Rodriguez Part B2 BILIAWAY

ERC Advanced Grant 2017

Research proposal [Part B2] 1
(not evaluated in Step 1)

Part B2: The scientific proposal (max. 15 pages)

The development of a simultaneous liver and kidney failure is not a rare case in patients suffering long
dialysis treatment or in patients in recuperation after major chirurgical interventions. There are even
described not few cases of acute renal failure accompanying hepatitis A infection. These combined failure
cases are associated to an unacceptable level of severe morbidity and mortality, caused mainly by the lethal
levels of bilirubin reached in blood due the impossibility of being cleared by any of both organs. The
pathology associated with high level of bilirubin in blood is called hyperbilirubinemia. In these cases,
bilirubin is accumulated in the brain, causing an irreversible neurological failure (Kernicterus) 1. The only
way to guaranty a higher survival ratio to these patients is to have a quick, effective and selective way for
bilirubin reduction and removal from blood associated to the dialysis treatment.
This is the challenge of this research project, the possibility of having in a short time a definitive solution for
the depletion of bilirubin for those patients in critical state. The ground-breaking nature of this proposal
does not fall on the superior characteristics of the new adsorbent biomaterial developed, but on its vital
importance for life-saving. Unfortunately, too much patients could need the treatment with these kind of
particles to survive. The main challenge is to create a polymeric material fully compatible with the blood
tissue complexity. The main risks are not to find the way of immobilising the proteins maintaining its
biological activity and to have a proper flow of the blood through the particle cartridge without plugging or
coagulation. This is the high risk/high gain of the proposed research.
Additionally, there are others disorders related to the enzymes responsible for metabolizing bilirubin that can
cause hyperbilirubinemia: Gilbert's syndrome, Crigler-Najjar syndrome, Dubin-Johnson syndrome and
hemolytic anemia. These disorders are nowadays of great concern, since the people who are affected by such
critical failures are increasing in recent years. In fact, it is an enormous problem affecting thousands of
patients around the world in critical health situations: over 1 million people die annually from kidney failure
and more than two million patients worldwide are already in some type of dialysis 2. Of dramatic importance
is to find a solution for the jaundice in newborns, where there is a ubiquitous transitional morbidity in the
vast majority of them and hyperbilirubinaemia is the leading cause of hospitalisation in the first week of life
worldwide. While timely and effective phototherapy and exchange transfusion are well proven treatments for
severe neonatal hyperbilirubinaemia, inappropriate or ineffective treatment of hyperbilirubinaemia, at
secondary and tertiary hospitals, still prevails in many poorly-resourced countries accounting for a
disproportionately high burden of bilirubin-induced mortality and long-term morbidity.
In Europe, the use of dialysis has increased in the last 20 years, from 25 to 65 patients per 100,000 people.
During the same period, the number of patients living with a transplanted kidney functioning jumped from
15 % to 38 % per 100,000 people in the OECD, thanks to better technology for renal transplantation and the
improvement of immunosuppressive therapies. Unfortunately, kidney transplantation remains being a
solution for a minority of cases due to limited availability, the complexity and risks associated with
transplantation.3 Thus, dialysis demand is expected to continue growing, in line with the aging population
and obesity cases, since conditions of high blood pressure and diabetes are present in over a 60 % of cases of
patients requiring this treatment. Overall, the annual growth of dialysis is expected to be between 6 and
12%2. In fact, the European dialysis market has grown significantly over the past decades, looking for better
diagnosis, higher long-terms conditions and product innovation. The growing power of providers of private
international dialysis services is leading to assistance improvements and technical developments in this
attractive market. There was an average ratio of 28.11 centres per million population in the European Union
in 2013. In that year, there were 211 million of cases of dialysis and 73,000 machines worldwide were sold.
The growing dialysis market generated 3.6 billion in 2010 and this value increased to 5.5 billion dollars in
2013.3 Such piece of the market cake is attractive to investors at all levels and the developments in the field
are convenient for patients and society.

1
Instructions for completing Part B2 can be found in the ‘Information for Applicants to the Advanced Grant 2017 Call’.
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Rodriguez Part B2 BILIAWAY

Bilirubin is the yellow breakdown product of normal hemocatabolism, caused by the body's clearance of
aged red blood cells which containing haemoglobin. According to the metabolic mechanisms and bilirubin
biochemistry, three types of bilirubin can be defined: conjugated, unconjugated and no albumin-bound. 4
 Direct bilirubin or conjugated bilirubin: It is linked with glucuronic acid and then can be stored in
the gallbladder as part of the bile, for later disposal. The conjugation with the glucuronic acid is
catalysed by the Uridine DiPhosphate Glucuronyl Transferase enzyme (UDPGT) located in the
hepatocyte. Normal standard value is 0 to 0.3 mg/dl adults. It is hydrophilic.
 Bilirubin indirect or unconjugated bilirubin: It is bound to albumin protein in blood and still not
transferred to liver proteins to enter into the liver mechanism for disposal. Normal value is
approximately 0.1 to 0.5 mg / dl adults.
 No albumin-bound: It is bilirubin that is not linked to anything. It can cross the blood–brain
barrier (BBB) and cause brain damage (kernicterus). As has been commented, this bilirubin type is
the one that can cause severe brain disorders and even death.
In the conventional removal pathway, the bilirubin bound to plasma albumin arrives to the hepatocytes and it
binds 2 intra-cellular liver proteins. Inside the hepatocyte, it is transported to the endoplasmic reticulum for
conjugation with two molecules of glucuronic acid and products the di-glucuronated bilirubin (direct
bilirubin or conjugated bilirubin). Once conjugated, it is transported by the bile canaliculus through specific
transporters located in the hepatocyte canaliculus membrane and finally excreted in the urine as urobilin 4.
When the body regulation for bilirubin fails in patients the typical failure is usually a bad operation of the
liver which inhibits the normal process of the bilirubin conjugation. If the direct bilirubin is not produced the
levels of unconjugated bilirubin grow in the body of the patient. If this situation continues, the albumin can
be saturated because the bilirubin cannot be eliminated by the dregs. Due to this fact, no albumin-bound is
generated, deriving in a very dangerous situation.
The development of solutions for the bilirubin removal must consider the ways it is commonly excreted by
the body.
In early seventies, several kinds of therapeutic treatments to mitigate the acute liver failure or chronic
diseases began to be evaluated. These therapeutic techniques were haemodialysis, hemoperfusion,
hemoadsorption, exchange transfusion, plasmapheresis or a combination of several of them or filtering blood
through systems to remove toxic substances. Unfortunately, their efficacy has been proved to be limited and
there is still lack of consensus about their correct application conditions. 5,6
During last decade new methods based in complex procedures using hepatic cells, which are tried to be
incorporated into system to act as bio-artificial livers, have appeared. 7 These systems have a biological
component (pig or human hepatocytes obtained from cadaver livers not useful for liver transplantation) and
extracorporeal circulation system. Currently, these bio-artificial livers are in clinic tests to prove their
applicability and efficacy in patients with hepatic insufficiency. However, the preliminary results showed that
this procedure would have contraindications in patients who have an acute intoxication due to drugs such as
paracetamol and multiple immunological problems.7
In practice, nowadays, there are two main techniques to remove bilirubin in patients with very acute
jaundice: MARS® and PROMETHEUS®.
MARS® (molecular adsorbent recirculating system) began to be used in 1993 and is an established
technology. This system is currently a worldwide treatment to patients with hepatic insufficiency. Nowadays,
more than 9,000 patients in more than 150 clinic centres in 45 countries are attended by MARS ®.8 MARS®
system combines the usage of a non-selective adsorbent to remove molecular toxins bound to albumin and a
dialysis membrane. Blood extracorporeal circuit passes through MARS ® fibre dialyzer FLUXTM. It is
dialyzed with an albumin solution allowing passage of toxins, like the bilirubin, and water soluble due to the
presence of albumin in the dialysate, the substance bound to the plasma proteins. The albumin solution
loaded with toxins then passes through another dialyzer to remove the soluble toxins. The albumin solution is
depleted of toxins and many other non-harmful substances by two absorbent columns. One of them, contain
activated carbon and the other an anionic exchange resin. Thus, the regenerated albumin solution is ready for
a new uptake of toxins from the blood.9
In the case of Prometheus®, blood is drawn continuously from the patient by a dialysis catheter. The
Prometheus® system extracts albumin-bound and water-soluble toxins in two consecutive steps and then
returns the blood to the patient. At first, albumin is separated to the blood by an albumin-permeable
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membrane. After that, the plasma which only contains molecules with the same or slightly higher molecular
weight than albumin, goes to a secondary circuit where toxic substances or proteins are removed by two
different adsorption process: first, a neutral resin and, second, and anion exchanger. When the adsorption is
finished, a dialysis process take place: water-soluble and toxic substances such as urea, creatinine or
ammonia, which are not removed in the adsorption; are eliminated by hemodialysis. Purified-albumin
plasma, linked to the blood without toxic compounds, is finally returned to the patient. 10
MARS® and PROMETHEUS® work in serial with the circuit of the standard dialysis system, as an additional
treatment.
One of the most critical problems of these systems is the difficulty to predict the bilirubin removal, because
in both of them the clearance of bilirubin and the reduction ratio of bilirubin after a single session differ
between patients and sessions, probably because they are not selective to bilirubin. Additionally to this
problem, a reduction of depurative capacity is observer along a treatment, due to the saturation and reduced
efficiency of the adsorbent columns used by both of them. Besides, the main disadvantage of MARS ® system
is that it is required a huge amount of concentrated albumin to capture the bilirubin because it is a very
inefficient process. All these problems made them complex to applied and uneconomic; for example, Hessel
et al. calculated that the direct cost per patient were of 15,509 € for the MARS ® system in 2003.11 Moreover,
with respect to cytokines, both MARS® and PROMETHEUS® fail to reduce serum levels due to their low
removal capacity when compared to cytokine production rates.
In conclusion, none of both systems solves the problem of bilirubin accumulation in hepatic failure, since
both have a strong reduction of the bilirubine elimination rate along the treatment, and, besides, do not
resolve the problem of the cytokine removal. Additionally, They present important contraindications to their
use such as disseminated intramuscular coagulopathy, thrombacytopas or coagulopacy with INR>2.3. 9
Furthermore, they are uneconomic processes.
General aim
Considering the mentioned problems of the available commercial systems and the rest of drawbacks of the
mentioned studied solutions in the state of the art, an option to overcome most of the mentioned limitations
could be the creation of a totally new functional particulate material to capture selectively the bilirubin
(based on albumin) in an extracorporeal treatment of the patients’ blood or a system to conjugate the
bilirubin and later be dialyzed.
Thus, ideally, there are two ways to get this objective: the anchoring of albumin to a solid matrix for the
selective entrapment of bilirubin or the immobilization of the UDPGT enzyme that can conjugate the
bilirubin with glucuronic acid to make it water soluble for its dialyzation.
Specific aims
In this work, the exploration of both alternatives is proposed, on the basis of creation of particulate materials
to solve the problem of hyperbilirubinemia. For this purpose, the following specific aims are proposed:
1. Development of different functionalized particles that can act as support for the albumin or the
UDPGT enzyme.
2. Albumin pathway: immobilization of the albumin with a specific configuration onto the previously
developed supports. A ground-breaking option to solve the lack of selectivity of the commercial
techniques to try to remove the bilirubin from blood would be to increase the selectivity of the
albumin by the conformation that it acquires when is joined to a solid matrix.
3. Enzymatic pathway: immobilization of the UDPGT enzyme with a specific configuration onto the
previously developed supports. UDPGT is a human enzyme which allows bilirubin degradation
using glucuronic acid. By this way, bilirubin is made soluble and can be eliminated with urine or
bile. This pathway tries to reproduce the real process which take place in human body but as part of a
dialysis system and suppose a completely different alternative to the techniques used nowadays.
Additionally, the enzyme performance is expected to be enhanced by the immobilization. 12,13
4. Analysis of the final developed systems (matrix-albumin or matrix-enzyme) efficiency in the
bilirubin removal from synthetic samples and human blood.
5. Design of the cartridge for the particulate material application in dialysis.

Potential impact
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With the figures presented in the state of the art in mind, it is estimated a market of nearly 1 MM of cartridge
units per year containing the developed material of this project to remove the no albumin-bound bilirubin. If
each catheter contains 200 g of selective microcapsules, this means that the production of microcapsules
should be at least of 200 Tons/year. As this is a High Added Value dispositive, the price of each filter should
not be less than 100 €, what means a business of more than 100 MM €; employing around 200 persons
permanently.
But beyond the economic impact, the most important issue is the amount of human lives that could be saved
and the improvement of the life quality of many people by the employment of such simple cartridge together
with the dialysis treatment. This system would be a more selective and economic alternative than the
MARS® and PROMETHEUS® systems (which treat more than 9000 patients per year) and could reach a
major number of patients.
Additionally, the principal investigator have lead more than 10 PhDs in the development of new materials
and the duration and volume of work of the proposed project will allow to form several new PhDs in the
field of the material science and chemical engineering. Scientific contributions in scientific journals indexes
in JCR and the attendance to scientific congresses will also contribute to stablish collaborations with another
European research groups to impulse the achievement of a successful solution for the hyperbilirubinemia
problem.

Section b. Methodology
In order to achieve the aims of the project, the following tasks will be developed:
Task 1. Literature review.
The PI investigator and the rest of the group will carried out during the whole project a revision of the novel
advances and methodologies that can appear in literature related to topic of research, looking also for
possible patents in the field.
Task 2. Experimental set-up and analytical techniques acquisition and adaptation
Task 2.1. Experimental set-up acquisition and adaptation
Among the facilities of the research group of the principal investigator, there are different equipment that can
be adapted for the synthesis and functionalization of the supports such as:
- Glass reactors with different volume from 0.2 liters to 100 liters. All these ones have been previously
use for the synthesis of microcapsules with shells from polystyrene (PS), polystyrene-methyl
methacrylate, polystyrene-divinylbenzene, polyurethane, silicone, etc. In this project, different
recipes will be tried in order to get directly functionalized microparticles what can involve changes
in the reactor tap for feeding reagents in the course of the reaction. Besides, the stirrer shape and
dimensions and a new feeding system provided with a special membrane must be acquire for the
control of the particle size of the microparticles.
- Pressure reactors BERGHOF 3000 BR 300 and BÜCHI BEP 280 with volumes of 0.3 and 0.6 liters,
respectively. These are commonly used for the synthesis of functionalized polyols for the further
production of polyurethanes. A system for the data acquisition and control of the main variables
(pressure and temperature) and the necessary elements for the automatization of the monomers
feeding to the reactor will be acquire for a better control and reproducibility of the obtained products.
- Microwave equipment for reaction performance of the union between the albumin and enzyme to the
functionalized supports. As will be described later, one of the proposed reactions for the union of the
albumin or the enzyme to the developed supports is a click chemistry reaction which can be
performed at room temperature and under atmospheric pressure but the reaction rate is sometimes
significantly increased by microwave assistance; thus, the acquisition of a microwave equipment is
proposed.
Task 2.2. Analytical techniques acquisition and setting-up.
The developed supports will be characterized by Fourier Transform Infrared Spectroscopy (FT-IR)
Thermogravimetric analyses (TGA) in the facilities of the research group and will be sent to be analyzed by
nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-

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TOF) mass spectrometry (MS) in order to determine the base composition and the incorporation of the
functional groups.
Scanning electron microscope (SEM) and low angle laser light scattering (LALLS) techniques will be used
for the surface shape characterization and particle size and particle size distribution determination. Besides,
the EDAX from the SEM equipment will allow to confirm the linkage of the albumin or the enzyme by the
determination of the amount of nitrogen in the particle surface. Additionally, a new module of the SEM
equipment will be adapted for the indentation hardness evaluation of the supports since it is a key property
for the suitability of the support in dialysis catheters in order to avoid an excessive pressure drops and system
clogging.
UV-visible spectra will be performed in order to confirm the bilirubin retention by the developed materials
constituted by the albumin or enzyme immobilized onto the different supports. Besides, thermogravimetric
analysis (TGA) can also be used to determine the material composition, observing different degradation steps
or weight percentages of each step for the support, the albumin or enzyme and the case of presence of
albumin after the removal tests in the matrix.
To identify in blood metabolites and the relative amounts of unconjugated bilirubin and sugar mono-
and di-conjugates of bilirubin liquid chromatography positive ion electrospray ionization tandem
mass spectrometry (LC/ESI-MS/MS) has to be employed.
Task 2 deliverable: report with the equipment and characterization techniques acquisition and setting-up.
Regarding the scientific approach, it will be divided in the two main pathways commented above, with a first
common step to obtain the matrix support adequate for both alternatives:
Task 3. Development of suitable matrixes for albumin or UDPGT enzyme immobilization
As indicated in the general aim, the matrix for the immobilization of the albumin or the enzyme must be a
functional particulate material suitable to be installed into a cartridge for the extracorporeal blood treatment,
similar to that conventionally executed in dialysis systems. Thus, the particle size and hardness will be key
issues in order to avoid high pressures drops and system clogging. To achieve this aim, particles with narrow
size distributions and sizes between 100 and 500 m will be explored, what should be enough for avoiding
large blood molecules withholding. Regarding hardness, it will be tested by an indenter module of the SEM
equipment and depending on that results, the particles will then tried in a cartridge testing that they do not
collapse when the blood flux come into contact with their bed.
For the support development, silica, polyurethane and polystyrene matrixes would be tested. The
employment of some component based on renewable source for example bio-polymers in the development of
these matrix will be also considered.
On the other hand, in a second step, or directly during its synthesis, the matrix will be functionalized. To this
aim, the presence of free reactive groups in the different kind of matrixes will be used to bond the albumin or
the UDPGT enzyme. In Table 1 are shown different functional groups that can be anchored to the matrix
since they are present on proteins or enzymes.

Table 1. Reactive Functional Groups in Natural Amino Acids

Amino
Reactive groups Amino acid Reactive groups
acid
-NH2 Primary amine Lysine Phenol Tyrosine
Glutamic
- Carboxylic acid
-CH2-S-CH3 Thioether Methionine
COOH acid Aspartic
acid
-SH Thiol Cysteine Imidazole Histidine

Serine
-OH Hydroxyl Guanidino Arginine
Threonine

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Task 3.1 Development of matrix from polystyrene.

The PI’s research group has a wide experience in the development of functionalized polystyrene
microcapsules by means of suspension like polymerization 14-20 even at a large scale 21. Considering the
previously commented requirements and the experience in this process, the following properties will be
studied and optimized:
- Particle size and distribution. The optimization of the particle size and distribution will be carried out
by the control of the stirring rate, a surfactant agent or by a proper membrane in the feed system 22.
- Material hardness. The hardness of these polystyrene microparticles type is expected to be enough to
avoid the system collapse once implemented on the cartridge, on contrary, it can be modified by
adding crosslinking agents such as divinylbenzene, ethylene glycol dimethacrylate (EGDMA) or
methyl methacrylate14,15,23,24.
The polystyrene microparticles will be obtained by the method described in the reference of Sanchez et al.
(2010)14 using or not co-monomers to modify the physical properties of the microparticles. As the functional
groups of the particles has to be located mainly in the surface of the macropores, a specific strategy for the
incorporation of pendant chain in the skin of the particle. To get this objective, once the identity point (final
size of the particles) is reached a pendant chain functionalised with a reactive group (hydroxyls, halogens,
terminal alkynes, etc.) will be added to the reaction media. This method would supposed a ground-breaking
system for functionalization the particles in a single step without an important effect on the rest of their
properties, especially the particle size and mechanical resistance.
Task 3.2. Development of matrix from polyurethane.
Polyurethanes are synthesized mainly from a polyol and a polyisocyanate and their properties depends on
these two main raw materials characteristics and also on the different additives incorporated in the recipe
(catalysts, surfactants, etc.). The PI’s research group has demonstrated experience in the synthesis of rigid
polyurethane (RPU) foams and even in the synthesis of polyols with the desired characteristics (molecular
weight, hydroxyl number, hydrophilicity, functional groups, etc.), even from waste materials or natural
resources25-38.
In this project, the proposed tasks for the development of the polyurethane matrix are:
- Synthesis from novel co-polyols containing functional groups such as alkyne or azide group, that
will by further used for the albumin or enzyme. Two different routes will be explored:
 Ring opening copolymerization in a single or sequential steps of ethylene and propylene oxides
with different co-monomers incorporating the functional groups such as glycidyl propargyl ether
(GPE), based on the work from Velencoso el al. (2013) 36
 It is known that polyols can be obtained from renewable sources by the epoxidation of natural
oils39. Furthermore, polyols derived from the oils epoxidation can be decorated with different
functionalities by opening the oxirane ring. In this research, the ring-opening reaction with
sodium azide will be explore as possible pathways for the polyol functionalization, following a
similar method to that proposed by De Haro et al. (2016) 34. Besides, the oxirane ring can also be
open for the direct union of amine groups present in proteins and enzymes.40
- Rigid polyurethane (RPU) recipe optimization. Once obtained the polyols, the albumin or enzyme
will be linked to them and then, the recipe and system for the RPU synthesis will be also optimized
to achieve proper particle size and mechanical properties varying the reagents (chain extenders,
crosslinkers of different functionalities, isocyanates, etc.) and the operating conditions (stirring at
high temperatures and cooling, suspension media, etc).
Task 3.3. Development of matrix from silica.
Silica and biosilica matrixes with different functionalities and configurations (spheres, gels, etc.) have
proven to be excellent supports for enzymes immobilization. 12,41-46 The proposed silica support on this
research, consist on an aldehyde activated silica matrix. A diol bonded silica will be initially formed by
reaction between silica and 3-glycidoxypropyltrimethoxy silane. Both compounds will be shaken and heated
up above 100ºC during 5 hours. Then, this intermediate product formed will be mixed with acetic acid water
mixture under reduced pressure to produce the aldehyde activated silica support.

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The development of each one of the functionalized supports is a key intermediate goal, since they can be
applied for the immobilization of a huge amount of molecules of interest or for instant as carriers for
controlled delivery of drugs, etc.

Task 3 deliverable: some grams of the different synthesized supports and a report with their properties
characterization.

At this point, the method to link the albumin or the UDPGT enzyme to the developed matrix will be selected.
There are different methods for protein immobilization, among which stand out the following ones due to the
fact that they present more effective results:
- Physical adsorption: the protein or enzyme is retained by different types of union sites located in the
surface of an inert material. 40,47 This kind of immobilization usually prevents significant changes of
the protein structure but it presents the disadvantage of easy loss of the protein by lixiviation. 40
- Covalent link: this method involves the permanent union between the protein or the enzyme and an
activate group from the support. Nowadays, it is the most commonly used method for proteins or
enzymes immobilization since it presents the big advantage of minimizing the leakage of the
immobilized molecule.40 For this reason, this will be the proposed linkage between albumin and the
UDPGT enzyme in this research work.
The covalent process can vary depending on the case of immobilizing the albumin or the UDPGT enzyme:
Albumin pathway
Task 4. Linkage between the support functional groups and the albumin.
The immobilization of the human albumin has been previously performed in different supports, such as
polystyrene or polyamides, in order to use it as reagent for the purification of antibodies to itself, for
studying the complex forming properties of human serum albumin, etc. However, most of the studies use
indirect approach that can give distorted results.48
The plasma albumin molecule is composed of three structurally homologous helical domains (I, II and III),
each one of which is divided into two subdomains, A and B. 49,50 Despite the internal structural symmetry, the
three domains have different capacities for binding fatty acids, thyroxine and drugs. 49,51,52 Thus, to determine
the best domain for bilirubin capture is essential. Besides, albumin-bilirubin links are reversible and in some
special conditions anions displace bilirubin from the albumin; what will be also studied in the project. Thus,
the albumin’s orientation once linked is really important because its function depends on its 3-D structure. As
indicated before, a covalent immobilization using functional groups of the albumin and in the surface of the
matrix has been selected as the most appropriate way to achieve this objective. The reaction conditions will
change depending on the type and chemistry of the substrate, its hydrophobicity or hydrophilicity, and the
coupling conditions such as temperature, pH, and ionic strength of the solution. It is necessary to assure that
the functional group, which will be joined to the bilirubin, does not react with the matrix. On the other hand a
key issue will be the pH control in order to avoid the albumin denaturalization since proteins are quite
sensible to pH changes.
The proposed chemical linkage between the matrix and the albumin will be a “click” reaction. The “click”
reactions are a set of ‘near-perfect’ bond-forming reactions useful for quickly assembly of molecules with
desired function. The previously mentioned functional groups of the matrix (Cl, OH, oxirane rings, etc.) can
be modified to get an azide group or an alkynyl group. For example the sodium azide can be introduced in
halogenated molecules or matrixes.27,36,53 Then, by means of the copper-catalyzed Huisgen 1,3-dipolar
cycloaddition (CuAAC) between azides and terminal alkynes, a triazole ring between both parts will be
formed, being a very stable link.54
The studied and optimized variables will be:
- Solvent mixture to facilitate the contact between reagents. The solvents must be easily removal
under smooth conditions to avoid the protein denaturalization.
- Reaction temperature. Most of CuAAC reactions take place at room temperature but the influence of
smooth temperatures in the reaction rates will be analysed.

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- Microwave effect. The CuAAC will be carried out under microwave conditions since it can
significantly reduce the reaction rate.
- Catalyst removal. It should be taken into account that this reaction is catalysed by Cu (II) salts that
can be toxic to cells, therefore, a purification process for the total removal of the catalyst must be
developed. Fortunately, ion exchange resins usually allow to totally remove this kind of catalysts 36.

Task 4 deliverable: some grams of the different developed systems matrix-albumin and a report with the
results of the characterization of their properties.

Task 5. Analysis of the matrix-albumin systems efficiency in the bilirubin removal.

Finally, different experiments will be carried out in order to determine the efficiency and selectivity to
remove the bilirubin of different pairs of matrix-albumin. Different buffer solutions containing albumin and
bilirubin will be prepared. The buffer solution might be phosphate at pH = 7.4.
The parameters to study will be:
- The amount of the matrix-albumin system per gram of synthetic solution containing bilirubin
- The bilirubin concentration in typical ranges of patients with hyperbilirubinemia.
- The contact time for further dynamic experiments. The dynamic experiments will consist in passing
through the final adsorbent material the bilirubin solution in different concentrations, similar to those
of patients with hyperbilirubinemia.
The best developed option in terms of bilirubin removal from the buffer solution will be compared with the
commercial technique MARS® or with anionic interchange resins. After that, the best conditions would be
tested with blood plasma to observe if the selected matrix-albumin system is selective in a real case.
Task 5 deliverable: a report justifying the best alternative from the developed matrix-albumin system chosen
for the removal of bilirubin.

Enzymatic pathway
Task 6. Immobilization of UDPGT.
Initially, the enzyme will be isolated from a fresh rat liver following the method described by Kim and
Wainer (2005)55. Then, a reaction between UDPGT and the functional groups of the support will be carried
out to immobilize the enzyme. Depending on the support the alternatives are:
- Polystyrene and polyurethanes. For these matrixes, the click chemistry reaction proposed for the
albumin pathway will be the main route to be explored.
- Silica support. For the UDPGT immobilization onto the developed aldehyde activated silica support, a
Schiff base method will be carried out and optimized parting form the results achieved by Kim and
Wainer (2005)55.
- It is also possible to incorporate silica sites in the polystyrene microparticles and join the enzyme by
the Kim and Wainer method, combining the advantages of both previously studied systems in a
promising system for the application of immobilized enzyme in the bilirubin removal in dialysis pre-
treatments.
Task 6 deliverable: some grams of the different developed systems matrix-UDPGT enzyme and a report with
the results of the characterization of their properties.

Task 7. Analysis of the matrix-enzyme systems efficiency in the bilirubin removal.

An exhaustive method to test the efficiency of each support will be carried out. Bilirubin and glucuronic acid
will be contained in the experimental solution that will flow throughout the bed that includes the support.
Glucuronic acid concentration will be set to blood concentration value. Bilirubin concentration will be varied
from common blood concentration to the highest concentration a person is able to endure.

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This solution will be introduced in an absorption batch bed that contains the activated support. The solution
will remain in the column for a fixed time length. Afterwards the bilirubin concentration will be measured by
using a liquid chromatograph. The higher difference between inner and outer bilirubin concentration, the
better the efficiency will be. The ratio bilirubin removal/g of UDPGT will be determined for each different
support in order to select the best alternative.
Task 7 deliverable: a report justifying the best alternative from the developed matrix-UDPGT system chosen
for the removal of bilirubin

Once the best support has been chosen, effects of human blood on conjugation reactions will be determined.
Optimization of design parameters in order to increase the removal efficiency and to prevent conditions of
deactivation will be studied. The parameters studied on the efficiency will be: temperature, amount of
enzyme, enzyme kinetics, deactivation enzyme conditions55.

Task 8. Design of the cartridge for the developed material application in dialysis.
At this point, the results of bilirubin removal from human blood will have indicated the most effective of the
developed materials. Besides, from the previously obtained results, it will be possible to design a commercial
catheter for the developed material application in dialyses treatments. The following calculation will be
done:
- The appropriate amount of material per catheter. An estimation of the suitable amount of material for
the daily dialysis treatment depending on the patients characteristics can be calculated and; with this,
determine the appropriate amount of material per catheter.
- Dimensions and design of the complementary dialysis device. From the amount of material and its
characteristics (particle size, hardness, swelling, etc) a first approach of the necessary devices for the
removal of the bilirubin as an additional part of traditional dialysis systems will be design.
- Besides, to avoid blood coagulation during the bilirubin removal, the addition of anticoagulant will be
probably necessary, trying to keep this way a proper viscosity for the blood flux through the bed of
particulate material. The type and amount of anticoagulant and the addition method will be also
studied.
Task 8 deliverable: report with the description of the developed device.

Table 2 shows the Gantt chart of the project with the main project tasks.

9
Rodriguez Part B2 BILIAWAY

Year Y1 Y2 Y3 Y4 Y5
Month 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
Literature update (Task 1)
Experimental set-up and
analytical techniques acquisition
and adaptation (Task 2)
Development of matrixes for
albumin or UDPGT enzyme
immobilization (Task 3)
Linkage between the support
functional groups and the
albumin (Task 4)
Analysis of the matrix-albumin
systems efficiency in the
bilirubin removal (Task 5)
Immobilization of UDPGT
(Task 6)
Analysis of the matrix-enzyme
systems efficiency in the
bilirubin removal (Task 7)
Design of the catheter for the
developed material application in
dialysis (Task 8)
Table 2. Gantt chart.

10
Applicant's last name Part B2 ACRONYM

As has been indicated through the whole scientific approach, the success of the research is supported by the
wide experience of the PI of the project in materials functionalisation, molecules modification and in
covalent union to polymer matrixes and in the study of the functionalised materials properties, the new
molecules configuration, the union between the molecules and the matrixes and the final properties of the
new materials 16,33,34,36,38,56,57.

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2. W. G. Couser, G. Remuzzi, S. Mendis, M. Tonelli. Kidney International 2011, 80, 1258-1270.
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4. J. Fevery. Liver International 2008, 28, 592-605.
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6. N. Inoue, M. Yoshiba, Z. Yamazaki, T. Sakai, K. Sanjo, K. Okada, T. Oda, T. Wada, T. Inoue, In Artificial Liver Support;
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10. K. Rifai, T. Ernst, U. Kretschmer, M. J. Bahr, A. Schneider, C. Hafer, H. Haller, M. P. Manns, D. Fliser. Journal of
Hepatology 2003, 39, 984-990.
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Technology 2007, 40, 1451-1463.
14. L. Sánchez-Silva, Á. Alcázar, A. De Lucas, M. Carmona, J. F. Rodríguez. Journal of Chemical Technology and
Biotechnology 2011, 86, 437-446.
15. A. Alcázar, A. De Lucas, M. Carmona, J. F. Rodríguez. Reactive and Functional Polymers 2011, 71, 891-898.
16. Á. Alcázar, I. Garrido, E. M. García, A. De Lucas, M. Carmona, J. F. Rodriguez. Separation and Purification
Technology 2015, 154, 255-262.
17. A. Alcazar, M. Carmona, A. M. Borreguero, A. de Lucas, J. F. Rodriguez. Journal of Microencapsulation 2015, 32,
642-649.
18. L. Sánchez-Silva, J. Tsavalas, D. Sundberg, P. Sánchez, J. F. Rodriguez. Industrial and Engineering Chemistry
Research 2010, 49, 12204-12211.
19. L. Sánchez-Silva, J. F. Rodríguez, P. Sánchez. Colloids and Surfaces A: Physicochemical and Engineering Aspects
2011, 390, 62-66.
20. L. Sánchez, P. Sánchez, A. de Lucas, M. Carmona, J. F. Rodríguez. Colloid and Polymer Science 2007, 285, 1377-
1385.
21. L. Sánchez-Silva, M. Carmona, A. De Lucas, P. Sánchez, J. F. Rodríguez. Journal of Microencapsulation 2010, 27,
583-593.
22. L. Sánchez, P. Sánchez, M. Carmona, A. de Lucas, J. F. Rodríguez. Colloid and Polymer Science 2008, 286, 1019-
1027.
23. L. Sánchez-Silva, J. F. Rodríguez, A. Romero, A. M. Borreguero, M. Carmona, P. Sánchez. Chemical Engineering
Journal 2010, 157, 216-222.
24. C. Araneda, C. Fonseca, J. Sapag, C. Basualto, M. Yazdani-Pedram, K. Kondo, E. Kamio, F. Valenzuela. Separation
and Purification Technology 2008, 63, 517-523.
25. A. M. Borreguero, J. L. Valverde, T. Peijs, J. F. Rodr{\'i}guez, M. Carmona. Journal of Materials Science 2010, 45,
4462-4469.
26. A. M. Borreguero, J. F. Rodr{\'i}guez, J. L. Valverde, R. Arevalo, T. Peijs, M. Carmona. Journal of Materials Science
2011, 46, 347-356.
27. A. M. Borreguero, P. Sharma, C. Spiteri, M. M. Velencoso, M. S. Carmona, J. E. Moses, J. F. Rodr{\'i}guez. Reactive
and Functional Polymers 2013, 73, 1207-1212.
28. A. M. Borreguero, J. F. Rodriguez, J. L. Valverde, T. Peijs, M. Carmona. Journal of Applied Polymer Science 2013,
128, 582-590.
29. D. Sim{\'o}n, A. M. Borreguero, A. De Lucas, J. F. Rodr{\'i}guez. Polymer Degradation and Stability 2014, 109, 115-
121.

11
Applicant's last name Part B2 ACRONYM

30. D. Sim{\'o}n, A. M. Borreguero, A. de Lucas, C. Molero, J. F. Rodr{\'i}guez. Journal of Material Cycles and Waste
Management 2014, 16, 525-532.
31. D. Sim{\'o}n, A. M. Borreguero, A. De Lucas, J. F. Rodr{\'i}guez. Polymer Degradation and Stability 2015, 116, 23-35.
32. D. Sim{\'o}n, A. M. Borreguero, A. De Lucas, J. F. Rodr{\'i}guez. Polymer Degradation and Stability 2015, 121, 126-
136.
33. A. M. Borreguero, M. Mu{\~n}oz, J. C. De Haro, M. Carmona, J. F. Rodr{\'i}guez. Reactive and Functional Polymers
2016, 101, 1-8.
34. J. C. de Haro, J. F. Rodríguez, Á. Pérez, M. Carmona. Journal of Cleaner Production 2016, 138, 77-82.
35. M. M. Velencoso, C. Gutierrez, M. J. Ramos, J. C. García-Martínez, A. De Lucas, J. F. Rodriguez. Journal of
Macromolecular Science, Part A: Pure and Applied Chemistry 2011, 48, 569-576.
36. M. M. Velencoso, A. S. Gonzalez, J. C. García-Martínez, M. J. Ramos, A. De Lucas, J. F. Rodriguez. Polymer
International 2013, 62, 783-790.
37. M. M. Velencoso, M. J. Ramos, J. C. Garcia-Martinez, A. De Lucas, J. F. Rodriguez. Journal of Macromolecular
Science, Part A: Pure and Applied Chemistry 2013, 50, 905-913.
38. M. M. Velencoso, M. J. Ramos, A. Serrano, A. de Lucas, J. F. Rodríguez. Polymer International 2015, 64, 1706-1714.
39. A. Guo, Y. Cho, Z. Petrovic. Journal of Polymer Science Part a-Polymer Chemistry 2000, 38, 3900-3910.
40. J. Křenková, F. Foret. Electrophoresis 2004, 25, 3550-3563.
41. A. Rollett, M. Schroeder, K. P. Schneider, R. Fischer, F. Kaufmann, R. Schöftner, G. M. Guebitz. Chemosphere 2010,
80, 922-928.
42. J. Ma, Z. Liang, X. Qiao, Q. Deng, D. Tao, L. Zhang, Y. Zhang. Analytical Chemistry 2008, 80, 2949-2956.
43. G. Yang, J. Wu, G. Xu, L. Yang. Colloids and Surfaces B: Biointerfaces 2010, 78, 351-356.
44. T. N. Nwagu, B. N. Okolo, H. Aoyagi. African Journal of Biotechnology 2011, 10, 15989-15997.
45. C. R. Matte, M. R. Nunes, E. V. Benvenutti, J. D. N. Schöffer, M. A. Z. Ayub, P. F. Hertz. Journal of Molecular
Catalysis B: Enzymatic 2012, 78, 51-56.
46. G. Bayramoglu, A. Akbulut, M. Yakup Arica. Journal of Hazardous Materials 2013, 244-245, 528-536.
47. K. Nakanishi, T. Sakiyama, Y. Kumada, K. Imamura, H. Imanaka. Current Proteomics 2008, 5, 161-175.
48. S. R. Usmanov, I. T. Yakubov, M. M. Rakhimov. Chemistry of Natural Compounds 1998, 34, 496-498.
49. U. Kragh-Hansen, H. Watanabe, K. Nakajou, Y. Iwao, M. Otagiri. Journal of Molecular Biology 2006, 363, 702-712.
50. J. Nilvebrant, S. Hober. Computational and Structural Biotechnology Journal 2013, 6.
51. R. Spinella, R. Sawhney, R. Jalan. Hepatology International 2016, 10, 124-132.
52. L. M. Gartner, I. M. Arias. New England Journal of Medicine 1969, 280, 1339-1345.
53. E. W. Elliott, A. L. Ginzburg, Z. C. Kennedy, Z. Feng, J. E. Hutchison. Langmuir 2017, 33, 5796-5802.
54. A. Mandoli. Molecules 2016, 21.
55. H. S. Kim, I. W. Wainer. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
2005, 823, 158-166.
56. A. M. Borreguero, P. Sharma, C. Spiteri, M. M. Velencoso, M. S. Carmona, J. E. Moses, J. F. Rodriguez. Reactive &
Functional Polymers 2013, 73, 1207-1212.
57. E. Gracia, M. T. García, A. M. Borreguero, A. De Lucas, I. Gracia, J. F. Rodríguez. Journal of CO2 Utilization 2017,
20, 20-26.

12
Applicant's last name Part B2 ACRONYM

Section c. Resources (including project costs)

Cost Category Total in euro

PI3 300037

Senior Staff 301000

Personnel Postdocs 175600

Students 300000

Other

i. Total Direct costs for Personnel (in euro) 1076637

Direct
Costs2 Travel 140000

Equipment 200000

Consumables 200000
Other
Publications (including Open Access fees),
goods and 20000
dissemination activities, etc.
services
Other (financial audit) [2] 8000

ii. Total Other Direct Costs (in euro) 568000

A – Total Direct Costs (i + ii) (in euro) 1644637

B – Indirect Costs (overheads) 25% of Direct Costs4 (in euro) 411159

C1 – Subcontracting Costs (no overheads) (in euro)

C2 – Other Direct Costs with no overheads5 (in euro)

Total Estimated Eligible Costs (A + B + C) (in euro) 2055796

Total Requested Grant (in euro) 2055796

Working environment
BILIAWAY will be carried out at the ITQUIMA installations, a UCLM centre of excellence in Chemical and
Environmental Technologies, leaded by PI Juan Francisco Rodriguez (http://www.itquima.uclm.es). This
research centre will provide state-of-the-art facilities and also help to attract prominent researchers.
The applicant is currently the Head of the Research Institute and the leader of the Research Group of
Laboratory of Polymerization and Separation Technologies. With about 40 researchers, ITQUIMA is one of
the reference research Institutes in the Region of Castilla-La Mancha in funding, number of projects,

2
An additional cost category 'Direct costing for Large Research Infrastructures' applicable to H2020
can be added to this table (below ‘Other Goods and services’) for PIs who are hosted by institutions
with Large Research Infrastructures of a value of at least EUR 20 million and only after having
received a positive ex-ante assessment from the Commission's services (see ‘Information for
Applicants to the Advanced Grant 2017 Call’ for more details).
3
When calculating the salary, please take into account the percentage of your dedicated working time to run the ERC
funded project (i.e. minimum 30% of your total working time).
4
Please note that the overheads are fixed to a flat rate of exactly 25%.
5
Such as the costs of resources made available by third parties which are not used on the premises
of the beneficiary (see ‘Information for Applicants to the Advanced Grant 2017 Call’ for details).
13
Applicant's last name Part B2 ACRONYM

scientific publications and technology transference. More than 70 scientific publications on average per year
and 180 participations in international congress validate the quality of the applied research work performed
in ITQUIMA. Three H2020 projects are running in ITQUIMA right now, being us the coordinators of
NANOLEAP.
As a whole, the Institute have received several distinctions: the best research centre of the Region and the
best “spin-off” of La Mancha. Several large pilot plant facilities including one for heterogeneous
polymerization and another one for spray drying preparation of particulate materials are now in operation in
ITQUIMA.
The assays with human blood, some of the bilirubin analysis and several additional biomedical tests will be
performed in the University Hospital of Ciudad Real where the Translational Medicine Group has a well-
equipped laboratory with a long collaboration trajectory with the applicant.

Human resources

The Principal Investigator will dedicate 50% of his time to the project.
The rest of the ITQUIMA Senior Staff will devote an average of 30 % of their time. A brief description of the
CVs and expertise of the members of my research group is provided in extra Annex 1. Basically is formed by
1 Full Prof (Dr. Antonio de Lucas) and 7 Permanent Staff as Associated Professors (Borreguero, Carmona,
Ramos, Perez, Garrido, Garcia and Gracia).
The team at the Translational Medicine Group at the Hospital is formed by 7 Permanent Staff headed by the
Associate Professor Dr. Redondo.
It’s also estimated needed to engage under a direct contract a postdoc (at least with one year experienced)
and 5 doctoral students in two steps (3 at the starting moment and 2 that will be incorporated after two year
running) during the 5 years of project duration.
External collaborations with preeminent experts in the different fields are foreseen in the proposal, aimed at
maximizing its impact and additional considerations. Specially, the collaboration with Prof. Steve Brocinni
from the Faculty of Pharmacy of the University College of London in the field of polymer functionalization
will be especially useful. Also the close relation that I have with the Bulgarian Institute of Polymers specially
with the Head, Dr. Nely Koseva will be especially useful for polymer characterization. The experience of
Prof. Filomena Barreiro and Isabel Ferreira, from the Instituto Politecnico du Braganza will be especially
useful for the development of the particulate material and analysis of bioactive substances. Finally, the long
term relation maintained with a specialist in drug design and click chemistry as Prof. John Moses of
University of Nottingham (now in temporal leave in Australia) will be required.

Other project direct costs

Travel costs and related subsistence allowances for the researchers are estimated at 140,000 € over the 5
years for 14 researchers, so, the cost per year per researcher is 2000 €.
Equipment 200,000€.
To identify in blood metabolites and the relative amounts of unconjugated bilirubin and sugar mono-
and di-conjugates of bilirubin liquid chromatography positive ion electrospray ionization tandem
mass spectrometry (LC/ESI-MS/MS) has to be employed.
The type of analytical that has to be acquired would be a LC-MS apparatus equipped with a quadrupole time-
of-flight (Q-TOF) mass spectrometer equipped with an ESI source.
Consumables 200,000 €.
Crucibles and other materials for TG: 5,000 €; Crucibles and other materials for DSC: 6,000€; Tools and
spare parts for the measurement of thermal conductivity and density of solids: 5,000€; Chromatography
columns and other consumables: 12,000€; Supports and other consumables for SEM: 10,000 €; Automatic
titration flasks and other non-reusable components: 4,000€; Laboratory glassware: 15,000€; Laboratory non-
reusable devices (pipettes, etc): 8,000 €; Laboratory gases: 10,000€; Laboratory chemicals: 13,000€; Bulk
Chemicals for pilot scale demonstration (spray-drying and/or polymerization experiments):40,000€.
Dissemination activities: 10,000€¸ Equipment Maintenance: 50.000 €, Analytical Standards: 12,000 €
Publications 20,000€. The UCLM participates in the OpenAIRE Project - Open Access Infrastructure for
Research in Europe (https://www.openaire.eu) throughout its repository RUIDERA (https://ruidera.uclm.es)
facilitating publications availability in open access

14
Applicant's last name Part B2 ACRONYM

Financial audit 8,000 €

Request for additional funding above Justification

EUR 2 500 000 for

Keep only that category(ies) that apply to the

project.

(a) covering eligible 'start-up' costs for a PI

moving from another country to the EU or an
Associated Country as a consequence of
receiving an ERC grant and/or,
(b) the purchase of major equipment and/or,
(c) access to large facilities.

Please indicate the duration of the project in months:6 60

Please indicate the % of working time the PI dedicates to the project over the period of
50 %
the grant:

I will be able to dedicate 50% of my total working time to the ERC project, because I will be released from
any teaching duty at UCLM (UCLM directive for PI of ERC grants), and I am to finish in my role as
Researcher of the H2020 RIA NANOLEAP by August 2018.

Request for additional funding above Justification

EUR 2 500 000 for

Keep only that category(ies) that apply to the

project.

(a) covering eligible 'start-up' costs for a PI

moving from another country to the EU or an
Associated Country as a consequence of
receiving an ERC grant and/or,
(b) the purchase of major equipment and/or,
(c) access to large facilities.

6
The maximum award is reduced pro rata temporis for projects of a shorter duration (e.g. for a
project of 48 months duration the maximum requested EU contribution allowed is EUR 2 million).
Additional funding to cover major one-off costs is not subject to pro-rata temporis reduction for
projects of shorter duration (e.g. with additional funding it is possible to request a maximum EU
contribution of EUR 3 million for a project of 48 months duration).
15