ULUSLARARASI SİSTEME TEORİK BAKIŞLAR 62-

Todos os dados dos experimentos realizados foram expressos como

média ± desvio padrão. Foi utilizado o teste de análise paramétrica de análise

de variância (ANOVA) seguido do teste de Tukey (Nível de significância de

p<0,05) como GraphPad InStat® Software versão 3.05 para Windows 95

(GraphPad Software Incorporation, San Diego, CA, EUA).

5. ARTIGOS PRODUZIDOS

5.1. Artigo 1 (SUBMETIDO)

Evaluation of potential antitumor activity of fucan nanogel

Periódico: Marine Drugs

Nanotechnology (The reference number for the article is NANO-102955)

Fator de impacto: 3.978

ISSN: 1660-3397 (Printed version)

ISSN: 1660-3397 (Online version)

Qualis: Medicina II – A2

Indexada: PubMed – indexado por MEDLINE

5.2. Capítulo de livro

Chapter 6 – Application of Marine Polysaccharides in Nanotechnology

Periódico: Marine Medicinal Glycomics

Biotechnology in Agriculture, Industry and Medicine Biochemistry

Research Trends

In: Vitor Hugo Pomin. (Org.). Marine Medicinal Glycomics. 1ed.New York: Nova

Science, 2013, v. 01, p. 65-114.

Binding: ebook

ISBN: 978-1-62618-649-1

5.3. Artigo 2

Evaluation of acute and subchronic toxicity of a non-anticoagulant, but

antithrombotic algal heterofucan from the Spatoglossum schröederi in

Wistar rats

Periódico: Brazilian Journal of Pharmacognosy

Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 21(4): Jul./Aug. 2011

Fator de impacto: 0.68

ISSN: 0102-695X (Printed version)

ISSN: 1981-528X (Online version)

Qualis: Medicina II – B3

Indexada: SCOPUS, SciELO, EMBASE e GEOBASE

5.4. Artigo 3

Evaluating the possible genotoxic, mutagenic and tumor cell proliferation-

inhibition effects of a non-anticoagulant, but antithrombotic algal

heterofucan

Periódico: Journal of applied toxicology : JAT.

J Appl Toxicol. 2010 Oct;30(7):708-15. doi: 10.1002/jat.1547.

Fator de impacto: 2.597

ISSN: 0260-437X (Printed version)

ISSN: 1099-1263 (Online version)

Qualis: Medicina II – B1

5.1. ARTIGO 1 (SUBMETIDO)

Mar. Drugs 2014, 12, 1-x manuscripts; doi:10.3390/md120x000x

marine drugs

ISSN 1660-3397

www.mdpi.com/journal/marinedrugs

Article

Evaluation of potential antitumor activity of fucan

nanogel

Jailma Almeida-Lima

1,2

, Arthur Anthunes Jacome Vidal

1

, Dayanne Lopes

Gomes

1,2

, Ruth Medeiros Oliveira

1

, Leonardo Thiago Duarte Barreto Nobre

3

,

Mariana Santana Santos Pereira Costa

1

, Nednaldo Dantas-Santos

2

, Helena

Bonciani Nader

3

, Francisco Miguel Gama

4

, Edda Lisboa Leite

1

, Hugo Alexandre

Oliveira Rocha

1,2*

1

Laboratory of Biotechnology of Natural Polymers (BIOPOL), Department of

Biochemistry, Federal University of Rio Grande do Norte (UFRN), Natal-RN 59078-

970, Brazil; E-Mails: [email protected] (J.A.-L.); [email protected]

(A.A.J.V.); [email protected] (D.L.G.); [email protected]

(R.M.O.); [email protected] (M.S.S.P.C); [email protected] (E.L.L);

[email protected] (H.A.O.R)

2

Graduate Program in Health Sciences, Federal University of Rio Grande do Norte

(UFRN), Natal-RN 59078-970, Brazil; E-Mails: [email protected] (N.D.-S.)

3

Department of Molecular Biology, Department of Biochemistry, Federal University

of São Paulo - UNIFESP, São Paulo-SP, 04044-020, Brazil; E-Mails:

[email protected] (L.T.D.B.N); [email protected] (H.B.N)

4

IBB—Institute for Biotechnology and Bioengineering, Centre for Biological

Engineering, Minho University, Braga 4704-553, Portugal; E-Mails:

[email protected] (F.M.G.)

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +55-84-3215-3416 (ext. 207); Fax: +55-84-3211-9208.

Received: / Accepted: / Published:

Abstract: Fucan is a term that defines a family of homo- and

heteropolysaccharides containing sulfated L-Fucose. In this work a

heterofucan (Fucan A) from seaweed Spatoglossum schröederi was

thiolated and treated by ultrasonication, giving rise to intermolecular

disulfide bonds and to the formation of fucan A nanogels. Fucan A nanogel

decorated with polyethylene glycol (FucA:PEG15) was stable for over one

month and showed an average diameter of 187 ± 11 nm in aqueous solution

and a zeta potential of -23.1 ± 2.0 mV, as measured by dynamic light

scattering. This nanogel inhibits the proliferation of 786-0 renal

adenocarcinoma cells in a dose-dependent way. Flow cytometric analysis

showed that FucA:PEG15 induces apoptosis through caspase and caspase-

independent mechanisms. The nanogel labeled with FITC was completely

taken up by 786-0 cells after 1 hour. When we stopped the cell endocytosis,

the FucA:PEG15 antiproliferative effect was abolished. In additon,

FucA:PEG15 has antioxidant activity and also exhibits antiangiogenic

activity . These data show that fucan A nanogel exhibits several activities

(antiproliferative, antioxidant, and antiangiogenic) and therefore its potential

for cancer therapy should be further investigated.

Keywords: fucan; sulfated polysaccharide; brown seaweed; nanogel;

cytotoxicity

1. Introduction

The term cancer is used to refer to more than one hundred different types of diseases

that have in common the characteristic uncontrolled proliferation of anaplastic cells.

These cells, at some point, will invade other tissues and organs. It is estimated that more

than 21 million people will contract cancer and 13 million deaths are expected by 2030.

Although cancer accounts for around 13% of all deaths in the world, more than 30%

could be prevented by modifying or avoiding key risk factors [1].

The use of chemotherapy for the treatment of cancer raises concerns about the

debilitating side effects of this form of treatment; on the other hand, poor cellular

internalization and insufficient intracellular drug release reduces its efficacy [2].

The development of nanomedicine aims to solve the issues associated with the

systemic administration of toxic pharmaceuticals. Thus, chemotherapeutic agents have

been encapsulated, conjugated, entrapped, or loaded into nanoformulations, resulting in

the site-specific drug delivery, thereby reducing the systemic toxicity [3].

Among the available nanosystems, nanogels are particularly attractive since they are

easy to produce, are affordable, and may effectively incorporate a variety of drugs,

including biopharmaceuticals [4]. Nanogels are composed of cross-linked three-

dimensional polymer chain networks that are formed via covalent linkages or self-

assembly processes. An increased interest has been witnessed recently for smart

nanogels allowing site-specific and controlled drug release, paving the way for the

development of improved cancer therapeutic formulations [5].

Among the numerous polymers that have been proposed for the preparation of

nanogels, polysaccharides have a number of advantages over the synthetic polymers,

which were initially employed in the field of pharmaceutics [6]. Fucans are

polysaccharides containing substantial amounts of L-fucose and sulfate ester groups,

expressed by brown seaweed [7] and some marine invertebrates (sea urchins and sea

cucumbers) [8]. Algal fucans showed several biological/pharmacological activities such

as antithrombotic, antiviral, anticoagulant, antioxidant, and anti-inflammatory

[9,10,11,12]. In addition, fucan anti-cancer activities have been reported frequently in

recent years, and the potential mechanisms of action were also investigated

[13,14,15,16].

Among the seaweed fucans, we can highlight those extracted from Spatoglossum

schröederi (Dictyotaceae). This brown seaweed is found along almost the entire

Brazilian coast (about 8000 km). The permanent availability of this organism in large

amounts makes it an excellent choice for prospecting bioactive compounds. It

synthesizes three fucans and the one obtained in the larger quantity was named fucan A.

We showed that fucan A is not toxic in vivo [17], but it demonstrated antiproliferative

activity against several tumor cell lines [18]. However, this activity was observed only

for high concentrations, probably because fucan A, due its ionic nature, binds onto a

great variety of proteins, becoming unavailable to perform the bioactivity of interest.

Another kind of sulfated polysaccharide known as heparin binds several proteins [19]

including extracellular matrix proteins [20]. In order to diminish this inactivating

effect, Bae and colleagues synthesized a heparin nanogel cross-linked with disulfide

linkages. When B16-F10 tumor cells were treated with heparin nanogel, their

proliferation was inhibited by about 50%, whereas free heparin alone inhibited the cell

growth to a much smaller extent (~10%) [21].

Previously, we chemically modified fucan A by grafting hexadecylamine to the

hydrophilic backbone. The obtained amphiphilic material self-assembled into fucan A

nanogel, which showed antiproliferative activity against human renal tumor cells (786-0

cells) [22]. In this study fucan A was covalently linked to thiol groups and then cross-

linked with disulfide linkages to produce fucan A nanogel for efficient cellular uptake.

This way, a fully hydrophilic nanogel is obtained. In addition, we investigated the

antiproliferative effect of the newly developed fucan A nanogels using 786 tumor cell

lines. The data obtained indicated that the fucan A nanogel exhibits high stability and is

a more powerful inhibitor of cell growth than free fucan A.

2. Results and Discussion

2.1. Synthesis of the fucan A nanogels

In the present work, fucan A was chemically modified with thiol groups and then

cross-linked with disulfide linkages to produce reducible fucan A nanogels. For this

cysteamine by reductive amination, yielding thiolated fucan A. Then, the nanogels

were synthesized by a method that comprises two steps: Thiolated fucan was initially

co-dissolved in DMSO with PEG, enabling the interaction between the two polymers,

presumably by hydrogen bonding, resulting in the spontaneous formation of nanosized

complex particles in the DMSO phase. In the second step, thiolated fucan A monomers

within the inner core of the complexes were covalently linked together by disulfide

linkages. This was achieved by ultrasonic treatment, which promotes the formation of

free radicals, which in turn accelerate the oxidation reaction of the thiol groups of the

cysteamine linked to fucan A. The crosslinked fucan nanogels were finally obtained by

withdrawing the DMSO and free PEG through exhaustive dialysis (Figure 1A).

Figure 1. A) Scheme of the synthesis of fucan A nanogels. B)

Transmittance of solution containing nanogels of fucan A prepared using

various weight ratios of PEG (0, 2.5, 5.0, 10, 15, 30) at 400 nm.

The effect of the fucan:PEG ratio on the size of nanogels was investigated. The fucan

A concentration was kept constant and PEG content increased at the ratio of 2.5, 5.0,

10, 15, and 30 times the concentration of fucan A; hence, the obtained samples were

called FucA:PEG2.5, FucA:PEG5.0, FucA:PEG10, FucA:PEG15, and FucA:PEG30,

respectively. We can observe (Figure 1B) that in the absence of PEG the transmittance

value was kept at 100%, i.e., similar to the control of dissolved fucan A. However, with

increasing PEG content, the transmittance gradually decreases to about 60%, for the

higher ratio of FucA:PEG 30, indicating the formation of colloidal particles, the

nanogels, as shown in the following section.

2.2. Characterization of the nanogels

The average hydrodynamic diameters of nanogels were measured at a concentration

of 1 mg/mL in water by dynamic light scattering (DLS). The effect of various fucan

A:PEG weight ratios on nanogel size, polydispersity (PDI), and zeta potential was

analyzed. As we can see in Table 1, a weight ratio of FucA:PEG of 30, originates a very

large nanogel (917.18 ± 83.12 nm), whereas the others showed a size ranging from

186.95 ± 10.62 to 301.44 ± 2.26. Bae and colleagues [21], using the same method to

obtain heparin nanogels, elected a weight ratio of heparin:PEG of 15 as the best one

because it would favor stronger interactions between PEG and heparin, leading to the

formation of compact nanogels. This observation is in agreement with ours, since

FucA:PEG15 showed the smaller size (186.95 ± 10.62 nm) among the tested

combinations. However, the size of FucA:PEG15 colloidal micelles is in the same order

of magnitude of other nanogels obtained with fucan A using a different synthetic route

[22], and of others obtained using different acidic polysaccharides and the same

synthesis approach. For instance, heparin nanogels resulted in a stable structure with an

average diameter of 248.7 ± 26.8 nm [21] and acetylated hyaluronic acid gave rise to

nanogels ranging from 275 ± 4 to 447 ± 8 nm [23].

The size of nanomaterials is an extremely important factor determining its fate in

vivo—biodistribution and pharmacokinetics—since it affects namely the phagocytosis

and ability to cross biological barriers. According to Dong and Mumper [24],

nanoparticles with a size around 220 nm are ideal targets for passive targeting of tumors

since the majority of solid tumors exhibit vascular pore cut-offs between 380 and 780

nm. Thus, using the size as a parameter of choice, all fucan A nanogels, except

FucA:PEG30, have potential application against tumor cells. However, other parameters

should be evaluated to confirm this statement.

Table 1. Physicochemical characteristics of nanogels obtained using

different ratios of the polyethylene glycol (PEG) determined by DLS.

Samples

Diameter

(nm)

Polydispersity

(PDI)

Conductance

(µS)

Zeta

Potencial

(mV)

FucA:PEG2.5

301.44 ± 2.26

0.69 ± 0.03

193

-28.12 ± 1.55

FucA:PEG5.0

297.15 ± 30.97

0.48 ± 0.09

254

-28.61 ± 0.15

FucA:PEG10

277.10 ± 17.41

0.49 ± 0.03

548

-26.11 ± 0.05

FucA:PEG15

186.95 ± 10.62

0.54 ± 0.06

347

-23.08 ± 1.96

FucA:PEG30

917.18 ± 83.12

0.84 ± 0.09

339

-22.18 ± 0.13

The zeta potential was negative for all nanogels, ranging from -22.18 ± 0.13 to -

28.61 ± 0.15 mV. These results can be explained by the negative charge of fucan A due

to the presence of ionized carboxyl and sulfated groups. This is in good agreement with

the zeta potential values found in previous studies for nanoparticles prepared with other

fucans [25], and with chitosan with a blend dextran sulfate [26].

Furthermore, nanogel, as evidenced by the polydispersity index (PDI), ranged

between 0.48 ± 0.09 to 0.84 ± 0.09. The PDI is dimensionless and scaled such that

values smaller than 0.05 are rarely seen other than with highly monodisperse standards.

Values greater than 0.7 are indicative of samples with a very broad size distribution and

probably not suitable for analysis using the dynamic light scattering (DLS) technique

[27,28]. Thus, the best nanogels regarding the heterogeneity of size distribution were

FucA:PEG5.0, FucA:PEG10, and FucA:PEG15.

Size and surface morphology of fucan A nanogels were evaluated by SEM. Figure 2

shows that not all formulations presented a spherical shape, in the case of FucA:PEG2.5

nanoparticles were not detected, while in the case of FucA:PEG30 particles with a

fibrous shape were observed. It can be speculated that some phase transition may have

occurred during the drying of the samples, since in the case of FucA:PEG2.5 nanosized

particles were detected by DLS. Nonetheless, these results demonstrate the critical

relevance of the balance of fucan A vs PEG, suggesting that the arrangement of the two

polymers determines the organization of the nanogel. On the other hand, FucA:PEG5.0,

FucA:PEG10, and FucA:PEG15 showed a spherical shape with an average diameter of

113.33 ± 0.23 nm, 101.85 ± 0.43 nm, and 98.25 ± 1.69 nm, respectively (Figure 2F).

The diameter measured by DLS was slightly larger than the one obtained from SEM,

presumably due to the swelling of fucan A nanogels in water (taking into account that

carbohydrate polymers are highly hygroscopic), since the samples needed to be dried

for analysis by SEM, as noted by other authors [21,23].

Figure 2. SEM photograph of nanogels with various weight ratios of

fucan A:PEG (A) 2.5, (B) 5, (C) 10, (D) 15, (E) 30, and F) average

diameter of the nanogels. The diameters of particles in SEM images were

measured by comparing them with the size bar.

2.3. FTIR of the fucan A nanogels

FTIR spectra of fucan A, FucA:PEG10, and FucA:PEG15 are depicted in Figure 3.

Characteristic sulfate absorptions were identified in the FTIR spectra: bands around

1265 cm

−1

for asymmetric S=O stretching vibration and around 1041 cm

−1

for

symmetric C–O vibration associated with a C–O–SO

3

group [18]. The bands at 813–850

were caused by the bending vibration of C–O–S. At 3200–3500 cm

−1

, fucan A and

fucan A nanogels showed bands from the stretching vibration of O–H [9]. The band

around 2900 cm

−1

corresponds to stretching vibrations of CH

2

, which is higher in fucan

nanogel spectra due to the presence of stretching vibrations of CH

2

in cysteamine

residues [29], as further confirmed by the band at 1468 cm

−1

, featuring a higher

intensity in fucan nanogel spectra. This band corresponds to C–H symmetric

deformation vibration [30]. A band around 1410 cm

−1

was identified in all spectra and

was assigned to symmetric vibration of COO

−

of glucuronic acid. The presence of

glucuronic acid was also confirmed by the antisymmetric stretching vibration of COO

−

at 1618 cm

−1

[31], which overlaps with the vibration of water. The H

2

O molecule has

strong IR absorbance with three prominent bands around 3400 (O–H stretching), 2151

(water association), and 1618 cm

−1

(H-O-H bending) [32] in the fucan A spectrum.

However, the band intensity decreases in fucan nanogels. The absorption band due to S–

H stretching vibrations of the thiol group was also observed at 2600–2550 cm

−1

[33].

Figure 3. Infrared of fucan A and nanogels FucA:PEG10, FucA:PEG15,

Fuc:PEG30.

2.4. Nanogel Stability

The nanogels FucA:PEG10 and FucA:PEG15 showed the smallest particle size and

best morphological features, and therefore were chosen for further characterization. In

DLS. As presented in Figure 4, the fucan A nanogels were highly stable in aqueous

solution, suggesting that their structural integrity was preserved.

Figure 4. Stability of fucan A nanogels FucA:PEG10 and FucA:PEG15

accessed by DLS. The nanogels were stored at 4 °C for up to 42 days and

analyzed in DLS at temperature 25°C.

2.5. Cytotoxicity assay

The cytotoxic of FucA:PEG10, FucA:PEG15, and Fucan A upon 786-0 cells was

investigated for 24 h using a colorimetric MTT-based assay (Figure 5). Fucan A

displayed a dose-dependent inhibitory effect, which was quite expressive (by about 3

fold) in the case of nanogels. FucA:PEG10 and FucA:PEG15 also showed a dose-

dependent effect, reaching saturation at around 0.04 and 0.06 mg/mL respectively. In

addition, the data also indicate that FucA:PEG15 is slightly more efficient as an

antiproliferative compound than FucA:PEG10.

Dantas-Santos et al. [22], using fucan A nanogels grafting hexadecylamine,

evaluated the cell viability of various types of tumor cells. The 786-0 cell line was the

more susceptible one (inhibition of ~40%). However, this inhibition was obtained only

for a concentration as high as 500 µg/mL, ten times higher than the required using

FucA:PEG15.

Figure 5. Inhibition of proliferation of 786 cells incubated with Fucan A,

FucA:PEG10, or FucA:PEG15 nanogels at various concentrations for 24

hours. Data are expressed as means ± standard deviation. a,b,c,d,e The

different letters indicate significant difference between the concentrations

of the same compound (p < 0.05).

2.6. Apoptotic effect of fucan A nanogels

Since FucA:PEG15 was the most potent cell growth inhibitor, it was chosen for

mechanistic studies, namely to determine whether cell death for apoptosis was

responsible for the observed effect. For this purpose, 786-0 cells were treated with

FucA:PEG15 (64 µg/mL) for 24 hours, followed by flow cytometric analysis.

Cell apoptosis features the exposure of phosphatidylserine on the external side of the

cell membrane, which can be recognized by annexin V. On the other hand, necrotic

cells can be identified using propidium iodide (PI), which stains only necrotic cells

bearing a compromised, porous cell membrane. Generally, cells stained with annexin V

are indicative of early apotosis and stained cells with PI, indicative of necrosis, while

double labeling is indicative of late apoptosis [34].

In Figure 6 we can see the results of flow cytometry analysis of cells cultivated in the

presence and absence of nanogels (control group). For the control group (Figure 6A),

91.4% of the cells are negative for annexin V and PI. However, after the FucA:PEG15

treatment, this number dropped to 59.0%, whereas the percentage of cells stained with

annexin V increased from 7.8 to 39%. Furthermore, the percentage of cells stained with

PI did not change (Figure 6B). These data indicate that FucA:PEG15 inhibits

In order to determine the role of caspases in the FucA:PEG15 nanogel-induced

apoptosis, 786-0 cells were incubated with ZVAD-FMK. As can be seen in Figure 6B,

in the presence of this pan-caspase inhibitor, the percentage of cells positive for annexin

decrease from 39 to 27%. Additionally, in the presence of E64 (cysteine peptidase

inhibitors, mainly cathepsin and calpains specific) the effect also decreased to about

10% of inhibition (Figure 6D). These data indicate that FucA:PEG15 has a complex

mechanism of apoptosis induction.

Several fucans have been shown to induce apoptosis in different types of tumor cells,

an event often related to caspase activation [35,36,37]; however, other proteins involved

in cell survival pathways may be affected by the presence of fucans in the culture

medium, such as proteins from ERK1/2MAPK pathway [16,38] and PI3K/AKT

pathway [13], apoptosis-inducing factor (AIF) [10], JNK/c-Jun/AP-1 pathways, and

death receptor-mediated and mitochondria-mediated apoptotic pathways [14].

The analysis of the aforementioned data suggests that the mechanism of apoptosis

induction by fucans is very complex, in agreement with the results obtained in this work

using the fucan A nanogel. Further work will be dedicated to identifying mainly the cell

proteins involved in the FucA:PEG15 mechanism of action to induce apoptosis.

Figure 6. Cytometry with cells 786-0. A) Control (Anexin + PI); B)

FucA:PEG15; C) FucA:PEG15 + ZVAD and D) FucA:PEG15 +E64 all

with the same concentration (64 µg/mL).

2.7. FucA:PEG15 intracellular uptake

The intracellular uptake of nanogels was investigated by confocal microscopy and

flow cytometry. For this purpose, the nanogel was conjugated with FITC and used as

fluorescence probe, allowing the observation of its interaction with the 786-0 cells.

The antiproliferative effect (around 40% for a concentration of 0.06 mg/mL) was not

affected by the FITC labeling. It is important to mention that FITC alone has no activity

on 786-0 cell proliferation (data not shown). The confocal observation of 786-0 cells

after incubation with FucA:PEG15+FITC for 1 h allowed the detection of internalized

fucan nanogel (Figure 7B). In addition, the nanogel is observed in the perinuclear space

Belgede Bosna savasi ve Yugoslavya'nin parcalanmasi (sayfa 75-96)