are various in dependence of the species from which it is extracted, growth environment, season of harvesting and the method of extraction used [2,3]. 

Fucoidans isolated from the majority of brown algae are branched and have a backbone of alternating (1!3)- and (1!4)-linked -l-fucose residues. Sometimes fucose molecules are connected by 1!2 linkage

(Figure 1) [3,4]. Studies demonstrated that the most of echinoderms and some families of brown algae

( Laminariaceae and  Chordariaceae) are the source of fucoidan with linear chain built of (1!3)-linked

-l-fucose residues [3–5]. The negative charge of this biopolymer results from the presence of sulfate

groups, which are mainly substituted on C-2 and C-4 and occasionally on C-3 positions [2,4]. Besides

fucose, fucoidan may contain other sugars, for example: xylose, arabinose, rhamnose, glucose, galactose

and uronic acid or even protein and acetyl groups. Both molecular weight (in the range between 10

and 2000 kDa), as well as the varied percentage shared in sugar and non-sugar components makes the

Mar. Drugs 2019, 17, 458; doi:10.3390/md17080458 www.mdpi.com/journal/marinedrugs

Mar. Drugs 2019, 17, 458 2 of 20

analysis of fucoidan structure dicult [3,4]. Furthermore, the structure of fucoidan depends on the

species of algae, but it can be dierent even for the same species thus the term “fucoidan” does not refer

to one specific structure, but includes a diverse group of sulfated polysaccharides containing fucose.

Mar. Drugs 2019, 17, x 2 of 21

structure of fucoidan depends on the species of algae, but it can be different even for the same species

thus the term “fucoidan” does not refer to one specific structure, but includes a diverse group of

sulfated polysaccharides containing fucose.

Figure 1. Scheme of the fucoidan structure; R‐ carbohydrate substituents: xylose, arabinose,

rhamnose, glucose, galactose, or uronic acid and non‐carbohydrate substituents: sulfate or acetate

residues.

The process of fucoidan extraction consists of several stages. The first step involves cleaning the

algae, drying and grinding it or cutting fresh macroalgae into pieces and leaving in the dark, damp

place to get exudate. Then different solvents (for example acetone, ethanol, mixture of acetone and

ethanol or mixture of methanol, chloroform with water) are used to remove lipids, phenols and

proteins. Fucoidan could be extracted by traditional solvent extraction (with water, ethanol or diluted

acid) or using modern methods such as ultrasound‐assisted extraction (UAE), microwave‐assisted

extraction (MAE) or enzyme‐assisted extraction (EAE) [3,6]. New technique used to obtain fucoidan

is also an extraction with 0.5% ethylenediaminetetraacetic acid (EDTA) at 70 °C. The unquestionable

advantage of this procedure is that EDTA enables simultaneous removal of pigments and fucoidan

extraction with high yield [7]. Regardless of the method used for extraction to obtain a specific

polysaccharide, the next process is purification, most often by applying chromatographic methods or

using molecular cut off membranes [6]. The wide interest of fucoidan in medicine and pharmacy is

increasing due to its broad spectrum of biological activities. The bioactivity of fucoidan depends on

several factors, predominantly on the content of sulfate groups and the molecular weight of the

polysaccharide [3,4,8]. It was found that some fucoidans might exhibit antioxidant [9], anticoagulant

[10], antibacterial [11,12], antifungal, antileishmanial [12], anti‐inflammatory, and

immunomodulatory [13] activity. Positive fucoidan impact on the treatment of patients with cancer

diseases by improving the effectiveness of chemotherapy and reducing its side effects was also

reported [2,3,14,15].

Despite many publications about fucoidan multidirectional biological activity, and many

scientific reports on its use in drug delivery, there are still no registered drug products with fucoidan.

It is currently available only in cosmetics, functional foods and dietary supplements as a regenerative,

anti‐inflammatory, and anti‐cancer factor for patients with immune‐compromised, musculoskeletal,

cardiovascular, or gut diseases. However, fucoidan as synergistic anticancer agent has been subjected

to clinical examination. It was observed that co‐administration of fucoidan resulted in no significant

Figure 1. Scheme of the fucoidan structure; R- carbohydrate substituents: xylose, arabinose, rhamnose,

glucose, galactose, or uronic acid and non-carbohydrate substituents: sulfate or acetate residues.

The process of fucoidan extraction consists of several stages. The first step involves cleaning the

algae, drying and grinding it or cutting fresh macroalgae into pieces and leaving in the dark, damp

place to get exudate. Then dierent solvents (for example acetone, ethanol, mixture of acetone and

ethanol or mixture of methanol, chloroform with water) are used to remove lipids, phenols and proteins.

Fucoidan could be extracted by traditional solvent extraction (with water, ethanol or diluted acid) or

using modern methods such as ultrasound-assisted extraction (UAE), microwave-assisted extraction

(MAE) or enzyme-assisted extraction (EAE) [3,6]. New technique used to obtain fucoidan is also an

extraction with 0.5% ethylenediaminetetraacetic acid (EDTA) at 70 C. The unquestionable advantage

of this procedure is that EDTA enables simultaneous removal of pigments and fucoidan extraction

with high yield [7]. Regardless of the method used for extraction to obtain a specific polysaccharide,

the next process is purification, most often by applying chromatographic methods or using molecular

cut o membranes [6]. The wide interest of fucoidan in medicine and pharmacy is increasing due

to its broad spectrum of biological activities. The bioactivity of fucoidan depends on several factors,

predominantly on the content of sulfate groups and the molecular weight of the polysaccharide [3,4,8].

It was found that some fucoidans might exhibit antioxidant [9], anticoagulant [10], antibacterial [11,12],

antifungal, antileishmanial [12], anti-inflammatory, and immunomodulatory [13] activity. Positive

fucoidan impact on the treatment of patients with cancer diseases by improving the eectiveness of

chemotherapy and reducing its side eects was also reported [2,3,14,15].

Despite many publications about fucoidan multidirectional biological activity, and many scientific

reports on its use in drug delivery, there are still no registered drug products with fucoidan. It is

currently available only in cosmetics, functional foods and dietary supplements as a regenerative,

anti-inflammatory, and anti-cancer factor for patients with immune-compromised, musculoskeletal,

cardiovascular, or gut diseases. However, fucoidan as synergistic anticancer agent has been subjected

to clinical examination. It was observed that co-administration of fucoidan resulted in no significant

impact on plasma concentrations of anti-cancer drugs, but might play a role in reducing side eects and

Mar. Drugs 2019, 17, 458 3 of 20

in enhancing the therapeutic eects of conventional anti-cancer therapies [16,17]. The eect of oligo

fucoidan in patients with non-small cell lung cancer treated with platinum-based chemotherapy has

been tested in III-IV stage of clinical trials [18]. Fucoidan was also under clinical eval‎uation in patients

with metastatic colorectal cancer treated with folinic acid, 5-fluorouracil, irinotecan, and bevacizumab.

It was reported that fucoidan led to alleviate side eects of anti-cancer chemotherapy [19]. Fucoidan

impact on the metabolism of fatty liver and liver fibrosis was also examined, but results have not been

published yet [20].

2. Pharmaceutical Features of Fucoidan

Fucoidan is a pale brown or yellow powder with hygroscopic properties. It easily dissolves in

water but is not soluble in organic solvents. Fucoidan solutions are not highly viscous, so it is not

applied as thickening agent [21–23]. The viscosity of aqueous solutions of fucoidan is low and depends

on many factors, including molecular weight, concentration, number of sulfate groups, degree of

branching of the molecule, as well as temperature and pH [24].

In order to determine the basic properties of fucoidan obtained from  Laminaria japonica (Weihai

Century Biocom Seaweed Co., Ltd, Weihai, China), we examined its aqueous solutions (Table 1). The

molecular weight of the tested fucoidan was 10.5 kDa. Both the content of fucose and sulphate groups

were classified at a level higher than 27%. It was observed that with the increasing concentration

of polysaccharide, the pH values of the solutions decreased (5.23 and 4.45 for 1% and 30% solution,

respectively), while their viscosity was increasing and reached a value of 507 mPas for 30% solution,

and no gelation was noted.

Similarly, Jae-Geun et al. have shown that water solutions of fucoidan from  Laminaria religiosa,

 Undaria pinnatifida,  Hizikia fusiforme, and  Sargassum fulvellum possessed low apparent viscosity and

were characterized by pseudoplastic behavior [21].

Viscosity of fucoidan is also influenced by algae species, presence of ions, or additional molecules.

Comparing fucoidan solutions from  Saccharina longicruris,  Ascophyllum nodosum, and  Fucus vesiculosus, the highest viscosity was observed when fucoidan isolated from  Fucus vesiculosus was applied [25].

Viscosity of fucoidan solutions obtained from  Cladosiphom okamuranus increased linearly with an

increase of polymer concentration (up to 2%), and with the addition of sodium chloride, calcium

chloride and sugar. It was stable at pH range from 5.8 to 9.5, indicating that fucoidan molecules are

stable under acidic and alkaline conditions [22]. Contrary to other polysacharides, fucoidan does not

show gelation ability [4,24,26], but upon fucoidan mixing with polymers with opposite net charge,

based on electrostatic interactions, gels might be created.

It should be also emphasized that due to the ability to interact with growth factors, macrophages,

cytokines and P-selectin, inhibition of the P-glycoprotein pump and -glucosidase, as well as to open

tight junctions in intestinal Caco-2 cell monolayer, fucoidan seems to be a valuable adjuvant in the

pharmaceutical technology [2–5]. These properties allow not only to protect the drug substance, but

Mar. Drugs 2019, 17, 458 4 of 20 also to strengthen drug activity, and they are described in more detail in further subsections devoted to

specific formulations.

3. Toxicity of Fucoidan

Fucoidan utilizing in the pharmacy and biomedicine is possible because it is a non-toxic,

biodegradable, and biocompatible compound [4,24]. Fucoidans from  Undaria pinnatifida and  Fucus

vesiculosus were approved in the USA by Food and Drug Administration (FDA) as Generally

Recognized As Safe (GRAS) category as food ingredients at levels up to 250 mg/day [27]. In Europe

preparations containing  Fucus vesiculosus are registered in Austria, Belgium, France, Poland, Spain,

and the United Kingdom [28].

No changes occurred in rats receiving fucoidan from  Laminaria japonica for 7 days, even at a dose

of 4000 mg/kg body weight (b.w.). Subchronic toxicity studies conducted over 6 months have reported

that no significant adverse eects were observed when fucoidan was orally administered to animals at

the dose of 300 mg/kg b.w. per day, but when the dose was increased to 900 and 2500 mg/kg b.w. per day, clotting time significantly increased [29]. Nevertheless, low molecular weight fucoidan (LMWF)

from  Laminaria japonica did not lead to changes in b.w., water and food consumption, or hematological

parameters after 28 days oral administration, even when the dose was 2000 mg/kg b.w. per day.

Dierences were observed only in two biochemical parameters—creatinine (CRE) and triglyceride

(TG) levels were significantly lower than in rats receiving 0.9% saline solution. In histopathological

examination of organs, including brain, heart, kidneys and liver, no discrepancies were observed

between the control group and fucoidan treated rats [30]. However, when the source of fucoidan

was  Cladosiphon okamuranus, the dose that did not cause significant changes was 600 mg/kg b.w. per

day and the anticoagulant activity of fucoidan was observed at higher doses (1200 mg/kg b.w. per

day) [31]. No dierences were observed in nutrition, b.w., hematological and biochemical parameters,

as well as in organs subjected to necroscopy and microscopic examination in rats receiving up to

1000 mg/kg b.w. per day fucoidan from  Undaria pinnatifida. Increasing the dose to 2000 mg/kg b.w.

per day caused significant changes in mean corpuscular hemoglobin concentration (MCHC), alanine

transaminase (ALT), TG, and high-density lipoprotein (HLD) levels [32]. Observed dierences between

the above-described studies indicate that the potential toxicity of fucoidans depends on the species

from which it is obtained, as well as fucoidan molecular weight.

The safety of oral ingestion of fucoidan has also been studied in humans. Twenty healthy, adult

volunteers received 4.05 g per day of mozuku fucoidan (isolated from  Cladosiphon okamuranus) for

two weeks. The blood samples showed a significant decrease in total cholesterol and low-density

lipoprotein cholesterol. In turn, the concentration of chloride ions increased significantly. There were

no changes in the parameters describing the liver and kidneys [33]. Similarly, it was shown in patients

with osteoarthritis that application of fucoidan from  Fucus vesiculosus at the dose 300 mg or from  Fucus vesiculosus,  Macrocystis pyrifera, and  Laminaria japonica at 100 mg and 1000 mg did not aect the blood tests results or these changes were clinically negligible [34,35].

Safety of fucoidan was also eval‎uated at subcutaneous administration. Fucoidan from  Fucus

vesiculosus and  Laminaria japonica (molecular weight 10–300 kDa) administered subcutaneously and

orally did not cause negative changes in dogs with hemophilia A. Moreover, it also led to improvement

of their hemostasis [36]. Similar conclusions about safety of fucoidan have been drawn from a study

conducted on horses. The solution containing 2500 mg of fucoidan administered intraperitoneally

during abdominal surgery did not have a negative impact on the results of laboratory tests. Most of

the parameters did not dier between the test and the control groups, and the observed dierences

in the amount of leukocytes, neutrophils, antithrombin III, value of hematocrit, or prothrombin time

were within the normal range [37].

Furthermore, the genotoxicity of high and low molecular weight fucoidan was also investigated

with using both in vitro and in vivo model. In bacterial reverse mutation and chromosome aberration

tests fucoidan from  Undaria pinnatifida (regardless of the molecular weight) did not show toxic eect

Mar. Drugs 2019, 17, 458 5 of 20

in concentrations 5000 g/plate and 5000 g/mL, respectively. Moreover, in micronucleus assay

conducted in ICR mice, fucoidan at dosages up to 2000 mg/kg b.w. per day did not display evidence

for genotoxicity [38,39].

4. Fucoidan Application in the Pharmaceutical Technology

Fucoidan plays an important role in the design of various dosage forms (Figure 2), especially

nanoparticles and microparticles, films, or hydrogels [4,5,24]. The wide application of fucoidan in

the pharmaceutical technology is due to its ionic nature. Negatively charged polysaccharide allows

the formation of complexes with other, oppositely charged molecules. Polyelectrolyte complexation

is the most commonly used technique for obtaining particles utilizing fucoidan. Other methods are

coacervation, ionic cross-linking, self-assembly, and spray-drying [2]. Fucoidan-based particles served

as carriers of various substances, including drugs (ciprofloxacin [40], gentamicin [41], doxorubicin [42],

isoniazid and rifabutin [43]), growth factors (basic fibroblast growth factor—bFGF [44], proteins (bovine

serum albumin [45], -lactoglobulin [46]), and genes [47]. Interestingly, fucoidan is used not only as an

excipient responsible for drug delivery, but also as a proper substance with therapeutic eects [12,48].

Mar. Drugs 2019, 17, x 5 of 21

conducted in ICR mice, fucoidan at dosages up to 2000 mg/kg b.w. per day did not display evidence

for genotoxicity [38,39].

4. Fucoidan Application in the Pharmaceutical Technology

Fucoidan plays an important role in the design of various dosage forms (Figure 2), especially

nanoparticles and microparticles, films, or hydrogels [4,5,24]. The wide application of fucoidan in the

pharmaceutical technology is due to its ionic nature. Negatively charged polysaccharide allows the

formation of complexes with other, oppositely charged molecules. Polyelectrolyte complexation is

the most commonly used technique for obtaining particles utilizing fucoidan. Other methods are

coacervation, ionic cross‐linking, self‐assembly, and spray‐drying [2]. Fucoidan‐based particles

served as carriers of various substances, including drugs (ciprofloxacin [40], gentamicin [41],

doxorubicin [42], isoniazid and rifabutin [43]), growth factors (basic fibroblast growth factor—bFGF

[44], proteins (bovine serum albumin [45], β‐lactoglobulin [46]), and genes [47]. Interestingly,

fucoidan is used not only as an excipient responsible for drug delivery, but also as a proper substance

with therapeutic effects [12,48].

Figure 2. Schematic presentation of pharmaceutical dosage forms designed with fucoidan

utilization.

4.1. Nanoparticles

Nanoparticles are usually spherical particles with diameters from 10 to 1000 nm. Depending on

the composition and the method of locating active substance in the nanoparticle, nanocapsules and

nanospheres can be distinguished. In nanospheres the drug is homogenously dissolved or suspended

in a polymer matrix. In turn, nanocapsules are the systems in which the drug is enclosed in a reservoir

surrounded by a polymer membrane. Nanoparticles are valuable multicompartment carriers in

modern pharmaceutical technology as they provide protection of the drug substance against

chemical and enzymatic degradation, and enable controlled or targeted drug release. As a

consequence, lower concentration of active substance is required to achieve a therapeutic effect,

which contributes to the reduction of side effects [4]. Examples of fucoidan‐based nanoparticles are

presented in Table 2. To obtain nanoparticles with fucoidan utilization, self‐assembly, and ionotropic

cross‐linking is commonly used. Self‐assembly method is a process in which participating molecules,

as a result of interactions between them, organize themselves into specific structures without

introducing additional elements [49]. The ionotropic cross‐linking is based on the interaction of the

negatively charged fucoidan with the positively charged polymer molecule (e.g. chitosan) [50].

Figure 2. Schematic presentation of pharmaceutical dosage forms designed with fucoidan utilization.

 4.1. Nanoparticles

Nanoparticles are usually spherical particles with diameters from 10 to 1000 nm. Depending

on the composition and the method of locating active substance in the nanoparticle, nanocapsules

and nanospheres can be distinguished. In nanospheres the drug is homogenously dissolved or

suspended in a polymer matrix. In turn, nanocapsules are the systems in which the drug is enclosed

in a reservoir surrounded by a polymer membrane. Nanoparticles are valuable multicompartment

carriers in modern pharmaceutical technology as they provide protection of the drug substance

against chemical and enzymatic degradation, and enable controlled or targeted drug release. As a

consequence, lower concentration of active substance is required to achieve a therapeutic eect, which

contributes to the reduction of side eects [4]. Examples of fucoidan-based nanoparticles are presented

in Table 2. To obtain nanoparticles with fucoidan utilization, self-assembly, and ionotropic cross-linking

is commonly used. Self-assembly method is a process in which participating molecules, as a result of

interactions between them, organize themselves into specific structures without introducing additional

elements [49]. The ionotropic cross-linking is based on the interaction of the negatively charged

fucoidan with the positively charged polymer molecule (e.g. chitosan) [50].

Mar. Drugs 2019, 17, 458 6 of 20

Table 2. Characteristic of selected fucoidan-based nanoparticles.

Fucoidan

(Source/Modification/Molecular

Weight)

Copolymer/Positive

Charge Donor Drug Method of Obtaining Application Route of

Administration Ref.

Acetylated fucoidan

(Fucus vesiculosus) - Doxorubicin Self-assembly and

dialysis

Anticancer therapy

and immunotherapy NA1 51

Fucoidan

(Laminaria japonica, 80 kDa) Protamine Doxorubicin Self-assembly Anticancer therapy Intravenous 52

Fucoidan (Fucus vesiculosus) Polyethyleneimine Doxorubicin Polyelectrolyte

complexation method Anticancer therapy Intravenous 53

Fucoidan (Fucus vesiculosus) Gold nanoparticles Doxorubicin Electrostatic

physisorption Anticancer therapy Ocular 42

Fucoidan (Fucus vesiculosus) Polyallylamine

hydrochloride Copper sulfide Layer-by-layer Anticancer therapy Intratumoral 54

Fucoidan (200–400 kDa) Polyallyamine

hydrochloride Methotrexate Self-assembly Anticancer therapy NA1 55

Fucoidan (Fucus vesiculosus) Chitosan - Coacervation Thrombolytic therapy Oral 56

Fucoidan

(Fucus vesiculosus, 50–190 kDa) Chitosan Methotrexate Self-assembly Skin inflammation Topical (ear skin) 57

Fucoidan Chitosan Curcumin Self-assembly Anticancer therapy Oral 58

Fucoidan (Fucus vesiculosus) O-carboxymethyl chitosan Curcumin Ionotropic crosslinking Penetration enhancer Oral 59

Thiolated fucoidan

(THL-fucoidan) Arginine-modified chitosan Dextran/rhodamine/

curcumin Self-assembly NA1 Oral 60

Fucoidan (Fucus vesiculosus) Chitosan Gentamicin Self-assembly Pulmonary diseases Pulmonary 41

Fucoidan (Fucus vesiculosus) Chitosan Gentamicin Ionotropic crosslinking Pulmonary diseases Pulmonary 50

Fucoidan (Fucus vesiculosus) Chitosan Silver nitrate Self-assembly Antibacterial and

anticancer therapy NA1 61

Fucoidan (20–200 kDa) TPP crosslinked chitosan Ciprofloxacin Self-assembly Infections of

Salmonella NA1 40

Fucoidan

(Fucus vesiculosus, 57.26 kDa) Chitosan Poly-l-lysine Layer-by-layer Antibacterial therapy NA1 62

Fucoidan

(Fucus vesiculosus, 5–50 kDa) Trimethyl chitosan Insulin Self-assembly Diabetes Oral 63

Fucoidan

(Fucus vesiculosus, 80 kDa) Chitosan Basic fibroblast

growth factor Ionotropic crosslinking Neurite extension Nerve tissue 44

Mar. Drugs 2019, 17, 458 7 of 20

Table 2. Cont.

Fucoidan

(Source/Modification/Molecular

Weight)

Copolymer/Positive

Charge Donor Drug Method of Obtaining Application Route of

Administration Ref.

Fucoidan (104 kDa) Isobutylcyanoacrylate Recombinant tissue

plasminogen activator

Redox radical emulsion

polymerization Thrombolytic therapy Retro-orbital

(C57BL/6 mice) 64

Fucoidan (Sargassum cymosum) Isobutylcyanoacrylate -

Anionic emulsion

polymerization and

redox radical emulsion

polymerization

Immunotherapy NA1 65

Fucoidan

Poly(lactide-co-glycolide)

and poly-l-ornitine

(core-shell)

- Layer-by-layer Anticancer therapy NA1 66

Fucoidan (Fucus vesiculosus,

20–200 kDa) - Cisplatin Self-assembly Anticancer therapy

and immunotherapy

Colonic drug

delivery system 67

Fucoidan (Spatoglossum

schr˝oederi, 21 kDa) Hexadecylamine - Self-assembly Anticancer therapy NA1 68

1NA—No data available.

Mar. Drugs 2019, 17, 458 8 of 20

As shown in Table 2, various nanoparticles were tested to determine fucoidan potential in

anticancer drug delivery. As model antitumor drugs, doxorubicin (DOX), copper sulfide (CuS),

methotrexate (MTX), curcumin (CUR), silver nitrate (AgNO3), and cisplatin (CSN) were studied.

In 2013, Lee et al. developed self-organized nanoparticles with acetylated fucoidan from  Fucus

vesiculosus. DOX was introduced into the nanoparticles through dialysis and it was released according

to the first order kinetics. Fucoidan was tested as a drug carrier due to its immunomodulatory

properties, ability to produce anticancer cytokines and drug eux pump inhibition [51]. In turn, Lu et

al. created nanoparticles by applying electrostatic interactions between the negatively charged fucoidan

and the cationic peptide–protamine. The self-assembled complex was stable at pH 7.4 (corresponding

to blood pH). When pH decreased to 4.5 (tumor cell pH) or 1.5 (stomach pH), electrostatic interactions

became weaker, which led to increased diameter of nanoparticles and release of more than 90% of

anticancer agent. The release of a smaller amount of DOX in the blood prevents side eects and

increases its concentration in tumor cells. This pH-dependent release profile makes intravenous

injection the best route for administration of these nanoparticles. Furthermore, using fucoidan that

possesses the ability to interact with P-selectin present in the breast cancer cells (MDA-MB-231),

resulted in the improved cell internalization and better inhibitory eect of designed nanoparticles

than free DOX [52]. The polyelectrolyte complexing method (PEC) was used to combine fucoidan

with polyethyleneimine (PEI). Similarly to the previously described study, when the medium pH was

decreased, the increased release rate of DOX was noted. In vivo studies were performed using BALB/c

mice with induced breast tumor. The results demonstrated that intravenously injected nanoparticles

with DOX were characterized by stronger antiproliferative properties and higher tumor inhibitory

activity than free DOX. The beneficial eects on the antitumor activity of the obtained nanoparticles

were caused probably by immunomodulatory properties of fucoidan, which were confirmed in the

in vitro studies [53]. In addition, fucoidan is characterized by the ability to improve the stability

of metallic nanoparticles thereby contributing to the reduction of their toxicity. Fucoidan-coated

gold nanoparticles with DOX caused a significant decrease in rabbit squamous carcinoma cells (VX2)

viability. The most significant decrease in viability of the examined cells was observed after using both

the discussed nanoparticles and laser irradiation. Similar conclusions were drawn from the in vivo

studies conducted in rabbits with eye tumor. Only in the group treated with nanoparticles and laser,

complete eradication of the tumor was observed after 6 days, and most importantly, there was no

relapse after 14 days [42]. In turn, Jang et al. used the layer-by-layer technique for the synthesis of CuS

nanoparticles alternately coated with fucoidan and polyallylamine hydrochloride (PAH). Based on

the obtained results, it was found that the applied nanoparticles showed stronger anticancer eect

than their components used separately. Obtained nanoparticles not only improved the intracellular

transport of fucoidan, which possesses the ability to induce apoptosis, but it also provided favorable

photothermal features. This was confirmed in the in vitro studies using human cervical cancer cells

(HeLa) and human lung adenocarcinoma cells (A549), and in thein vivo studies in mice injected with

these cells [54]. The same ingredients (fucoidan and PAH) were used to create nanoparticles with

MTX via self-assembly method. MTX was released according to the first order kinetics and it showed

stronger in vitro inhibition of HeLa and MCF-7 cell proliferation than unbounded drug. It should be

noted that fucoidan and PAH complex was biocompatible and did not aect cells viability [55].

In nanoparticles the combination of fucoidan and chitosan or its derivatives is very commonly

used, which is related to the properties of both polysaccharides. The positive charge of chitosan

allows electrostatic interactions with sulfate groups of fucoidan, leading to the formation of complexes.

In addition, chitosan is characterized by antifungal and antibacterial activity. Due to its ability to

muco-adhesion and overcoming epithelial barriers, it facilitates the transport of active substances.

Importantly, similar to fucoidan, it is biocompatible and its acquisition costs are relatively low [40,56,57].

In 2012, Silva et al. compared the activity of fucoidan–chitosan nanoparticles with the eect of fucoidan

solution. Nanoparticles obtained by coacervation method were biocompatible with human epithelial

cells (Caco-2) from colon adenocarcinoma and had the ability to open tight junctions between them.

Mar. Drugs 2019, 17, 458 9 of 20

As a result, nanoparticles showed better permeability and stronger anticoagulant eect than the

fucoidan solution per se [56]. Applying self-assembly method, Barbosa et al. produced nanoparticles

and as the positive charge provider, chitosan was used. MTX loaded nanoparticles were nontoxic for

both fibroblasts and keratinocytes when drug concentration did not exceed 50 g/mL. The strongest

anti-inflammatory activity manifested by the inhibition of Il1-, Il-6, and TNF- production and the

greatest penetration of the active substance through the pig’s ear skin provided formulation with the

highest fucoidan:chitosan ratio (5:1) [57]. Using the same electrostatic interactions between fucoidan

and chitosan, Huang et al. created pH-sensitive nanoparticles for oral administration of CUR, which

is characterized by limited bioavailability due to its poor solubility and sensitivity to environmental

conditions of the gastrointestinal tract. Whereas at pH 1.2 CUR release was inhibited, at pH 7.0

significant increase in drug release was observed. Hence, the discussed nanoparticles can be considered

as a carrier enabling oral administration of CUR, providing protection against degradation in the

stomach and adequate bioavailability after absorption in the intestine [58]. Similar conclusions were

also drawn from the studies in which CUR was placed in nanoparticles obtained from fucoidan and

o-carboxymethylchitosan by ionic crosslinking. Controlled release of CUR in the gastrointestinal tract,

its reduced cytotoxicity to mouse fibroblasts cells (L929), and increased cellular uptake by Caco-2

cells constitute the positive eects resulting from CUR encapsulation in nanoparticles [59]. By using

thiolated fucoidan and arginine-modified chitosan it was also possible to obtain nanoparticles with

dextran (DTX) or CUR. Due to the fact that utilized polysaccharides possess the ability to open

tight junctions in intestinal Caco-2 cell monolayer, and thiolated fucoidan inhibits the activity of the

P-glycoprotein pump, the penetration of both drugs were significantly better. Encapsulation of CUR as

model hydrophobic compound into nanoparticles improved its water solubility and stability [60].

To deliver antibiotics to the lungs, fucoidan and chitosan composed nanoparticles via simple

self-assembly method were designed. Antioxidant activity of the obtained nanoparticles (confirmed

by the test of reactive oxygen species with lipopolysaccharides and by the method of scavenging

1,1-diphenyl-2-picrylhydrazyl radicals), stability in phosphate buered saline, no negative eect on

the viability of A549 cells at a concentration from 0.37 mg/mL to 3 mg/mL and controlled release of

gentamicin make them an auspicious pulmonary drug delivery system [41]. Similar conclusions were

drawn from the study in which nanoparticles with gentamycin were obtained by ionotropic crosslinking

and applied intratracheally. The biphasic antibiotic release profile, as well as the antibacterial and

antioxidant properties of fucoidan, resulted in stronger growth inhibition of  Klebsiella pneumoniae,

simultaneously limiting the potential nephrotoxic and ototoxic eects of gentamicin [50].

The protective properties of fucoidan were used to provide stability for silver nanoparticles. The

fucoidan–chitosan complex coated silver nanoparticles, produced by self-assembly method, showed

antibacterial activity both against Gram-positive ( Staphylococcus aureus) and Gram-negative ( Escherichia

coli) bacteria. Moreover, the use of nanoparticles at a concentration of 50 g/mL showed considerable

cytotoxicity to HeLa cells [61]. In turn, Elbi et al. investigated the activity of ciprofloxacin loaded into

chitosan nanoparticles obtained by ionic crosslinking using tripolyphosphate and subsequently coated

with fucoidan. As fucoidan reacts with scavenger macrophages receptors, the obtained nanoparticles

were characterized by better cellular uptake and thus led to more eective eradication of  Salmonella.

Research conducted on infected by  Salmonella Drosophila melanogaster confirmed that ciprofloxacin

(in the form of free drug, chitosan nanoparticles, or fucoidan coated chitosan nanoparticles) at

concentrations four times higher than its minimum inhibitory concentration (MIC) did not aect

the survival of the flies. However, the use of antibiotics at a concentration four times higher than

minimum bactericidal concentration (MBC) caused the greatest survival rate of flies treated with the

drug in the form of particles coated with fucoidan. Moreover, in this group the greatest decrease in

bacterial load was noted, which confirmed the stronger antibacterial activity of ciprofloxacin enclosed

in fucoidan coated chitosan nanoparticles, both in comparison to the free drug and to the form of

chitosan nanoparticles [40]. By using the layer-by-layer method, Pinheiro et al. received nanoparticles

made of alternating layers of chitosan and fucoidan deposited on polystyrene core. After removing

Mar. Drugs 2019, 17, 458 10 of 20

the polystyrene element, nanoparticles were filled with poly-l-lysine (PLL), which is characterized

by antibacterial properties. The satisfactory encapsulation eciency (about 45%) obtained by the

adsorption of PLL on the core before its removal, as well as the pH-dependent release of the active

ingredient from the nanoparticles, make them a promising drug carrier [62].

The peptide therapy is becoming increasingly important in medicine and pharmacy. However, due

to poor bioavailability, which results from peptide degradation in digestive tract and poor absorption,

oral administration is limited. Tsai et al. have recently developed nanoparticles with trimethyl chitosan

and fucoidan as carrier for insulin. Greater insulin protection against pH changes and better penetration

across the intestinal epithelium was observed. It was also shown that fucoidan possesses the ability to

inhibit -glucosidase activity. Obtained nanoparticles at concentration of 2 mg/mL inhibited activity of

this enzyme at ratio of 33.2% [63]. Additionally, the combination of fucoidan and chitosan provided

protection for the basic fibroblast growth factor (bFGF). Nanoparticles were obtained by the ionotropic

cross-linking. The eectiveness of encapsulation (EE), due to the ability of fucoidan to bind amino acids

on the surface of bFGF, increased with increasing fucoidan content (for nanoparticles prepared from

chitosan and fucoidan in a ratio of 10:1 and 1:10, EE was 80.9% and 90.4%, respectively). Moreover, the

same relationship regarding the contribution of fucoidan was observed with respect to the protection

of bFGF by enzymatic degradation and temperature inactivation. Nanoparticles at a concentration of

125 ng/mL showed no toxicity to pheochromocytoma PC12 cells, and due to the fucoidan and chitosan

content therein, the amount of bFGF required to neurite extension was significantly lower than free

bFGF [44].

It is worth noting that biological activity of nanoparticles built from fucoidan depends on the

properties of this polysaccharide and the form in which it was used. In 2017, Juenet et al. obtained

by redox radical emulsion polymerization (RREP) fucoidan-based nanoparticles. Due to the use of

fucoidan, which has an anity for P-selectin present on thrombocytes, the ability of nanoparticles

to bind to the activated plates was greater. The fluorescence intensity observed after perfusion of

both the fucoidan coated nanoparticles without recombinant tissue plasminogen activator (rt-PA) and

with it was significantly higher than those in which no fucoidan was used. In addition, nanoparticles

containing rt-PA and fucoidan showed the strongest thrombolytic activity in studies conducted in

C57BL/6 mice. The thrombus density after their application decreased to about 30%, whereas after

injecting nanoparticles without fucoidan, the value of this parameter was about 66%. It was found

that nanoparticles not only protected rt-PA, but because of the ability of fucoidan to imitate P-selectin

glycoprotein ligand-1, they improved its thrombolytic activity [64].

In turn, Lira et al. compared the properties of isobutylcyanoacrylate (IBCA) nanoparticles coated

with fucoidan, which were obtained by two dierent methods. Anionic emulsion polymerization (AEP)

allows the creation of stable formulations without the addition of DXT, whereas in the case of REEP

only those containing up to 25% of fucoidan in the polysaccharide mixture were stable. Cytotoxicity

was assessed after 48 h of nanoparticles incubation with macrophages J774 or fibroblasts NIH-3T3.

The method of obtaining nanoparticles had a major impact on the cytotoxicity of nanoparticles only

in relation to macrophages J774, because the IC50 value for nanoparticles formulated by AEP was

1.3  0.2 g/mL, and by REEP 9.6  0.5 g/mL. Interestingly, distribution of nanoparticles in these cells

was dependent not on the method used for their production, but on fucoidan presence. Fucoidan did

not aect the interaction of nanoparticles with fibroblast cells, but it also modulated nanoparticles

uptake and distribution within macrophages [65]. The safety of using nanoparticles was assessed

by Cai et al. Core of nanoparticles was made of poly(lactic-co-glycolic) acid (PLGA) and then it was

covered with alternating layers of poly-L-ornithine (PLO) and fucoidan using the layer-by-layer (LbL)

technique. Even at concentration of 100 g/mL, they did not aect the morphology and proliferation

ratio of breast epithelium (MCF-10A) cells. In addition, the hemolysis test on fresh blood from rabbits

proved that by closing the PLGA in the capsule made of fucoidan and PLO, its biocompatibility

increased (hemolysis rate for LbL and PLGA nanoparticles at concentration 100 g/mL was 2.67%

and 3.85%, respectively). Destructive eects on the liver, changes in nutrition and weight were not

Mar. Drugs 2019, 17, 458 11 of 20

observed in mice after intraperitoneal injection of these nanoparticles at concentration of 10 mg/mL.

The obtained results confirmed that such connection of fucoidan and PLO, not only protected active

ingredient, but also improved its biocompatibility [66].

Interestingly, Hwang et al. prepared nanoparticles for colonic drug delivery by combining CSN

and pure fucoidan. In order to obtain nanoparticles, two methods were used, diering in the order of

dissolution of the components. Appropriate amounts of fucoidan were dissolved in 2 mg/mL CSN

solution, or appropriate amounts of CSN were dissolved in 10 mg/mL fucoidan solution. Fucoidan

nanoparticles tested as CSN carrier not only decreased toxicity of CSN against RAW264.7 macrophage

cells, but they also improved its cytotoxicity in HCT-8 tumor cells [67].

Fucoidan was used to form shell of nanostructures dispersible in aqueous media. The source

of polymer composed of (1-3)-glucuronic acid substituted at the C4 position with fucose was the

brown seaweed  Spatoglossum schroederi. Chemical modification with hexadecylamine allowed to

produce amphiphilic nanoparticles stable in water for up to 70 days. In order to determine the use

of nanoparticles in the treatment of tumors, their eect on the proliferation and viability of various

cell lines at 50, 100, and 500 g/mL was eval‎uated by MTT test. It is based on the reduction of the

water-soluble MTT dye, which is a triazole salt (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium

bromide) to an insoluble dark blue formazan, the amount of which is proportional to the number of

viable cells. This study showed that the produced nanoparticles did not aect Chinese hamster ovary

(CHO) and mouse monocyte macrophage (RAW) cells. Antiproliferative activity was noted against

rabbit aorta endothelial cells (RAEC) and all tested tumor cells: human renal cell carcinoma (786),

human hepatocellular carcinoma cells (HepG2) and human marrow stromal cells (HS-5) [68].

 4.2. Liposomes

Liposomes are specific type of structures, which size usually does not exceed 100 nm. The core

formed by the aqueous environment is surrounded by the phospholipid bilayer. The main reason for the

use of liposomes is the possibility of targeted therapy, which in addition to the improved eectiveness

of the treatment, allows to reduce drug side eects [69]. The antitumor eect of LMWF encapsulated in

liposomes was compared to the activity of fucoidan with molecular weight 2–10 kDa and 80 kDa using

human osteosarcoma cells (143B). It was shown that apoptosis induction by activating caspase pathway

was significantly stronger than by native fucoidan (95% and 25%, respectively). Similar conclusions

were observed in studies in C3H mice that were previously injected with murine osteosarcoma cells

(LM8). In the group receiving orally for 28 days liposomes with fucoidan or unprocessed fucoidan in

the amount of 100 mg/kg per day, there was a significant decrease in both volume and weight of the

tumor compared to mice receiving only water. However, better results were noted when fucoidan

liposomes were applied. Furthermore, liposomes were characterized by better penetration through the

Caco-2 cell layer. In turn, native fucoidan more strongly inhibited spontaneous metastases to the lungs.

None of the forms (unprocessed fucoidan and fucoidan liposomes) led to negative changes in animal

weight [70].

 4.3. Microparticles

The term microparticles refers to particles with sizes ranging from 1 m to 1000 m. Analogously,

as in the nanoparticles discussed above, among these group of particles microspheres and microcapsules

can be distinguished. Microspheres prepared with fucoidan utilization are termed ‘fucospheres’.

The morphology of fucospheres developed by our research team is shown in Figure 3. They were

successfully obtained by the spray drying of 3% aqueous fucoidan solution (Table 1) using the Mini

Spray Dryer B-290 (Büchi, Flawil, Switzerland) with experimental parameters set as follows: inlet

temperature 122 C, outlet temperature 62 C, pressure 60 mmHg, aspirator blower capacity 100%,

feed rate 2.1 mL/min.

Mar. Drugs 2019, 17, 458 12 of 20

Mar. Drugs 2019, 17, x 12 of 21

(a)

(b)

Figure 3. Scanning electron microscope (SEM) pictures of fucospheres: (a) magnification °ø5000; (b)

magnification °ø20,000.

According to the literature, preparation of fucoidan‐based microparticles is usually performed

with additional copolymers or positive charge donors (Table 3). In 2006, Sezer et al. proposed the use

of fucospheres obtained by interaction between the negatively charged fucoidan and positively

charged chitosan and, as a model protein, bovine serum albumin (BSA) was used. It was noted that

with increasing fucoidan content in the fucospheres, BSA encapsulation capacity increased and the

value of zeta potential decreased. Formulated microspheres provided three‐phasic BSA release

profile [45]. Interactions between fucoidan and chitosan were also used to create fucospheres for the

treatment of dermal burns in rabbits. As in the previous study, the size of the fucospheres obtained

by polyion complexation increased with the increase in the polymer concentration. Wounds that were

treated with fucospheres, were characterized by the highest epithelial thickness. The most relevant

difference was observed on day 7th, when the value of epithelial thickness was 193, 121, 118, and 111

μm, for fucospheres, fucoidan solution, chitosan microspheres and untreated group, respectively.

Similarly, epithelial lengths increased with time, and on the 14th day of therapy the highest values

were observed in the group treated with fucospheres (2733, 2086, 2316, and 1950 μm, as above). The

fastest skin regeneration after burns was noted in the group treated with fucospheres due to the effect

of fucoidan on the migration of fibroblasts, release of growth hormones and cytokines involved in reepithelization.

In addition, based on this study, it was concluded that fucoidan and chitosan have a

synergistic effect on the treatment of skin burns [48].

Li et al. designed poly(alkylcyanoacrylate) (PACA) microcapsules with fucoidan layer on their

surface. The use of fucoidan was due to its ability to detect thrombosis by binding to P‐selectin. The

microparticles produced by emulsion–evaporation polymerization process were filled with contrast

agent (perfluorooctylbromide—PFOB) used in various imaging diagnostics (ultrasonography, The safety of microparticles application was

determined 3T3 mouse fibroblast cells. Even at concentration of 5 g/L, there was

no difference in cell viability when using microcapsules with and without fucoidan. Additionally,

two in vitro studies were performed to eval‎uate their binding capacity to P‐selectin. Both in the static

binding on human activated platelets and in the flow chamber assay, microparticles with fucoidan

showed significant adhesive properties. Moreover, designed microcapsules were administered

intravenously to healthy Wistar rats and rats with abdominal aortic aneurysm. The strongest

fluorescence was observed in the animals treated with microcapsules with fucoidan but only in the

affected area. Weaker bonds of both types of microparticles were noted in healthy blood vessels. It

was concluded that fucoidan microcapsules seem to be valuable targeting carrier candidate for drug

Figure 3. Scanning electron microscope (SEM) pictures of fucospheres: (a) magnification 5000;

(b) magnification 20,000.

According to the literature, preparation of fucoidan-based microparticles is usually performed

with additional copolymers or positive charge donors (Table 3). In 2006, Sezer et al. proposed the use of

fucospheres obtained by interaction between the negatively charged fucoidan and positively charged

chitosan and, as a model protein, bovine serum albumin (BSA) was used. It was noted that with

increasing fucoidan content in the fucospheres, BSA encapsulation capacity increased and the value of

zeta potential decreased. Formulated microspheres provided three-phasic BSA release profile [45].

Interactions between fucoidan and chitosan were also used to create fucospheres for the treatment of

dermal burns in rabbits. As in the previous study, the size of the fucospheres obtained by polyion

complexation increased with the increase in the polymer concentration. Wounds that were treated

with fucospheres, were characterized by the highest epithelial thickness. The most relevant dierence

was observed on day 7th, when the value of epithelial thickness was 193, 121, 118, and 111 m, for

fucospheres, fucoidan solution, chitosan microspheres and untreated group, respectively. Similarly,

epithelial lengths increased with time, and on the 14th day of therapy the highest values were observed

in the group treated with fucospheres (2733, 2086, 2316, and 1950 m, as above). The fastest skin

regeneration after burns was noted in the group treated with fucospheres due to the eect of fucoidan

on the migration of fibroblasts, release of growth hormones and cytokines involved in re-epithelization.

In addition, based on this study, it was concluded that fucoidan and chitosan have a synergistic eect

on the treatment of skin burns [48].

Li et al. designed poly(alkylcyanoacrylate) (PACA) microcapsules with fucoidan layer on their

surface. The use of fucoidan was due to its ability to detect thrombosis by binding to P-selectin.

The microparticles produced by emulsion–evaporation polymerization process were filled with

contrast agent (perfluorooctylbromide—PFOB) used in various imaging diagnostics techniques

(ultrasonography, magnetic resonance imaging). The safety of microparticles application was

determined by MTT test using 3T3 mouse fibroblast cells. Even at concentration of 5 g/L, there was no

dierence in cell viability when using microcapsules with and without fucoidan. Additionally, two

in vitro studies were performed to eval‎uate their binding capacity to P-selectin. Both in the static binding

on human activated platelets and in the flow chamber assay, microparticles with fucoidan showed

significant adhesive properties. Moreover, designed microcapsules were administered intravenously to

healthyWistar rats and rats with abdominal aortic aneurysm. The strongest fluorescence was observed

in the animals treated with microcapsules with fucoidan but only in the aected area. Weaker bonds

of both types of microparticles were noted in healthy blood vessels. It was concluded that fucoidan

Mar. Drugs 2019, 17, 458 13 of 20

microcapsules seem to be valuable targeting carrier candidate for drug substances or contrast agents

for the treatment or imaging of diseases that are accompanied by overexpression‎ of P-selectin [71].

Fucoidan microparticles are also examined as potential drug carriers in the cancer treatment.

In 2017,Wang et al. used the layer-by-layer self-assembly technique to develop microparticles with

DOX. After coating the core made of calcium carbonate with PLO and fucoidan, they were loaded

with DOX at a high, nearly 70% encapsulation eciency. Designed microparticles were characterized

by biocompatibility—they did not aect the survival of C2C12 mouse myoblasts (at concentration of

400 g/mL cell viability was about 90%). In addition, the hemolysis rate at 200 g/mL was lower than

3%. A study conducted at pH 7.4 showed prolonged DOX release, and antitumor tests on breast cancer

cells (MCF-7) confirmed its eective release and ability to inhibit cells more than free DOX [72].

Fucoidan microparticles are also tested as antibiotics carriers. Sezer et al. compared the properties

of fucospheres and chitosan microspheres, obtained by polyion complexation and precipitation

methods, respectively. Ofloxacin (OFL) encapsulation eciency (EE) was greater in fucospheres than

chitosan microparticles (EE values 63.9%–94.8% and 48.8%–89.2%, respectively). The use of fucoidan

caused a slower OFL release, consistent with the Higuchi kinetics model [73]. Recently, Cunha et al.

conducted studies on the use of fucoidan-based microparticles in the treatment of tuberculosis. This is

the result of fucoidan ability to recognize macrophages in the alveoli, which allows the delivery of

drugs to the aected areas and leads to an increase in the eectiveness of the therapy. The microparticles

containing isoniazid (INH) or rifabutin (RFB) produced by the spray-drying with high yield (75%–85%)

were characterized by the appropriate aerodynamic properties measured using the Andersen cascade

impactor (ACI). The mass median aerodynamic diameter of particles with isoniazid was about 3.78 m,

and with rifampicin 1.99 m. The fraction of fine particles tested with the dry powder inhaler type

RS01, showing the most eective penetration to the lungs, was 39% and 55%, respectively. Due to the

wrinkled surface of the microparticles and the good solubility of fucoidan, the total release of both

drugs occurred in a short time (about 15 minutes), regardless of the medium pH and the solubility of

the free drug. It is worth noting that MTT tests proved that microencapsulation reduces the toxicity

of loaded RFB, both in relation to human alveolar epithelium (A549) and human monocytic (THP-1)

cells. The viability of A549 and THP-1 cells after 24 h incubation with RFB at a concentration of 0.05

mg/mL was 50% and 49%, respectively. In turn, the use of the microparticles with RFB increased cells

viability after 24 h incubation even up to 90% (A549) and to around 70% (THP-1). However, unloaded

fucoidan-based microparticles, free fucoidan, and free INH did not show cytotoxicity [43]. Very similar

results were noted with reference to fucoidan-based microparticles being a combination of INF and

RFB. The spray drying technique allowed to obtain product with a yield of over 80%. By using the

RS01 inhaler in the ACI, it was found that product exhibited proper aerodynamic properties. In studies

in vitro it was reported that microparticles did not aect the viability of A549 cells and their viability

was classified above 70%, regardless the incubation time. THP-1 cells showed greater sensitivity and

at concentration 1.0 mg/mL after 24 h of exposure, the viability decreased to 65%. Viability of A549

and THP-1 cells after 3 h incubation with free RFB (0.05 mg/mL) was 68% and 56%, whereas with

microparticles (1.0 mg/mL) containing INH and RFB at mass ratio 1:0.5, 84% and 77%, respectively.

It is worth noting that the significant uptake of microparticles by macrophage-like cells (up to 87%)

along with their simultaneous stimulation for the production of cytokines involved in the fight against

pathogen was associated with the presence of fucoidan [74].

 4.4. Semi-Solid Formulations

Hydrogels are three-dimensional formulations made of hydrophilic polymers with appropriate

structure and properties. They are widely used in biomedicine, for example as wound dressings,

implants, drug delivery systems, or tissue regeneration materials [75].

Mar. Drugs 2019, 17, 458 14 of 20

Table 3. Characteristic of selected fucoidan-based microparticles.

Fucoidan (Source/Molecular

Weight)

Copolymer/Positive

Charge Donor Drug Method of Obtaining Application Route of

Administration Ref.

Fucoidan

(Fucus vesiculosus, 80 kDa) Chitosan Bovine serum albumin Ionotropic cross-linking Peptide and protein

delivery NA 1 45

Fucoidan

(Fucus vesiculosus, 80 kDa) Chitosan - Polyion complexation Treatment of dermal burns Topical 48

Fucoidan Poly(alkylcyanoacrylate)

and dextran Perfluorooctylbromide Emulsion-evaporation

polymerization Targeting carrier Intravenous 71

Fucoidan (200–400 kDa) Poly-l-ornithine (shell);

calcium carbonate (core) Doxorubicin Layer-by-layer

self-assembly Anticancer therapy NA 1 72

Fucoidan

(Fucus vesiculosus, 80 kDa) Chitosan Ofloxacin Polyion complexation Antibiotics carriers NA 1 73

Fucoidan

(Laminaria japonica,

598.4 Da–0.598 kDa)

- Isoniazid or rifabutin Spray-drying Tuberculosis therapy Pulmonary 43

Fucoidan (Laminaria japonica) - Isoniazid and rifabutin Spray-drying Tuberculosis therapy Pulmonary 74

1 No data available.

Mar. Drugs 2019, 17, 458 15 of 20

Unique properties, such as anticoagulant and anti-inflammatory activity make fucoidan a useful

component in the formation of hydrogels. Sezer et al. proposed a hydrogel obtained by combining

fucoidan with an oppositely charged chitosan. An important feature of hydrogels is the ability to

absorb exudate and to provide adequate moisture. It turns out that formulations with fucoidan

addition were characterized by greater possibility of water absorption and higher swelling ratio than

pure chitosan gels. Electrostatic interactions between polymers significantly aected the hardness of

the gels, which increased with the increase of fucoidan concentration. Similarly to the parameters

discussed above, formulations containing the highest concentrations of polymers (0.75% fucoidan

and 2% chitosan) showed the greatest cohesiveness and adhesion values. Stronger mucoadhesive

properties were also associated with the content of fucoidan. Hydrogels containing only chitosan

and chitosan with the addition of 0.25% or 0.75% fucoidan showed the following values of work of

adhesion 23, 62, and 142 J/cm2, respectively. The eectiveness of wound healing using the designed

hydrogels has also been eval‎uated in vivo. In a study conducted on New Zealand rabbits statistically

significantly higher values of the length and thickness of the epithelium were observed in wounds

treated with fucoidan–chitosan gel. In addition, the number of rete pegs, responsible for fixing the

epidermis in the dermis and organized nuclear regions (NOR), which confirmed cell proliferation,

were also the highest in this group. Complete cure within 21 days was observed only with the use of a

complex hydrogel, arming the synergistic eect of fucoidan and chitosan on the healing process [76].

In turn, Murakami et al. proposed hydrogels made of chitosan/chitin, fucoidan and alginate.

Hydrogels obtained by crosslinking with ethylene glycol diglycidyl ether were safe and showed no

cytotoxicity to human dermal fibroblast (DFC) and dermal microvascular endothelial (DMVEC) cells.

In comparison to the commercially available product (Kaltostat—calcium alginate fiber), they were

characterized by better—gradually increasing exudate absorption capacity, without maceration for 18 h.

In vivo studies in rats with mitomycin C-induced, impaired wound healing confirmed the positive

eects of fucoidan—its ability to interact with growth factors (FGF-1 and FGF-2) and with cytokines

involved in the reconstruction of the epidermis and angiogenesis processes. The stimulation of the

granulation and capillary formation observed after 7 days of treatment with hydrogel contributed

to the best results after 18 days of application, including eective division of epidermal stem cells

located in the intact adjacent epidermis and the strongest wound closure. Interestingly, the eect of the

hydrogel on the course of wound healing without the factor inhibiting cell proliferation (mitomycin

C) was insignificant [77]. Hydrosheets made of the same polymers (fucoidan, chitin/chitosan, and

alginate) were compared with two dierent hydrogel dressings (Kaltostat and DuoACTIVE). The

obtained results indicated an advantage in the treatment of mitomycin C-impaired wounds with the

proposed dressings than with the currently available products or plastic wrap used as a control (in

the study group within 14 days there was a reconstruction of the epidermis with significantly higher

wound closure rate). It should be added that the use of all these dressings had no significant eect on

the wounds which were not treated with mitomycin C [78].

As fucoidan is characterized by the ability to interact with growth factors and possesses lower

than heparin anticoagulant activity, it might be utilized to create drug carrier for tissue regeneration.

The main limitation in designed carriers for protein compounds, is their short half-life in vivo, resulting

from sensitivity to temperature and enzymes. Nakamura et al. formed a micro complex-hydrogel

composed of chitosan and fucoidan loaded with fibroblast growth factor-2 (FGF-2). The gradual release

of FGF-2 along with progressive degradation of the hydrogel contributed to statistically significantly

stronger neovascularization in the test group compared to mice injected with growth factor alone

or hydrogel placebo [79]. In turn, vascular endothelial growth factor (VEGF) and the influence of

fucoidan molecular weight on its activity in micro- and macroporous 3D scaolds were eval‎uated by

Purnama et al. Hydrogels obtained by crosslinking with sodium trimetaphosphate (STMP) pullulan

and DXT were control group, and those with fucoidan (with low, medium, and high molecular

weight—LMWF/MMWF/HMWF) addition were test groups. It was shown that significant decrease

in the release rate of VEGF compared to the control, regardless of the pore size in the hydrogel,

Mar. Drugs 2019, 17, 458 16 of 20

guaranteed the addition of fucoidan with medium molecular weight (39 kDa). A positive aspect is

also the significant increase in the number of human endothelial progenitor cells and their extent of

proliferation. The synergism of MMWF and VEGF was confirmed by observations after subcutaneous

injection of hydrogels to C57/BL6J mice. Both the neovessel area and density in the group treated with

hydrogels obtained from MMWF and VEGF were statistically higher compared with control group or

group treated scaolds with single substance [80].

In order to obtain hydrogel to ocular applications made of poly(2-hydroxyethyl methacrylate)

(pHEMA), fucoidan and methacrylic acid (MAA), Lee at al. used ethylene glycol dimethacrylate

(EGDMA) as crosslinking agent. Developed hydrogels were tested in terms of water absorption

capacity, adsorption of proteins and antibacterial properties. It was noted that water content of

hydrogels and adsorption of proteins (especially lysozyme) did not depend on the concentration of

fucoidan and increased only with MAA increase. The predicted antibacterial activity of fucoidan with

regard to  S. aureus and  E. coli turned out to be infinitesimal, and its potentiation was caused by the

addition of MAA [81].

5. Conclusions

Fucoidan is the important ingredient in various pharmaceutical formulations. It can be used

either as a substance with a specific therapeutic eect or as an excipient, allowing to obtain a drug

form with appropriate properties. However, utilization of fucoidan in the pharmaceutical industry

involves many challenges. Diculties appear already at the acquisition stage. Dierences in the

growth conditions of algae, pollution of the marine environment, as well as a wide range of species

that are the source of fucoidan contribute to the heterogeneity of its properties. This, in turn, leads to

the necessity of matching extraction and purification methods to allow obtaining the raw material with

desired characteristics [82–84]. Fucoidan features are also influenced by sugar composition and sulfate

content, and by dierential molecular weight (10–2000 kDa). Other disadvantage is lack of gelation

ability, important feature in designing drug dosage forms. This limitation can be overcome by fucoidan

combining with polymers (chitosan and its derivatives) or other compounds that provide a positive

charge (protamine, polyethyleneimine, polyallyamine hydrochloride, poly(isobutylcyanoacrylate),

poly(lactide-co-glycolide), poly-l-orithine, and hexadecylamine, poly(alkylcyanoacrylate)). The broad

spectrum of biological activity of this polysaccharide per se contributes to the fact that application

of designed drug carriers, like nanoparticles, liposomes, microparticles or semi-solid forms is very

dierential and using of fucoidan not only provides protection of the loaded active substance, but can

also increase its potency and improve the eectiveness of the treatment. Due to diversified properties

and potential broad area of application, the interest in fucoidan utilization in the biomedicine and

pharmaceutical industry will be probably extended.

Author Contributions: Conceptualization, A.C. and K.W.; designing and performing experiments, A.C. and M.S.;

writing—review and editing, A.C. and K.W.; visualization, A.C.; supervision, K.W.; funding acquisition, K.W.

Funding: This research was funded by Medical University of Bialystok grant number SUB/2/DN/19/004/2215.

Acknowledgments: We gratefully acknowledge A. Basa for SEM pictures made using the scanning electron

microscope(Inspect.S50, FEI Company, Hillsboro, OR, USA), which is a part of Center BioNano Techno equipment,

supported partly by EU founds (projectnumber POPW.O1.03.00-20-004/11).

Conflicts of Interest: The authors declare no conflict of interest.

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