Preparation of cubic-shaped sorafenib-loaded nanocomposite using well-defined poly(vinyl alcohol alt-propenylene) copolymer

Article abstract of DOI:10.1016/j.ijpharm.2019.03.008

Vinyl alcohol (VA) copolymers having fine tunable polarities are emerging materials in drug delivery applications. VA copolymers rendering well-defined molecular architecture (C/OH ratio = 2, 4, 5 and 8) were used as carriers for model drug compound, fluorescein, which exhibited significantly different release characteristics depending on the polarity of the polymers. Based on the preliminary drug release tests the well-defined VA copolymer having C/OH = 5 ratio, poly(vinyl alcohol alt-propenylene) copolymer (PVA-5) was selected for nanocomposite synthesis. Sorafenib anticancer drug was embedded into PVA-5 (C/OH = 5 ratio) nanoparticles by nanoprecipitation resulting in nanoparticles exhibiting unusual cubic shape. The sorafenib-loaded nanocomposites showed continuous release during a day and concentration-dependant cytotoxicity on HT-29 cancer cells. This might be interpreted by the sustained release of the drug.

Full text of DOI:10.1016/j.ijpharm.2019.03.008

Accepted Manuscript
Preparation of cubic-shaped sorafenib-loaded nanocomposite using well-de-
fined poly(vinyl alcohol alt-propenylene) copolymer
Tivadar Feczkó, Gabriella Merza, György Babos, Bernadett Varga, Eszter
Gyetvai, László Trif, Ervin Kovács, Robert Tuba
PII:
S0378-5173(19)30184-X
https://doi.org/10.1016/j.ijpharm.2019.03.008
IJP 18190
DOI:
Reference:
International Journal of Pharmaceutics
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 September 2018
3 March 2019
4 March 2019
Please cite this article as: T. Feczkó, G. Merza, G. Babos, B. Varga, E. Gyetvai, L. Trif, E. Kovács, R. Tuba,
Preparation of cubic-shaped sorafenib-loaded nanocomposite using well-defined poly(vinyl alcohol alt-
propenylene) copolymer, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.
2019.03.008
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Preparation of cubic-shaped sorafenib-loaded nanocomposite using well-defined
poly(vinyl alcohol alt-propenylene) copolymer
Tivadar Feczkó,a,b Gabriella Merza,a György Babos,a,b Bernadett Varga, a,b Eszter Gyetvai,a László Trif, a Ervin Kovács,a Robert
Tubaa*
a.
Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences
Magyar tudósok körútja 2., H-1519 Budapest, P.O. Box 286, Hungary.
b.
Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprém, Egyetem utca 10., H-8200,
Hungary.
Vinyl alcohol (VA) copolymers having fine tunable polarities are effects.(Long et al., 2017) Consequently, the development of
emerging materials in drug delivery applications. VA copolymers sustained drug delivery systems to improve the bioavailability
rendering well-defined molecular architecture (C/OH ratio = 2, 4, 5 of sorafenib is an important nanopharmaceutical task.
and 8) were used as carriers for model drug compound, fluorescein
exhibited significantly different release characteristics depending
on the polarity of the polymers. Based on the preliminary drug
Insert Figure 1 here
release tests the well-defined VA copolymer having C/OH = 5 ratio, Polyvinyl alcohol (PVA) has been widely applied in drug delivery
poly(vinyl alcohol alt-propenylene) copolymer (PVA-5) was application in various forms, such as films,(Stampella et al.,
selected for nanocomposite synthesis. Sorafenib anticancer drug 2013),(Gan et al., 2016) hydrogels(Sakai et al., 2013),
was embedded into PVA-5 (C/OH = 5 ratio) nanoparticles by nanofibers,(Mahanta
nanoprecipitation resulting in nanoparticles exhibiting unusual membranes(Manasco
and
et
Valiyaveettil,
al., 2014)
2012)
and
cubic shape. The sorafenib-loaded nanocomposites showed nanocomposites.(George et al., 2012) In composite
continous release during a day and concentration-dependant nanoparticles generally hydrophilic drugs are incorporated by
cytotoxicity on HT-29 cancer cells. This might be interpreted by the PVA.(Malarvizhi et al., 2014) In another approach sorafenib, the
sustained release of the drug.
anticancer
drug
was
loaded
into
PVA-coated
superparamagnetic iron oxide nanoparticles.(Tom et al., 2018)
To our knowledge lipophilic PVA has not been used for the
preparation of nanomedicines. Drug delivery applications can
benefit from tuning the polarity of PVA copolymers so that they
can be used in the regulation of controlled drug release.(Santos
et al., 2018) The polarity of the well-defined PVA copolymers
can be adjusted by changing the density (i.e. C/OH ratio) of the
hydroxyl groups along the polymer chain.(Su, 2013),(Tuba et al.,
2016)
The polymer hydration properties can be modified by the
introduction of polar functionalities on the polymer backbone.
The type and the density of the polar substituents can
significantly affect the hydration properties and so indirectly the
embedded drug release rate.(Ghori and Conway, 2015)
Different polar substitution density allows different water
mobility thus different drug release characteristics.(Rajabi-
Siahboomi et al., 1996) The substituents of a polymer side chain
alter its polarity and its melting point. For instance,
substitutions of the side-chain groups by more polar groups
result in a reduction in the crystallinity of the polymer, which is
reflected in a decrease in its melting point. This affects the
solubility of the polymer in water.(Sarkar and Walker, 1995) The
number of hydroxyl groups has an impact on the polarity of the
polymers used as drug carrier which can have a great effect on
the drug release, as well.
1. Introduction
Physiological processes at the molecular level occur at specific space
and time scales, which are different for every tissue predetermining
the need for the carriers that allow tunable drug release.(Uskoković
and Ghosh, 2016) Metals, polymers, ceramics and bio-derived
materials have been studied and used as medical implants.(Prakasam
et al., 2017) Such materials can be designed for a wide range of
biomedical applications, e.g. controlled drug delivery devices (Park,
2014) and tissue engineering constructs.(Uskoković, 2015) However,
these materials should possess the ability to be tunable to exhibit
complex kinetics of therapeutic molecules’ release. Overall, there is
a need for the development of sensitive and smarter controlled
release carriers in pharmaceutics.
Fluorescein (Fig. 1. a) is a chemical dye having several medical
application. It has been approved by the Food and Drug
Administration (FDA) of the U.S. Department of Health and Human
Services since 2006.
Sorafenib (Fig. 1. b) is a multikinase inhibitor acting at tumor
angiogenesis and proliferation. It is the exclusive drug approved
by the FDA against advanced hepatocellular carcinoma (HCC)
which is the fifth most common malignant cancer in the world
and the third most frequent trigger of cancer-related
death.(Hsu et al., 2017) HCC resistance to chemotherapeutic
agents is especially high. The efficiency of sorafenib on tumor
elimination is low most likely because of drug resistance that
has substantially limited the overall therapeutic effect of cure.
In addition, many patients suffer from multiple side
PEVA (poly (ethylene-vinyl acetate)) as copolymer of ethylene
and vinyl acetate is often used in biomedical engineering
applications as drug delivery matrices. The synthesis of PEVA via
radical co-polymerization of ethylene with vinyl acetate leading

to VA copolymer derivatives are well-known.(Okaya, 1993)
Cationic group-transfer polymerization(Mori et al., 1994) and
of
a
“temporary” strained
cis-4-cyclopentene-1,3-diol
poly(vinyl alcohol2-alt-methylene) was synthetized under
forcing conditions.(Scherman et al., 2002)
palladium-catalyzed
ethylene−vinyl
acetate
copolymerization(Ito et al., 2009) are also reported. These
reactions result in polymers exhibiting randomly distributed
polar and non-polar dyads. However, reports on the synthesis
of VA copolymers exhibiting well-defined molecular
architecture are rare.(Li et al., 2013; Scherman et al., 2002; Tuba
et al., 2014; Valenti and Wagener, 1998)
In this study well-defined VA-copolymers (Fig. 3.) with fine-
tuneable polarity have been investigated as potential drug
delivery materials for pre-programmed drug release. VA
alternating copolymers have been synthetized via olefin
metathesis reactions by the ROMP of cyclooct-4-ene-1-
ol(Martinez et al., 2014a) and cyclooct-5-ene-1,2-diol
(Scherman et al., 2005) and ADMET of 1,6-heptadiene-4-ol
The specific structural characteristics of PVA and VA copolymers
originating from the sequencing (primary structure) of
monomeric units, as well as the configurational and according to standard literature procedures.(Tuba et al., 2014)
conformational assembly of the secondary structure, affect the
drug delivery properties of the carrier material. The key aspect
in these cases is the relative balance between the functional
groups (structure, relative concentration, and position in the
repeating unit) and the overall hydrocarbon content of the
repeating units, as well as their arrangement in homopolymer
or copolymer structures.(Pajak et al., 2010) The biodegradation
of PVA was investigated by several research teams using
different types of microorganisms over the past decades.
Pseudomonas microorganisms can degrade the polymer by the
mechanism through secondary alcohol oxidase (SAO) and β-
diketone hydrolase (BDH), using specific dehydrogenase of PVA
observed in Alcaligenes faecalis KK314 strain.(Chandra and
Rustgi, 1998; Chiellini et al., 2003; Pajak et al., 2010) The
biodegradation of VA copolymers is fostered by hydroxyl
functionalities of the polymer chain.(Chandra and Rustgi, 1998),
(Pajak et al., 2010)
The formed polyolefins were hydrogenated by Pd on carbon
giving the target VA copolymers in quantitative yield. The yields,
the polymers’ molecular weights (Mw) and dispersities (Ð)
summarized in Table 1. correlate well with the data found in
the literature.(Tuba et al., 2014)(Martinez et al.,
2014b)(Scherman et al., 2005)
Insert Figure 3 here
C/O
ratio
Tg
Name
PVA
Structure
Mw
Ð
[°C]
2
4
2000
-
85
PVA 4
-*
1.3 34.4
When the VA-copolymers are synthetized by olefin metathesis
polymerization the distribution of the OH groups along the well-
defined VA-copolymer chain can be predetermined unlike in
case of the random structured PEVA produced by radical
polymerization.
PVA 5
PVA 8
5
8
1340 1.3 -**
6500 5.7 6.8
Well-defined VA copolymers can be synthesized by ring opening
metathesis polymerization (ROMP) or acyclic diene metathesis
polymerization (ADMET) reactions using well-defined Grubbs,
ruthenium-based metathesis catalyst systems (Fig. 1. c). ROMP
is a chain-growth polymerization process where strained cyclic
olefins are converted to a polymeric material. ROMP reactions
are driven by the release of ring strain from the monomer(s)
(e.g. cycloolefins). ADMET is a step-growth polymerization,
which involves the elimination of small molecules such as
ethylene (Fig. 2.).(Sutthasupa et al., 2010), (Wagener et al.,
2006)
Table 1 The GPC analysis (Mw, Ð) and glass transition point Tg values of the PVA
co-polymers used in controlled drug release experiments *PVA-4 was insoluble in
**
THF, no GPC data was obtained. PVA-5 has crystalline structure, no Tg data was
obtained
Herein, we report the sustained release of fluorescein and
sorafenib using well-defined vinyl alcohol copolymers having
high content of precisely-spaced vinyl alcohol functionalities.
The fluorescein model compound has been selected for
preliminary drug release investigations, as its logP value (3.4) is
similar to that of the sorafenib (3.8). In the preliminary
experiments fluorescein has been embedded into a series of
well-defined VA copolymers. Based on the drug release tests,
the most promising VA copolymer was selected as a nanocarrier
for sustained sorafenib anticancer drug release. VA copolymer-
sorafenib nanoparticles have been synthetized by
nanoprecipitation and subjected to in vitro studies including
biorelevant drug release test, cellular uptake - and cytotoxicity
investigations with HT-29 human cancer cell line.
Insert Figure 2 here
In general, molybdenum and tungsten catalysts are relatively
sensitive to oxygen-containing functional groups including
alcohols, aldehydes and carboxylic acids. Nevertheless,
ruthenium metathesis catalysts are rendering high functional
group tolerances.(Balla et al., 2016) Earlier studies reported the
ruthenium catalyzed ROMP of hydroxyl functionalized
cyclooctene resulting in linear hydroxyl-functionalized
polyoctenamers.(Hillmyer et al., 1995) The synthesis of well-
defined ethylene-vinyl alcohol copolymers was reported by the
ADMET of two vinyl acetate-containing symmetrical -diene
monomers (Li et al., 2013) and also introduced primary,
secondary and tertiary hydroxyl groups in precise intervals into
the polyolefin chain.(Valenti and Wagener, 1998) By the ROMP
2. Materials and methods
Poly(vinyl alcohol)- (PVA, Mw=30,000-70,000, 87-90%
hydrolysed), polyoxyethylene sorbitan monooleate- (Tween 80)
and Pluronic F68 emulsifiers; ethanol, acetone, dimethyl

sulfoxide
dimethylaminopropyl)
hydroxysuccinimide (NHS) and 3-(4,5-dimethylthiazol-2-yl)-2,5- stirring overnight at room temperature and 1 bar. Blank PVA-5
diphenyltetrazolium bromide (MTT), abs. toluene, nanoparticles were prepared similarly using Pluronic F68
(DMSO),
sodium
azide,
1-ethyl-3(3- aqueous solution containing PVA, Pluronic F68 or Tween80
carbodiimide
(EDC), N- emulsifier. The organic solvent was evaporated by magnetic
tripropylamine, p-toluenesulfonyl hydrazide, xylenes, BHT, 1,4- emulsifier, though without sorafenib active agent. To prepare
dioxane, TFA, abs. THF, LiAlH4, abs. diethyl ether, N- sorafenib crystals, the drug was precipitated by the same
methylmorpholine
N-oxide,
OsO4,
n-hexane,
1,5- method, but without adding the encapsulating polymer. The
cyclooctadiene, m-chloroperbenzoic acid, 1,6-heptadiene-4-ol, nanocomposite dispersions were centrifuged by a Hermle Z216
palladium on carbon, ethyl vinyl ether and fluorescein sodium MK microcentrifuge (Germany) with 15,000 rpm for 20 min,
salt were obtained from Sigma Aldrich, ruthenium catalyst G2 washed and redispersed in equal volume of MilliQ water three
was from Materia Inc. Sorafenib (free base) was purchased from times.
LC Laboratories (US). Cyanine 5 amine was the product of Characterization of nanoparticles
Lumiprobe Gmbh (Germany).
Polymer preparation
The nanocomposites were imaged with FEI Apreo scanning
electron microscope (SEM, Thermofisher, USA) at 20 kV. The
PVA-4 was synthetized (Fig. 3. a) by the ROMP of acetal- size distribution of the NPs was measured by dynamic light
substituted cyclooctene (COE), where acetal moiety worked as scattering method with a Zetasizer Nano ZS (Malvern
a protecting group for 5,6-dihydroxy-COE to increase the Instruments, Malvern, UK). The particles were characterized by
solubility of the polymer. Acetals were polymerized with G2 the intensity mean diameter and polydispersity index (PDI). Zeta
catalysts (Fig. 1. a). Saturation of the polymer backbone, potential was measured also by Zetasizer Nano ZS (Malvern
followed by deprotection, generated perfectly alternating Instruments, Malvern, UK) after washing and redispersion of
copolymers of vinyl alcohol and ethylene.(Scherman et al., the nanoparticles in MilliQ water. Thermal behavior of the
2005)
samples was investigated by differential scanning calorimetry
PVA-5 was synthetized (Fig. 3. b) by the ADMET of 1,6- (DSC) on a Setaram LabsysEvo system. The measurements were
heptadien-4-ol(Tuba et al., 2014) and PVA-8 (Fig. 3. c) by the performed in flowing (50 mL/min) high purity argon (99.999%)
ROMP of cyclooct-4-en-1-ol(Martinez et al., 2014) with G2 atmosphere, in 25-300 °C temperature range with a scanning
catalyst (Fig. 1. a) in an open vial in a glovebox at ambient rate of 10 °C/min. An average of 4-5 mg sample was placed into
temperature. Hydrogenation of the resulting polymers were 100 μL aluminium crucible. The measurement results were
performed on palladium on carbon followed by atmospheric blank corrected. All measurement results were evaluated with
hydrogen exposure.(Tuba et al., 2014)
Calisto Processing (v2.01) software.
Polymer characterization
The encapsulation efficiency was determined after
1
Routine H NMR spectra were obtained on a Varian Unity dissolving 10% of the prepared and washed nanoparticles in 5
INOVA spectrometer operating at an equivalent 1H frequency of ml ethanol. The sorafenib concentration was measured by
500 MHz performed at 30 °C.
HPLC. The chromatography system was Young Lin YL 9100, YL
The GPC system was composed of a Waters 515 HPLC pump, Instruments Co., Ltd, South Korea. Chromatographic separation
a Waters 717plus Autosampler, a column system of 2 Waters was performed on a Zorbax SB-Aq column (150 mm × 4.6 mm,
Styragel HR columns (HR1 and HR4), a Jetstream thermostat 5 μm; Agilent, USA). The mobile phase from time zero till 5 min
and an Agilent Infinity 1260 Differential Refractometer was composed of 70% distilled water (0,025% tetrachloroacetic
detector. Distilled tetrahydrofuran was used as eluent with a acid) and 30% methanol. The percentage of methanol was
flow rate was 0.3 mL/min.
increased continuously to 100% over 22 min, and remained till
Thermogravimetric analyses were performed in air the time point 30 min. Then, the composition was changed back
(50 ml/min) using a Perkin Elmer Pyris 1 Thermogravimetric to distilled water (0,025% TFA)–methanol (70:30; v:v) within 5
Analyser over a temperature interval 30–600 °C at a heating min. The flow rate was of 1 mL/min throughout the 35-min run.
rate of 10 °C min−1.
PVA-fluorescein blend preparation
100 mg of polymer was dissolved in 5 ml of the appropriate
Chromatography was performed at 30 °C. The absorbance of
eluent was measured at a wavelength of 255 nm.
The concentration of sorafenib was calculated using a
calibration curve and the encapsulation efficiency was
solvent (H2O-PVA, DMSO-PVA-4, MeOH-PVA-5, THF-PVA-8
)
and 5 mg of fluorescein was separately dissolved in 3 ml of calculated as follows:
methanol then the solutions were mixed and stirred together
Encapsulation efficiency (%) = (mass of drug in NP / mass of
for an additional 20 minutes, then the solvent was removed by total loaded drug) × 100.
rotavapor. The sample was homogenized by mechanical mixing In vitro drug release
and 10 mg of the resulting solid material was pressed into a
During the pre-programmed drug release experiments
tablet by direct compression with a PerkinElmer Manual fluorescein as a model compound was embedded into polymers
Hydraulic Press (5 tons).
(PVA
, PVA-4, PVA-5 and PVA-8) synthesized with different
PVA-sorafenib nanoparticle preparation
polarities, from which fluorescein release was monitored by
The nanocomposites were formed by nanoprecipitation Metertech SP-8001 UV/Visible Spectrophotometer at 490
method. During the process 1 ml 10 mg/ml PVA-5 polymer nm.(Doughty, 2010) The physiological condition was ensured by
dissolved in ethanol and 0.1-0.2 ml 10 mg/ml sorafenib solution phosphate buffered saline aqueous solution (50 ml) and
in aceton were combined, and added (one-shot) to 10 ml incubation at 37 °C, continuous shaking at 200 rpm. Samples

were taken at the 0th, 5th, 10th, 20th, 30th minutes and 1st, 2nd, control).
(see
in
detail
https://www.antibodies-
4th, 6th hours of the experiments. In each case, the experiments online.com/resources/17/1247/what-is-flow-cytometry-facs-
were repeated at least three times, the standard deviation of analysis/)
the results was well within 10%.
MTT cytotoxicity assay based on a colorimetric reaction
The sorafenib-loaded nanocomposite was subjected to was applied to study the effect on viability of HT-29 carcinoma
biorelevant in vitro drug release experiments, that is, after their cells. Cells were seeded (10000 cells/well) in 96-well plates.
washing, 10 mg nanocomposites were re-suspended in 15 ml After 24 h pre-incubation, 200 μl fresh RPMI-1640 medium
human blood plasma containing 0.03% sodium azide containing 10% FCS and 50 g PVA-sorafenib nanoparticles
bactericide. The NPs in release medium were incubated at 37°C were pipetted to each well (8 parallel). The effect of four
in G24 Environmental Incubator Shaker (New Brunswick sorafenib concentration levels (0.5 μg, 1.0 μg, 2.5 μg and 5.0
Scientific Co. Inc., USA) and shaken by BIO RS-24 Mini-rotator μg per well). The positive control samples were also supplied
(Biosan, Latvia) for 2 days at 700 rpm. At predetermined by the same amount of free sorafenib in DMSO solution.
intervals, 0.5 ml of each sample was centrifuged (Hermle Z216 DMSO cytotoxicity (without drugs) was also investigated. After
MK microcentrifuge, Germany) for 20 min at 15,000 rpm, 12 h, 24 h and 48 h incubation 20 l/well MTT solution (5 mg
washed three times and the gained nanoparticles were MTT/ml) and 0.2 ml/well culture media were added and the
dissolved in 0.5 ml ethanol. The sorafenib concentration was incubation took 2 h. The supernatant was removed, and MTT
analyzed after HPLC separation as described above.
Fluorescent dye conjugation
lysis solution (DMSO, 1% acetic acid, 10% SDS) was pipetted
into each well. The cells containing the formed MTT formazan
For cellular uptake studies the PVA-sorafenib nanoparticles crystals were dissolved during 30 min, and, the absorbance of
were fluorescently labelled in order to make them visible for cell suspension was measured at 492 nm by a plate reader
fluorescent activated cell sorting (FACS). Briefly, washed (Robonik Readwell Touch, India). The viability of treated cells
nanoparticle (1 mg) dispersion that was resuspended in 0.5 ml was related to that of the untreated cells (negative control).
PBS (pH 7.4) was mixed with 0.1 ml PBS (pH 7.4) containing 10
mg EDC and 10 mg NHS, and incubated for 60 min at 25°C,
centrifuged and washed with MilliQ water, and redispersed in
1.0 ml PBS (pH 7.4). The prepared carbodiimide activated
3. Results and discussion
In vitro fluorescein release of PVA blends. Preliminary PVA-
fluorescein composite release tests were performed using
nanoparticle dispersion was added to 0.02 ml PBS (pH 7.4)
commercially available PVA. These experiments were used as
solution containing 0.5 mg/ml Cyanine 5 amine fluorescent dye,
and incubated 1 h at 25°C. Then, the suspension of
nanoparticles was centrifuged, washed three times and
redispersed in 1 ml PBS.
etalon tests for the comparison of the fluorescein release
properties of composites made from polymers with preadjusted
polarity (various C/OH ratios).
Fig. 4 shows clearly that polymers with different polarity can
affect controlled fluorescein release. From the water-soluble
PVA rapid fluorescein release was observed, as the embedded
compound was completely dissolved within the first 20 minutes
of the reaction. The experiences demonstrated that the greater
the C/OH ratio is in the polymer, the slower the release of the
drug. After 6 hours, the release of the fluorescein was only 67%,
51% and 30% for the poly(vinyl alcohol alt-ethylene) (PVA-4),
poly(vinyl alcohol alt-propenylene) (PVA-5) and poly(vinyl
alcohol alt-hexenylene) (PVA-8), respectively.
Cell cultures
The human cancer cell line HT-29 was grown in RPMI-1640
medium including 10% fetal calf serum (FCS) and 100 U/ml
penicillin. The cells were cultured at 37 °C in a humidified
atmosphere containing 5% CO2. They were trypsinized,
resuspended and precultured before use.
In vitro cellular uptake and cytotoxicity studies
HT-29 cellular uptake of the nanoparticles was investigated
by FACS. The cells were cultured in 24-well plates at a cell
density of 2x105 cells per well at 37 °C for 24 h. Then, 0.1 ml
Insert Figure 4 here
nanoparticle was added to each well (3 parallel) and incubated
for 24 h. The cells cultivated without the nanoparticles were
used as negative control. The cells were washed by PBS,
trypsinized and redispersed in PBS containing 2% BSA. Flow
cytometry was done by a BD FACSAria III Cell sorter (BD
Biosciences, San Jose, CA, US) at Ex/Em 633/660 nm
wavelength. Shortly, fluorescent light is emitted by fluorescent
molecules after excitation by a laser of suitable wavelength.
Fluorescent light may originate from naturally fluorescing
materials in the cell, or may originate from fluorescent dyes
used to label the cell. Using FACS one can physically sort a
heterogeneous mixture of cells into different populations. Usual
representation includes the intensity of a single channel
(horizontal axis) vs number of detected events (vertical axis).
Histograms can be used to compare a single parameter from
two different sample populations (e.g. experimental vs.
Considering the model drug release experiments, the most
promising sparingly water-soluble polymer, PVA-5 was selected
for further investigations. On Fig. 5 the rate of fluorescein
release is shown in a 6-day time interval. A relatively intense
fluorescein release was observed on the first day of the
experiment, it reached about 65%. However, the process has
slowed down and reached only about 85% for the 6th day of the
experiment with the standard deviation of the results within
10%.
Insert Figure 5 here
Physical and chemical properties of PVA-sorafenib
nanoparticles
. PVA-5 and PVA-8 were evaluated as having the
most favourable release kinetics with the model drug, they

sorafenib had optimally an average size of 225-271 nm (Table 2). It
means the polymeric nanocomposites also followed the shape of the
sorafenib crystals, however with substantially reduced size.
were examined, whether they are suitable for encapsulating
sorafenib anticancer drug. Meanwhile the PVA-8 showed highly
non-polar characteristics and was hardly soluble in most general
solvents, the PVA-5 exhibited sparing solubility in water and
good solubility in ethanol. In fact, PVA-8 was soluble exclusively
in hot tetrahydrofuran, and after nanoprecipitation in the
presence of different emulsifiers, nanoparticles could not be
prepared, a huge agglomerate formation was observed.
Insert Figure 8 here
Differential scanning calorimetry (DSC) investigation has been done
in order to confirm the SEM results. Fig. 9 draws the DSC curves of
pure-, precipitated sorafenib and PVA-5-sorafenib nanocomposite.
The endothermic peaks found in all of the three thermograms clearly
suggest crystal structure of each sample. Pure sorafenib and
precipitated sorafenib had very similar thermograms, as expected.
The two overlapping peaks show two different polymorphs: a more
stable with a high and narrow endotherm and a less stable with a
wide one. These peaks can be observed also in the PVA-5-sorafenib
nanocomposite, however, with a substantial shift that verifies the
formation of nanocrystals, which are visible also in the SEM image
(Fig. 8).
Hence, PVA-5 was selected to prepare nanotherapeutics by
nanoprecipitation method (Miladi et al., 2016). Three different
emulsifiers PVA, Tween 80 and Pluronic F68 were investigated during
the preparation process. Tween 80 emulsifier in the
nanoprecipitation process provided nanocomposites with large size,
while the use of PVA surface active agent resulted in submicron-sized
nanoparticles. Pluronic F68 provided the smallest nanocomposites
(
Fig. 6), thus, it was used to optimize the particle preparation.
Insert Figure 6 here
Pluronic F68 concentration was varied in the range of 0.5-5.0% in the
water phase to find out the optimal emulsifier concentration.
However, a minimum in the size was found at a surfactant
concentration of 2% (see Fig. 7 and Table 2) but a small peak in the
micron size range can be seen in the size distribution (Fig. 7). This is
also reflected in the PDI value (Table 2) that is higher than 0.2, which
can be attributed to the agglomeration of the nanoparticles or
separately precipitated sorafenib particles. The multimodal
distribution of PVA-5-sorafenib prepared using 5% Pluronic F68
emulsifier might result from the micelle formation beside the
mentioned reasons.
According to Fig. 7 and Table 2 it can be established that 1% Pluronic
F68 concentration is optimal for the nanocomposite preparation in
this process. Nanoparticle morphology was monitored by scanning
electron microscope (SEM) indicating the formation of particles
mostly of submicron size. This is in accordance with the results of
measurements achieved by dynamic light scattering method.
Insert Figure 9 here
The encapsulation efficiency reached its maximum at 2% Pluronic
F68 concentration (Table 3), and because the yield of the
nanoparticles did not change significantly by increasing the
emulsifier concentration, the loading degree was also the highest
(12.0 %) at this concentration of surfactant. The ratio of encapsulated
active agent reduced significantly, when the emulsifier concentration
was increased to 5 %, which can be explained by the elevated
solubility of the sorafenib due to the high emulsifier concentration.
Zeta potential value of PVA-5-sorafenib nanoparticles in MilliQ water
determined by Zetasizer (Malvern Instruments, UK) did not show
stabilization by charge of the nanoparticles (Table 2). However, the
nanoparticle suspensions did not show aggregation in the solution of
emulsifier during a day or in blood plasma during the release study.
Emulsifier
concentration (%)
Encapsulation
efficiency (%)
Yield (%) Loading degree (%)
Insert Figure 7 here
0.5
1
52.2
82.1
93.6
74.7
70.4
69.4
71.1
68.9
6.8
10.7
12.0
9.9
Zeta potential
Emulsifier
concentration (%)
Intensity mean
size (d.nm)
PDI
(mV)
2
5
0.5
1
306.1
271.2
225.2
430.0
0.179
0.142
0.216
0.376
- 0.18
- 0.45
- 0.53
- 0.92
Table 3 Encapsulation efficiency, yield and loading degree values of PVA-sorafenib
nanoparticles as a function of Pluronic F68 concentration.
2
Biorelevant drug release of PVA-5-sorafenib nanoparticles.
Sorafenib release of the prepared nanocomposites was investigated
under biorelevant condition in human blood plasma. The active
agent was released from the nanocomposites continuously within
one day (Fig.10.). This is substantially quicker, than that was found
for fluorescein. Nevertheless, the nanocomposites possess
significantly higher surface area compared to the PVA blends.
Moreover, the plasma release medium contains a lot of protein that
can behave as surfactant, thus via the solubilisation they can
5
Table 2 Intensity mean diameter, PDI and zeta potential values of PVA-5-sorafenib
nanoparticles as a function of Pluronic F68 concentration.
Interestingly, most of the particles exhibited cubic shape (Fig. 8. a)
which is rather unusual among the polymeric nanoparticles.(El-Enin
and AL-Shanbari, 2018; Karami and Hamidi, 2016; Salah et al., 2017)
The precipitation of sorafenib also produced cubic crystals (Fig. 8. b),
although their size was considerably larger, with an average diameter
of 1.45 m, while composites containing both the polymer and

accelerate the dissolution of sorafenib. Nevertheless, serum proteins
were proved to be prone to interact with sorafenib.(Shi et al., 2015)
experiments nor in the literature. The main significance of this
finding is that the encapsulating polymer followed the structure
of the drug, which is quite unusual, since by polymeric
microencapsulation typically amorphous composites can be
formed. Shape is critical parameter influencing cellular uptake
and/or the speed and site specific drug delivery from the
nanoparticles. Preferential interaction with targeted proteins
can be reached on proper shape selection of nanomaterials
(Ankamwar, 2012). This interesting feature of the well-defined
PVA polymer might be useful in some other application (e.g.
diagnostic or optical), where the shape gets more role in the
function of a composite. Thus, in the following studies it is
important to clarify, whether this surprising phenomenon can
be realized using this polymer in composite with other
components.
Insert Figure 10 here
Carcinoma cellular uptake and cytotoxicity of PVA-5-sorafenib
nanoparticles. In cellular uptake studies by HT-29 model carcinoma
cells, during the 24 h incubation time, all of the cells engulfed
fluorescently labelled sorafenib loaded PVA-5 nanoparticles (Fig. 11)
with a relative count of 16690 ± 1401. Fig. 10 shows the histograms
of control cells (A) and cells cured with fluorescent labelled PVA-5-
sorafenib nanoparticles. Comparing the two histograms, it can be
seen that there is not any overlap between the peaks, which means
all of the cells contained the fluorescent dye after incubation with
the labelled nanoparticles.
5. Acknowledgements
Insert Figure 11 here
This work was funded by grants provided by the National
Competitiveness and Excellence Program, Hungary (NVKP_16-
1-2016-0007) and by the BIONANO_GINOP-2.3.2-15-2016-
00017 project. Dr. Tivadar Feczkó acknowledges the funding of
Alexander von Humboldt Foundation (Ref. No.: 3.3-
UNG/1161203 STP and 3.3-1161203-HUN-HFST-E) and the
support of Hungarian Academy of Sciences via Bilateral Joint
Project with the Polish Academy of Sciences.
Cytotoxicity tests were performed on HT-29 cells using different
active agent concentrations. Sorafenib solution was added to the
cells in DMSO solution, thus the effect of the same concentration
(0.5% related to the medium volume) of DMSO was also investigated
and it did not exert significant toxic effect.
Blank PVA-5 nanoparticles without active agent neither decreased
the viability of the cells compared to the non-treated cells (negative
control) (Figure 12). As expected, sorafenib solution triggered
concentration-dependent cytotoxicity and this was also observed by
the nanocomposites. However, the viability was reduced to a smaller
extent, probably due to the prolonged release of the drug. 48 h
incubation with the nanocomposites resulted in the highest cytotoxic
effect. Although all of the drug was released within one day, the
burst effect was not too high. Thus, the available drug concentration
is substantially lower for some hours than in the case of sorafenib
solution. The rate of cellular uptake must have also influenced the
cytotoxicity of the nanoparticles.
6. Conflicts of interest
There are no conflicts to declare.
7. Notes and references
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Cl
F
F
F
HO
O
OH
N
N
O
Mes
Mes
HN
Cl
Cl
Ru
HN
O
O
PCy3
O
N
O
(a)
(b)
(c)
NH

ROMP
(a)
(b)
n
n
n
n
ADMET
n

monomer synthesis
OH
O
DCM
LiAlH4
mCPBA
DEE
43%
OsO4
MNO
OH
OH
H2O/THF/aceton
52%
polymer synthesis
(a)
O
O
O
OH
OH
G-II
Me2C(OMe)2
toluene
n
PPTS
acetone
O
50%
97%
xylenes
BHT
tosylhydrazide
tripropylamine
98%
PVA 4
OH
O
TFA/H2O
n
1,4-dioxane
n
OH
O
99%
(b)
OH
PVA 5
OH
OH
Pd/C, H2
G-II
THF
MeOH
n
n
n
94%
54%
(c)
OH
PVA 8
OH
OH
Pd/C, H2
G-II
THF
n
n
THF
88%
99%

100
80
60
40
20
0
PVA
PVA-4
PVA-5
PVA-8
0
1
2
3
4
5
6
Time [h]

100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
Time [day]

25
20
15
10
5
PVA
Tween 80
Pluronic F68
0
1
10
100
1000
10000
Size (d,nm)

18
16
14
12
10
8
0,005
0,01
0,02
0,05
6
4
2
0
1
10
100
1000
10000
Size (d,nm)

A
B

100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
25
Time [h]

120
100
80
60
40
20
-

Fig. 1 Structure of fluorescein model (drug
compound - a), sorafenib (anticancer drug - b), G-
II catalyst (ruthenium-based metathesis catalyst -
c).
Fig. 2 A generalized example of ring opening
metathesis polymerization (ROMP - a) and acyclic
diene metathesis polymerization (ADMET - b)
reactions.
Fig. 3 Synthesis of well-defined VA-copolymers
with different polarities (PVA-4, PVA-5 and PVA-
8).
Fig. 4 Rate of fluorescein release from PVA blends
as a function of carriers possessing different C/O
ratio.
Fig. 5 Rate of fluorescein release by PVA-5 blend
(3 independent experiments) in a 6-day time
interval.
Fig. 6 Size distribution of PVA-5-sorafenib
nanoparticles prepared by PVA, Tween 80 or
Pluronic F68 emulsifiers.
Fig. 7 Size distribution of PVA-5-sorafenib
nanoparticles prepared by varying concentrations
(0.5 % - blue line, 1.0 % - yellow dashed line, 2.0
% - green line, 5.0 % - red line) of Pluronic F68
emulsifier.
Fig. 8 Scanning electron microscopic image of
PVA-5-sorafenib nanoparticles (A) and
precipitated sorafenib crystals (B)
.
Fig. 9 Differential scanning calorimetry
thermograms of pure sorafenib (Sorafenib, green
line), precipitated sorafenib (SOR-BLANK, red
line) and PVA-5-sorafenib nanocomposite (SOR-
18, blue line).
Fig. 10 Cumulative sorafenib release of the PVA-5-
sorafenib nanocomposites in human blood plasma.
Fig. 11 Fluorescence-activated cell sorting (FACS)
diagram of control cells without treatment (A) and
cells treated with Cyanine5-labelled PVA-5-
sorafenib nanocomposites (B).
Fig. 12 Viability of HT-29 cells by MTT assay due
to DMSO, sorafenib in solution (‘SOR’, brown
columns) and PVA-5-sorafenib nanoparticles
(‘PVA-SOR’, green columns) after 12 h, 24 h and
48 h incubation related to the non-treated (negative
control) cells.

Graphical Abstract
onge
d
activ
e
agen
t
relea
se.
Sora
fenib
che
mot
hera
peut
ic
drug
VA-
copo
lyme
r
(C/O
H = 5
ratio
)
nano
com
posit
e
exhi
bitin
g
unus
ual
cubi
c
shap
e
sho
ws
prol