Journal of Controlled Release 273 (2018) 131–138

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Biopolymer strategy for the treatment of Wilson's disease T

a,⁎ b a a b
Miroslav Vetrik , Jana Mattova , Hana Mackova , Jan Kucka , Pavla Pouckova ,
Olivia Kukackovaa, Jiri Brusa, Sebastian Eigner-Henkec, Ondrej Sedlaceka, Ludek Sefcc,
⁎
Petr Stepaneka, Martin Hrubya,
a
Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic
b
Institute of Biophysics and Informatics, First Faculty of Medicine, Charles University, Salmovska 1, 120 00 Prague 2, Czech Republic
c
Center for Advanced Preclinical Imaging (CAPI), First Faculty of Medicine, Charles University, Salmovská 3, 120 00 Prague 2, Czech Republic

A R T I C L E I N F O A B S T R A C T

Keywords: Wilson's disease is a genetic disorder that causes excessive accumulation of copper in the body, leading to toxic
Wilson's disease damage, especially in the liver and nervous system. The current treatment cause burdensome side effects. We
Copper chelators describe the use of chemically modified biopolymer carriers based on microcrystalline cellulose and chitosan
Biopolymers containing the highly specific copper chelator 8-hydroxyquinoline as a new type of therapy for Wilson's disease.
Copper scavengers
The chelators can scavenges copper ions released from food during digestion and copper ions present in se-
treatment
cretions in the gastrointestinal tract. Because the chelator is covalently bound to indigestible biopolymer carriers
(crosslinked chitosan or modified cellulose), it is not taken up by the gastrointestinal tract and it can be
eliminated through the feces, avoiding unwanted side effects. This concept was tested on Wistar rats, which
received a radioactive 64CuCl2 solution together with the polymers with covalently bound 8-hydroxyquinoline
through a gastric probe. 64Copper complex uptake from the gastrointestinal tract was significantly inhibited by
both chelating polymers. With the modified polymers, the presence of 64Cu was detected mostly in the gas-
trointestinal tract, not in the internal organs. These findings indicate modified cellulose and crosslinked chitosan,
with covalently bound 8-hydroxyquinoline exhibited the potential to be excellent therapeutics for treating
Wilson's disease.

1. Introduction
Copper is an essential dietary nutrient needed for many important
processes in the human body [1]. It is involved in numerous processes,
and in particular, it is an essential constituent of enzymes, e.g., fer-
roxidases, lysyl oxidase, dopamine β-hydroxylase, copper/zinc super-
oxide dismutase, monoamine oxidase or tyrosinase [2]. Copper is also
needed for mitochondrial respiration, melanin biosynthesis, iron
homeostasis, connective tissue formation, dopamine metabolism and
peptide amidation [3]. Defects in copper homeostasis can cause several
human diseases, including Wilson's disease [4].
Wilson's disease is an autosomal recessive inherited disorder caused
by a genetic mutation in the ATP7B gene [5]. ATP7B is responsible for
copper transport into the trans-Golgi network and for the excretion of
copper from the cell [6]. Mutations of ATP7B genes lead to high copper
accumulation in the body, which causes oxidative-stress-related da-
mage in the liver, brain and other parenchymal tissues (Fig. 1) [7].
The clinical manifestation of Wilson's disease mainly affects the
liver and neurological system and can cause movement disorders,
psychiatric disease or hemolytic anemia [8]. The most typical disorder
is liver disease, which manifests as acute hepatitis or fulminant hepatic
failure, chronic hepatitis and cirrhosis [3].
Currently, the standard treatment combines several approaches [9].
First, daily copper intake must be prevented by a lifelong diet with low
copper contents. Therefore, the intake of nutrients with high copper
contents must be avoided such as mushrooms, nuts or seafood. The
average copper uptake for the normal human diet ranges from 0.5 mg
to 2.5 mg per day [10]. However, a low copper diet is not sufficient by
itself. Therefore, supplementing this diet with pharmacological treat-
ment is also necessary.
The pharmacological treatment of Wilson's disease is a lifelong
therapy based on low-molecular-weight copper-chelating agents e.g.,
penicillamine and trientine. These chelating agents increase copper
elimination through the urine [3,11]. Another low-molecular-weight
copper chelator used for the treatment is ammonium tetra-
thiomolybdate, which forms complexes with copper in the gastro-
intestinal tract (GIT). It also forms complexes with copper and albumin
in the blood. Ammonium tetrathiomolybdate seems to be useful for

⁎
Corresponding authors.
E-mail addresses: vetrik.miro@gmail.com (M. Vetrik), mhruby@centrum.cz (M. Hruby).

https://doi.org/10.1016/j.jconrel.2018.01.026
Received 28 November 2017; Received in revised form 16 January 2018; Accepted 23 January 2018
0168-3659/ © 2018 Elsevier B.V. All rights reserved.
M. Vetrik et al.
Fig. 1. The mechanism of copper accumulation in hepatocyte.
patients with neurological symptoms [12]. Along with the low-mole-
cular-weight chelator therapy, high doses of divalent zinc salts can also
be used as a maintenance therapy. Zinc (II) utilizes the same carriers for
absorption in enterocytes as copper, and therefore, high amounts of
zinc competitively decrease copper absorption [13].
Unfortunately, all these therapies have severe drawbacks. Even
when patients follow a strict low-copper diet, completely avoiding
copper intake is impossible. Main disadvantage of the pharmacological
treatment is its presence in the other organs of the patients' body.
Penicillamine has a high level of toxicity that causes severe side effects,
e.g., immunologically induced lesions, pyridoxine deficiency or inter-
ference with collagen and elastin formation [3]. Zinc therapy is ac-
companied by strongly adverse gastrointestinal effects due to the high
zinc uptake of 1200 mg/day, whereas normal daily intake of zinc is in
the range 8 mg/day to 15 mg/day, i.e., a hundred times less than that of
the therapy [14,15].
Copper enters the body with the food. Some copper is also secreted
into the GIT with digestion fluids and further taken up by the GIT. The
reuptake of copper from digestion fluids was reported to be even higher
than the overall copper content taken up from food by the GIT [16].
Therefore, we focused on the GIT as the main place where copper (re)
uptake occurs. The ideal copper scavenger should therefore be present
only in the GIT and should not be able to enter the bloodstream or other
inner organs of the body to avoid unwanted side effects. Such copper
scavengers can thus be constructed using insoluble and indigestible
polymer carriers for the chelators, which are in general non-resorbable
and biologically neutral [17–20].
The polymer materials studied in this paper should be resistant to
the action of pancreatic and faecal enzymes present in the GIT of human
beings [21–23]. Microcrystalline cellulose is biologically non-digestible
because the human body does not have the cellulolytic enzymes ne-
cessary to digest cellulose [24]. Modified celluloses such as ethylcel-
lulose, carboxymethylcellulose, methylcellulose and hydro-
xymethylcellulose are also not digested by the colonic bacteria present
in the GIT [25,26]. Moreover, microcrystalline cellulose is a common
ingredient used in pharmaceutical industry and can also be found in
many processed food products. The degree of digestion of different
cellulose structures in rat models showed that microcrystalline cellulose
was the least digestible of the tested materials [27]. However, different
species are well known to have different digestion capabilities due to
differences in the microflora of each organism.
The second examined biopolymer, crosslinked chitosan, was de-
monstrated not to undergo degradation by the action of colonic bac-
terial or pancreatic enzymes [22]. Crosslinked chitosan prevents
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Journal of Controlled Release 273 (2018) 131–138
subsequent degradation and dissolution in the stomach and fully allows
the movement of the chitosan hydrogel through the GIT.
Both microcrystalline cellulose and crosslinked chitosan were
modified by the covalently bound chelator 8-hydroxyquinoline (8HQ).
The covalent bond prevents release the 8HQ into the body and its
subsequent organ deposition. This chelator was chosen based on our
previous studies [18]. We found that 8HQ is able to complex copper in
wide range of pH. In vitro studies showed that Cu-8HQ complex is
stable in wide range of pH and in presence of competing zinc ions favors
Cu-8HQ complex over Zn. The abovementioned chelation properties of
8HQ are crucial because chelation occurs mostly in acidic pH (pH 2–4),
but as the Cu-8HQ complex moves thru the GIT the pH rises to higher
values. This pH shift can cause release of copper ions from Cu-8HQ
complex. Moreover, in the same study we investigated Cu-8HQ complex
chelation properties in the presence of free amino acids (stomach and
small intestine model) that may lead to significant leakage of the ab-
sorbed copper form the sorbent. The intestinal environment was si-
mulated by a solution of amino acids, with two of the most strongly
copper chelating amino acids (l-cysteine and l-histidine) added at
concentrations corresponding to their average daily uptake. The re-
maining amino acids were simulated with glycine at concentrations
corresponding to amounts that are consistent with an average daily
ingestion (50 g per 1.5 L).
We are describing completely new, advanced functional materials
with suitable properties for the maintain treatment of Wilson's disease,
which is a lifelong threatening disorder. This paper focuses on the de-
velopment of next-generation, non-digestible, non-resorbable, bio-
compatible chelating polymers derived from microcrystalline cellulose
and crosslinked chitosan particles for oral therapy for Wilson's disease.
This therapy should not have the abovementioned drawbacks of the
previous model systems, and it can potentially overcome all of the side
effects exhibited by current therapies. The prepared materials contain a
significantly higher content of chelating groups, and they do not ac-
cumulate in gastric mucosa, unlike our previous methacrylate system
[28]. Our materials pass through the GIT with digested food. In our
previous study we show and prove possess strong selective chelation
abilities for copper in the biological environment model of used che-
lator 8HQ. In vivo studies on Wistar rats proved that copper scavengers
based on cellulose and chitosan exhibit much higher copper chelating
properties in vivo than the previously described materials. Our in-
soluble crosslinked systems with covalently bounded 8HQ should not
be present in bloodstream or other organs. Therefore, it should not
elicit side effects caused by organ deposition.
Our treatment is design as maintaining treatment lowering stress of
the patients under current treatment. Application of prepared materials
is shifting levels of copper that is uptaken from the food.
2. Experimental section
2.1. Materials and methods
The instrumental setup, chemical preparation and biodistribution
information can all be found in the supplementary information. All
animal experiments described hereinafter have been performed in ac-
cordance with the corresponding legislative, Act on Experimental Work
with Animals (Decrees No. 311/97; 117/87 and Act No. 246/96 of the
Czech Republic), which is fully compatible with the corresponding
European Union directives.
3. Results and discussion
3.1. Polymer synthesis and characterization
3.1.1. Cellulose modification
Microcrystalline cellulose was modified via a series of reaction steps
resulting in the final product 8HQ-cellulose (4) shown in Fig. 2(A).
M. Vetrik et al. Journal of Controlled Release 273 (2018) 131–138

Fig. 2. A Schematic representation of the microcrystalline cellulose chemical modification. B Chemical modification of crosslinked chitosan with 8HQ.

The first part of the synthesis was adopted from the literature,
wherein glycidyl cellulose (2) was the final product [29]. In the first
step, cellulose was dissolved in N,N-dimethylacetamide with LiCl under
elevated temperature and stirring. The addition of allyl bromide to the
basic solution of cellulose resulted in introducing allyl groups
(Fig. 2(A)). The reaction progress was monitored by FTIR, wherein
cellulose was compared with allyl cellulose (1). The signal of the hy-
droxyl groups of cellulose decreased within the reaction at 3332 cm− 1,
and new signals belonging to the allyl group increased at 1624 cm− 1
(C]C) and 920 cm− 1 (]CeH), (Fig. S1). In 1H NMR spectra ethylene

133
M. Vetrik et al.
hydrogens are present at δ 5.17 (2H) this hydrogens are part of newly
formed allyl group. 13C NMR shows signals at 135.5, 116.4 that belong
to unsaturated ally carbons. The covalently bound allyl groups on cel-
lulose then underwent an oxidation reaction with peracetic acid. This
reaction gives glycidyl cellulose (2). The presence of epoxy groups was
confirmed by the FTIR spectrum (Fig. S2), wherein the peak at
907 cm− 1 is clearly visible, whereas the disappearance of allyl groups
at 1624 cm− 1 (C]C) and 920 cm− 1 (]CeH) is evident. In 1H NMR
the hydrogens bounded at oxiram ring are present at ethylene hydro-
gens are present at δ 3.1 with the (2H, m). Reaction progress is proved
also by bridging methylene group at δ 2.5 (2H, m). Disappearance of
ally hydrogens at δ 5.17 supports this statement. In 13C NMR signals
previous present at 135.5, 116.4 (C]C) disappeared. The epoxy groups
were reacted with the amino groups of methylamine to form N-me-
thylamino cellulose (3), as shown in Fig. 2(A). The FTIR is also sup-
ported by the 13C NMR where signal at 29.8 belongs to methyl group
bounded onto secondary amine. The disappearance of the epoxy group
signals indicates reaction progress, as shown in Fig. S3, wherein the
epoxy peak is no longer visible at 907 cm− 1. Additionally, the peak
visible at 3312 cm− 1 and a low-intensity band at 1550 cm− 1 prove the
presence of a secondary amine on the molecule. Moreover, in 1H NMR
signal with at δ 3.4 represent hydrogens of the methyl group. The re-
action between the epoxy groups and methylamine provide additional
crosslinking of the cellulose chains. This step introduces secondary
amino groups to cellulose molecules, which undergo a Mannich con-
densation reaction with 8HQ (4). The presence of covalently bound
8HQ on the modified cellulose was confirmed by the FTIR spectrum in
Fig. S5. We do not find any evidence of unbounded 8HQ molecules in
solutions after the washing process. Additional crosslinking of cellulose
chains make this crosslinked system insoluble in any solution what
guarantees its stability and non-degradability in gastrointestinal en-
vironment. Therefore, we analyzed our final products with the solid-
state NMR (see below).
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Journal of Controlled Release 273 (2018) 131–138
Fig. 3. 13C CP/MAS NMR spectra of 8HQ-chitosan and
8HQ-cellulose (*spinning sideband). The units from chitin
are labeled Chit and ethanol as Eth. C1–C6 are the numbers
of the carbohydrate unit and are numbered as usual (C1 –
anomeric carbon).
3.1.2. Chitosan modification
Spherical particles of chitosan were prepared by emulsifying an
aqueous chitosan solution into sorbitan monooleate and paraffin, thus
creating an emulsion of aqueous chitosan droplets in a continuous hy-
drophobic oily phase. The addition of glutaraldehyde into the homo-
genized emulsion mixture caused a crosslinking reaction between the
protonated free amine groups of the chitosan and the aldehyde groups
of the glutaraldehyde. The spherical chitosan particles were kept
floating in the emulsion using mechanical stirring, which provides time
for crosslinking. The crosslinking of chitosan prevents the chitosan
matrix from degradation in the GIT. Prepared beads were treated with
methanol and water for several days to remove unreacted glutar-
aldehyde. The liquid phase of the washing solutions was derivatized
with 2,4-dinitrophenylhadrazine and analyzed with the HPLC system
by the method described earlier [30]. The presence of free-unbound
glutaraldehyde in the final product was not detected. Glutaraldehyde is
used quite usual as a crosslinker for amine groups for chitosan. Similar
routine process is used for scaffolds preparation in tissue engineering
without any significant effect on cells [31]. Numerous studies used this
crosslinker for crosslinking chitosan. This crosslinker can be considered
as good candidate for the oral delivery of drugs [32]. The crosslinked
beads were modified using a Mannich reaction with formaldehyde and
8HQ (Fig. 2(B)). As a result, crosslinked chitosan particles with cova-
lently bound 8HQ were obtained (6). As in case of 4 we do not find the
presence of 8HQ in the solution after the washing steps. The crosslinked
system is insoluble in any of the solvent therefore other characteriza-
tion was done by solid-state NMR (see below).
3.2. Solid-state NMR analysis and 8HQ quantification
Solid-state NMR spectroscopy is an efficient tool to establish the
structure and chemical composition of various complexes and to pro-
vide information about the mode of their binding. We used a standard
13
C CP/MAS NMR experiment to characterize the structures of the
M. Vetrik et al.
prepared biopolymers 8HQ-chitosan (6) and 8HQ-cellulose (4), which
provides qualitative information on the primary structure of the applied
biopolymers and enables estimating the extent of their chemical mod-
ification.
The 13C CP/MAS NMR spectra of both the investigated systems
8HQ-cellulose (4) and 8HQ-chitosan (6), and the signals were assigned
using data from the literature [33–35] are demonstrated in Fig. 3.
In both cases, the presence of the 8HQ fragment in the biopolymer
matrix is clearly demonstrated by the signals of aromatic carbon atoms
(8HQ) resonating in the frequency region from 156 to 113 ppm. As
chitosan and cellulose are both derivatives of glucose, their 13C NMR
resonances show similar values (110–53 ppm). For instance, the signal
of anomeric carbon C1 is clearly apparent ca. 100 ppm. On the other
hand, the overall spectral pattern of cellulose considerably differs from
the pattern of the chitosan fragment. Consequently, the primary
structure of biopolymer matrices can be identified with absolute cer-
tainty, and the systems were easily discriminated from each other.
Moreover, in the sample of 8HQ-cellulose (4), the CH3 units from the N-
methylamino cellulose (3) complex resonate in the frequency range
from 43 to 41 ppm.
The low-intensity signals detected in the spectrum of 8HQ-chitosan
at 174 and 23 ppm reflect C]O and CH3 units, respectively. These
signals indicate the presence of chitin structure units. In this regard,
these signals were also found in the spectrum of neat chitosan used for
further modifications (Fig. S6). In the 8HQ-chitosan (6) sample, traces
of ethanol were also found, identified by the signals at 58 and 18 ppm,
resulting from the preparation process. Because these signals were
generated using the 1He13C cross-polarization process, the molecules of
ethanol must be strongly bound onto and incorporated into the bio-
polymer matrix.
The CH2 linking units between the 8HQ and the biopolymer matrix
resonate in the region from 52 to 48 ppm, according to the literature
[35]. When analyzing the recorded spectra, we found these expected
signals ca. 50 ppm for both complexes. These linking units are denoted
by C7 for 8HQ-chitosan (6) and C10 for 8HQ-cellulose (4). This finding
thus affirmed the chemical modification of both biopolymer matrices
and the substitution of chitosan and cellulose polymers by 8HQ frag-
ments.
In general, the quantitative analysis of 13C CP/MAS NMR spectra is
always complicated by the unequal rate of polarization transfer from
protons to neighboring carbon units. Consequently, the simple in-
tegration of detected signals provides inaccurate quantitative results,
and the amount of non-protonated carbon units is usually under-
estimated. However, when comparing compositions of structurally si-
milar systems and using identical experimental parameters, the ob-
tained 13C CP/MAS NMR spectra provide a suitable representation of
the relative amounts of individual components. Using Eqs. (1) and (2)
for modified cellulose (4) and chitosan (6), respectively, enables as-
sessing the amount of 8HQ binding onto the polymer matrices by in-
tegrating the corresponding signals (Fig. 3) [36]:
(I8HQ)/27
8HQ (mol%) = ∗100
ICellulose,C1 (1)
(I8HQ)/9
8HQ (mol%) = ∗100
IChitosan, C1 (2)
where I8HQ is the integral signal from carbons that belong to aromatic
rings of 8HQ with chemical shifts of 156–113 ppm, ICellulose, C1
(IChitosan,C1) is integral signal of anomeric carbon/chitosan. In the 8HQ-
cellulose (4) sample, the rate of substitution of cellulose units by 8HQ
was determined to be ca. 93% of the theoretical amount (1). In this
case, one cellulose unit can be assumed to be ideally substituted by
three molecules of 8HQ. For the 8HQ-chitosan (6) system, the rate of
substitution of chitosan units by 8HQ groups was calculated to be ca.
56% using Eq. (2) (in an ideal case, one 8HQ fragment per chitosan unit
is assumed). The lower degree of substitution of chitosan than that of
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Journal of Controlled Release 273 (2018) 131–138
cellulose can be attributed to the fact that part of the amino groups on
the chitosan participates in the crosslinking with glutaraldehyde.
The conversion of the reactions with 8HQ was determined using 13C
ss NMR and elemental analysis. The amount of 8HQ bound on the
cellulose particles (C8HQ) was calculated from the 13C ss NMR spectra of
8HQ-cellulose and elemental analysis. For the calculation, Eq. (3) was
used:
I8HQ ⎞ ⎛ ωc ⎞ ⎛ 1000 ⎞ ⎛ I8HQ ⎞
C8HQ = 1000 × ⎛ ⎜ ×⎜⎟ ⎟ × ⎜ ⎟ = × ωc × 9.2506
⎜ ⎟
⎝ IΣ ⎠ ⎝ ω8HQ ⎠ ⎝ M8HQ ⎠ ⎝ IΣ ⎠
(3)
where I8HQ is the integral signal from carbons that belong to aromatic
rings of 8HQ with chemical shifts of 156–113 ppm, I∑ is the sum of all
carbon integral signals from the sample, ωC is the weight ratio of the
carbon in the sample, ω8HQ is the weight ratio of carbons in 8HQ,
(ωC8HQ = 0.7447), and M8HQ (145.16) is the molecular weight of 8HQ
[mmol/g]. The content of 8HQ groups covalently bound onto the cel-
lulose polymer chain using Eq. (3) was determined to be 3.72 mmol/g.
The content of 8HQ groups [mmol/L] in chitosan was also determined
using Eq. (3). The content of 8HQ in the crosslinked chitosan was de-
termined to be 2.24 mmol/g. This content is approximately 10 times
higher than that in our previous studies, where poly(glycidyl metha-
crylate-co-ethylene dimethacrylate)-based beads were utilized as a
model carrier. The suitability of the materials described in this article is
much more promising.
Based on these experimental data, we can conclude that the content
of 8HQ groups is much higher than that of the previously used particles
based on poly(glycidyl methacrylate-co-ethylene dimethacrylate)-based
beads. This statement also is underpinned by the higher scavenge ca-
pacity of the copper by new carriers. Table 1 shows the chemical
modification properties and maximum scavenging capacity of the Cu of
the carriers.
3.3. Biodistribution of 64
Cu-biopolymers in Wistar rats
Two different in vivo experiments using Wistar rats were performed.
First, we observed biodistribution in vivo using μPET/CT-imagining
studies. Images were taken at 2 h and 8 h after oral application of 64Cu
and 64Cu-labeled carriers. The second ex vivo biodistribution experi-
ment was performed to measure 64Cu (model) and 64Cu-labeled carriers
uptake from the digestive tract into the internal organs in 8 and 24 h.
The use of radioactive isotopes is advantageous because biodis-
tribution in the body can be traced very precisely. The radionuclide
64
Cu also has an optimal half-life (12.7 h) for imaging and in vivo ex-
amination while passing through the GIT.
However, using animal models led to several obstacles that had to
be overcome by some approximation. The first approximation is that up
to date there are not animal models that can simulate Wilson disease.
The second approximation was that both types of carrier beads had to
be ground before they were administered orally using the gastric probe.
Grinding was done in ball mill in solid state. Particles were grinding for
3 h at r.t. Grinding was necessary because using a gastric probe for
administration into the rats' stomachs was impossible due to fast par-
ticle settling and probe plugging. However, particles well under 1 μm
are undesirable because they can be partly absorbed in the colon by
Table 1
Functional substitution and maximum complexation capacity of the carriers modified
with 8HQ.
Carrier 8HQ content Capacity Cu Substitution rate 8HQ per
[mmol/g] [mmol/g] pH 2/ [%] unit
pH 4
8HQ-chitosan 3.72 1.17/1.51 56 1
8HQ-cellulose 2.24 1.72/1.86 93 3
M. Vetrik et al.
Fig. 4. In vivo biodistributions of copper scavengers and coronal projections of Wistar rats μPET/CT scans. From left:
suspension - 2 (A, B), 64Cu-8HQ-Chitosan suspension - 3 (A, B). Each group has two time points, A - 2 h and B - 8 h.
Peyer's patches [37]. Therefore, both beads were ground on a ball mill
in dry stay and carefully washed with water and PBS before the ex-
periment. The particle size distribution after grinding was measured by
a particle size analyzer in the wet state. The administered 8HQ-cellulose
(4) and (6) particles had median diameters of 56.6 and 63.3 μm, re-
spectively. The size characteristics of both ground materials, the size
distributions of both particles and SEM microphotography can be found
in the supplementary information (Table S1, Figs. S7–S9). Before the
experiment carriers were swollen in PBS buffer for 5 days while re-
peatedly changing the buffer.
3.3.1. Imaging μPET/CT
3.3.1.1. 64CuCl2 control group. The animals treated with 64CuCl2
showed the expected pattern for the biodistribution of orally
administered free 64Cu2 + ions. Two hours after administration, the
64
Cu was mainly located in the stomach and intestines (Fig. 4-1A).
Some administered dose was already present in the blood (Fig. 4). After
8 h, the kidneys, lungs and stomach are clearly exhibited the presence
of 64Cu presence due to uptake into the bloodstream (Fig. 4-1B). We
recommend to see the video scans of all groups of animal models. The
video files can help to identify the position of the radioactive copper
during the biodistribution. This can be found in supplementary material
in video section.
3.3.1.2. 64Cu-8HQ-cellulose. Two hours after administration, the
majority of the activity was located in the stomach (Fig. 4-2A). Eight
hours after the administration approximately half of the administered
radioactivity was still located in the stomach (Fig. 4-2B), while
approximately the other half passed through up to the colon and into
the feces, as shown in the image.
3.3.1.3. 64Cu-8HQ-chitosan. The biodistribution using 64Cu-8HQ-
Chitosan shows that most of the radioactivity is located in the
stomach 2 h after administration (Fig. 4-3A). After 8 h, the
administered radioactivity had migrated through the digestive tract
and was located in the colon (Fig. 4-3B). At this time point, formation of
excrements in the colon is evident. These excrements show the high
radioactivity concentration that is indicator of the main clearance
pathway - feces. The radioactivity could be found in the lungs, which is
most likely an artifact of administration using the gastric probe, when
an animal may have accidentally aspirated some of the radioactive
mixture. This effect was sometimes also observed in our previous
studies [28]. However, we used just one animal model for each
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Journal of Controlled Release 273 (2018) 131–138
64
CuCl2 solution: 64
Cu-Control- 1 (A, B), 64
Cu-8HQ-Cellulose
evaluated carrier system, and because the μPET/CT imaging is very
difficult to quantify, we performed ex vivo experiments to gain precise
quantitative insights into the situation applying statistical methods.
3.4. Ex vivo biodistribution of 64
Cu after administration of biopolymers in
Wistar rats
The ex vivo biodistribution yields more quantitative insights into
the overall radioactive biodistribution. Copper absorbed from the in-
testine is delivered to the liver, where almost all copper is incorporated
via ceruloplasmin from the blood plasma. The liver plays a major role in
the regulation of copper excretion. Most copper excretion occurs
through the bile and enterohepatic cycle. Only small amounts of copper
are excreted via urine, sweat and menstrual blood [7]. Thus, the copper
content in the liver is the best marker to determine copper absorption
from the GIT. We followed the copper uptake of 64CuCl2 and the uptake
of the modified biopolymer carriers 8HQ-cellulose (4) and 8HQ-chit-
osan (6) from the digestive tract in Wistar rats (Fig. 5).
A much higher amount of copper was found in the livers of the
controls (8.3% of initial radioactivity) than in the groups with 8HQ-
cellulose (4) or 8HQ-chitosan (6) (0.7% and 1.1% of the initial radio-
activity, respectively). This indicates that much more copper was ab-
sorbed from the GIT in the controls than in the groups with the che-
lator-modified biopolymers. We measured a certain quantity of copper
radioactivity in the blood of the controls (1.8% of initial radioactivity)
Biodistribution of 64CuCl2 after 8 h from administration
Percentage of initial radioactivity
Controls
Cellulose + 8-HQ
Chitosan + 8-HQ
Fig. 5. The biodistribution of radioactive 64Cu in individual organs 8 h after adminis-
tration with 8HQ-Cellulose (4) and 8HQ-Chitosan (6).
M. Vetrik et al.
Table 2
The average biodistribution of radioactive 64Cu in selected organs 8 h after administra-
tion. ANOVA statistical values (P,F) were calculated for α = 0.01 against a control group.
Control 8HQ-Cellulose
Liver [%] 8.3 ± 2.7 0.7 ± 0.6
P _ 0.000046
F _ 46.68
Blood [%] 1.8 ± 0.9 0.1 ± 0.2
P _ 0.0019
F _ 17.36
Kidneys [%] 5.7 ± 2.8 0.1 ± 0.1
P _ 0.00071
F _ 23.19
Biodistribution of 64CuCl2 24 h after administration
Percentage of initial radioactivity
Controls
8HQ-cellulose
8HQ-chitosan
Fig. 6. The biodistribution of radioactive 64Cu in individual organs 24 h after adminis-
tration with 8HQ-cellulose (4) and 8HQ-chitosan (6). Fig. 6. shows all organs where
radioactivity was detected.
while the radioactivity was almost zero in the groups with the biopo-
lymers (0.1% of initial radioactivity for both 8HQ-cellulose (4) and
8HQ-chitosan (6)). We also found significant differences in the radio-
activity in the kidneys of the animals (5.7% of initial radioactivity for
controls, 0.1% of initial radioactivity for 8HQ-cellulose (4), and 0.7% of
initial radioactivity for 8HQ-chitosan (6)). The importance of these
results evidently lies in the fact that the modified polymer complexes
with 64Cu prevent its absorption into other organs. Thus, these results
indicate that the biopolymers 8HQ-cellulose (4) and 8HQ-chitosan (6)
can complex copper from food and simply passed through the digestive
tract with the chelated copper.
Very low radioactivity (less than 1% of initial radioactivity) was
measured in the hearts and spleens of the animals. We could also ob-
serve radioactive copper in the lungs of some but not all of the animals,
which could have been caused by the presence of blood or by the partial
aspiration of copper from the gastric probe during administration.
Table 2 shows numerous values of 64Cu in selected organs 8 h after
administration. Table 2 shows that all values are significantly different
from those of the animal control group according to α = 0.01.
Copper uptake of 64CuCl2 and uptake of the modified biopolymer
carriers 8HQ-cellulose (4) and 8HQ-chitosan (6) are shown in Fig. 6.
Twenty-four hours after administration, the majority of the radio-
activity was found in the appendix, small intestine and colon of the
control rats (altogether less than 10% of initial radioactivity).
Moreover, we measured a higher level of radioactivity in the livers and
kidneys of the control group than in the same organs of the groups
receiving chelating polymers. Therefore, the chelating polymer com-
plex 8HQ-cellulose (4) and 8HQ-chitosan (6) were able to prevent
copper ions to be uptaken into the internal organs. Only radioactive
parts (organs) are depicted in Fig. 6. Other organs and body parts (e.g.,
muscles, head, and limbs) were measured but the presence of radio-
active 64Cu was under the detection limit. Therefore, we suggest that
rest of the applied radioactivity is eliminated through the feces or urine
Journal of Controlled Release 273 (2018) 131–138
over 24 h. We also observed no symptoms of toxicity for any of the
administered polymers within the duration of the experiment. Twenty
four hours after the administration we measured radioactivity in the
8HQ-chitosan lungs only in control groups with average value 0.64%.
The results and observations from these experiments demonstrate
1.1 ± 0.7 the more advanced ability to chelate copper by 8HQ-cellulose (4) and
0.000078
8HQ-chitosan (6) than by our previous poly(glycidyl methacrylate-co-
40.96
0.1 ± 0.2 ethylene dimethacrylate)-based beads [7]. Moreover, cellulose and
0.0019 chitosan are biologically very well tolerated in humans, which indicates
17.48 that these biopolymers may be used more efficiently and can be less
0.7 ± 0.4 harmful to patients undergoing treatment for Wilson's disease.
0.0017
18.10
4. Conclusions
This study describes the development of biopolymer-based mate-
rials with possible excellent properties for the treatment of Wilson's
disease. The proposed treatment uses modified cellulose and modified
chitosan with covalently bound 8HQ as carriers for scavenging copper
directly from the GIT. The described scavengers are able to complex
copper from the model solution, hold complexed copper in the whole
GIT and help to eliminate it from the body. Both these carriers contain
more than 10 times more chelator groups than our previous model
based on poly(glycidyl methacrylate-co-ethylene dimethacrylate)
beads. These crosslinked insoluble carriers are non-digestible do not
react with bio-compartments and therefore they can be harmless. We
used the radioactive copper isotope 64Cu as a tracer to determine the
copper content in animal organs and tested these compounds in Wistar
rats. The animal groups that received copper with cellulose and chit-
osan with covalently bound 8HQ showed improved clearance of 64Cu
from the organism without organ deposition. After a 24 h interval, no
absorption of the polymer 64Cu complex was measured on the stomach
walls, as we observed with our previous synthetic material. The ex-
amined biopolymers pass through the digestive tract and can be fully
eliminated. All these properties prevent the harmful side effects that are
typical for low molecular therapeutics (that are present in all biological
system) currently in use. Avoiding these side effects can significantly
increase the comfort of patients in real life, who must face lifelong
treatment. Carriers are environmentally friendly and can be biode-
gradable after they are released from the body.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jconrel.2018.01.026.
Acknowledgment
Financial support from the Grant Agency of the Czech Republic
[grant # 16-02870S], the Ministry of Health of the Czech Republic]
grant # 15-25781a], the Ministry of Education of the Czech Republic
[grant # LM2015064] and from the National Sustainability Program I
[NPU I; project # POLYMAT LO1507].
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