Activity enhancement of selective antitumoral selenodiazoles formulated with poloxamine micelles

Article abstract of DOI:10.1016/j.colsurfb.2018.06.009

Selenium (Se) incorporated into organic frameworks has demonstrated anticancer activity against several cancer types. One of the drawbacks of most of these constructs is their poor solubility and bioavailability, which can be overcome with the use of suitable nanocarriers. We have synthesized a series of 5-substituted amide selenodiazoles, based on the parent structure of ebselen, an organoselenium drug with proven cytoprotective activity, and solubilized them in polymeric micelles of poloxamines, poly(ethylene oxide)-poly(propylene oxide) X-shaped tetrablock-copolymers. Scattering methods (SANS and DLS) were employed to characterize the micellar nanocarriers. MTT biological evaluation highlights the selectivity of the Se-compounds towards cancer cells, with MCF-7 standing as the most responsive line. The alkylation of the heterocycle with a 12-carbon hydrophobic tail displays the highest activity, showing a 100-fold increase with respect to ebselen. This compound also exhibits the greatest increase in solubility in poloxamine micelles, overall resulting in a one-fold increase in activity with respect to the non-formulated form, making it a hit compound for further optimization.

Full text of DOI:10.1016/j.colsurfb.2018.06.009

Accepted Manuscript
Title: Activity enhancement of selective antitumoral
selenodiazoles formulated with poloxamine micelles
´
´
Authors: Amaia Uriz, Carmen Sanmartın, Daniel Plano,
´
´
Cecile A. Dreiss, Gustavo Gonzalez-Gaitano
PII:
S0927-7765(18)30380-1
DOI:
Reference:
https://doi.org/10.1016/j.colsurfb.2018.06.009
COLSUB 9399
To appear in:
Colloids and Surfaces B: Biointerfaces
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Revised date:
Accepted date:
1-3-2018
21-5-2018
6-6-2018
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Please cite this article as: Uriz A, Sanmartın C, Plano D, Dreiss CA,
´
Gonzalez-Gaitano G, Activity enhancement of selective antitumoral selenodiazoles
formulated with poloxamine micelles, Colloids and Surfaces B: Biointerfaces (2018),
https://doi.org/10.1016/j.colsurfb.2018.06.009
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Activity enhancement of selective antitumoral selenodiazoles
formulated with poloxamine micelles
Amaia Úriz1,2, Carmen Sanmartín*2,3, Daniel Plano2,3, Cécile A. Dreiss*4 and Gustavo González-
Gaitano*1
1 University of Navarra, Faculty of Sciences, Department of Chemistry, Irunlarrea 1, E-31008, Pamplona, Spain.
2 University of Navarra, Faculty of Pharmacy and Nutrition, Department of Organic and Pharmaceutical Chemistry, Irun-
larrea 1, E-31008, Pamplona, Spain.
3 Instituto de Investigación Sanitaria de Navarra (IdiSNA), Irunlarrea 3, E-31008 Pamplona, Spain.
4 King’s College London, Institute of Pharmaceutical Sience, School of Cancer and Pharmaceutical Sciences, Franklin-
Wilkins Building, 150 Stamford Street, SE1 9NH London, UK
Corresponding Authors: Gustavo González-Gaitano, Carmen Sanmartín, Cécile A. Dreiss
Graphical abstract
Highlights





Tetronic micelles were used as nanocarriers for a new family of selenodiazoles
The micelles enhance the water-solubility and cytotoxicity of the Se-derivatives
The new Se constructs are selective towards cancer prostate and breast cancer cells
Increasing the length of the constructs’ hydrocarbon chain increases their activity
SE12 (side chain with 12 methylenes) shows a activity 100-fold higher tan ebselen
ABSTRACT:
Selenium (Se) incorporated into organic frameworks has demonstrated anticancer activity against sever-
al cancer types. One of the drawbacks of most of these constructs is their poor solubility and bioavaila-
bility, which can be overcome with the use of suitable nanocarriers. We have synthesized a series of 5-
substituted amide selenodiazoles, based on the parent structure of ebselen, an organoselenium drug with
proven cytoprotective activity, and solubilized them in polymeric micelles of poloxamines,
poly(ethylene oxide)-poly(propylene oxide) X-shaped tetrablock-copolymers. Scattering methods
(SANS and DLS) were employed to characterize the micellar nanocarriers. MTT biological evaluation

highlights the selectivity of the Se-compounds towards cancer cells, with MCF-7 standing as the most
responsive line. The alkylation of the heterocycle with a 12-carbon hydrophobic tail displays the highest
activity, showing a 100-fold increase with respect to ebselen. This compound also exhibits the greatest
increase in solubility in poloxamine micelles, overall resulting in a one-fold increase in activity with
respect to the non-formulated form, making it a hit compound for further optimization.
STATISTICAL SUMMARY: 5365 words (including references), 3 tables, 1 scheme, 4 figures/sub-figures
Activity enhancement of selective antitumoral selenodiazoles formu-
lated with poloxamine micelles
Amaia Úriz1,2, Carmen Sanmartín*2,3, Daniel Plano2,3, Cécile A. Dreiss*4 and Gustavo González-
Gaitano*1
1 University of Navarra, Faculty of Sciences, Department of Chemistry, Irunlarrea 1, E-31008, Pamplona, Spain. 2 University
of Navarra, Faculty of Pharmacy and Nutrition, Department of Organic and Pharmaceutical Chemistry, Irunlarrea 1, E-
31008, Pamplona, Spain.3 Instituto de Investigación Sanitaria de Navarra (IdiSNA), Irunlarrea 3, E-31008 Pamplona,
Spain. 4 King’s College London, Institute of Pharmaceutical Sience, School of Cancer and Pharmaceutical Sciences, Frank-
lin-Wilkins Building, 150 Stamford Street, SE1 9NH London, UK
KEYWORDS selenium, ebselen, polymeric micelles, Tetronics, poloxamines
ABSTRACT: Selenium (Se) incorporated into organic frameworks has demonstrated anticancer activity against several cancer
types. One of the drawbacks of most of these constructs is their poor solubility and bioavailability, which can be overcome with the
use of suitable nanocarriers. We have synthesized a series of 5-substituted amide selenodiazoles, based on the parent structure of
ebselen, an organoselenium drug with proven cytoprotective activity, and solubilized them in polymeric micelles of poloxamines,
poly(ethylene oxide)-poly(propylene oxide) X-shaped tetrablock-copolymers. Scattering methods (SANS and DLS) were employed
to characterize the micellar nanocarriers. MTT biological evaluation highlights the selectivity of the Se-compounds towards cancer
cells, with MCF-7 standing as the most responsive line. The alkylation of the heterocycle with a 12-carbon hydrophobic tail dis-
plays the highest activity, showing a 100-fold increase with respect to ebselen. This compound also exhibits the greatest increase in
solubility in poloxamine micelles, overall resulting in a one-fold increase in activity with respect to the non-formulated form, mak-
ing it a hit compound for further optimization.
1. Introduction
Selenium (Se) is a micronutrient for humans; low plasma levels have been linked to a high incidence of several cancers1,2,3,4. Never-
theless, outcomes from the SELECT trial (Selenium and Vitamin E Cancer Prevention Trial) have given rise to some controversy
about this relationship5. One of the main weaknesses of this trial was the chemical form used to deliver selenium (namely, L-
selenomethionine). This alone could explain the unexpected failure of the SELECT trial since it is well documented that Se specia-
tion is critical for its biological effects6,7,8. Indeed, some Se derivatives can be metabolized to form selenocysteine, a Se amino acid
that can be incorporated into 25 different selenoproteins. Most of the selenoproteins play essential roles in human health and low
levels have been reported in several diseases 9,10,11,12. At the same time, other Se derivatives can be metabolized to methylselenol
(CH3SeH) or to hydrogen selenide (H2Se); both present antitumor activities, different than those from selenoproteins6. The activity
of Se derivatives occurs through several mechanisms, some of them are not fully understood to date13. Taking into account all these
considerations, it is clear that the activity of Se is dependent on the specific constructs in which it is incorporated, and the failure of
some Se compounds should not be generalized to all Se derivatives. Recently, our group and many others have demonstrated that
the incorporation of Se into organic frameworks, in particular small molecules such as selenocyanate, diselenide, selenourea or
selenide, confers unique and very valuable properties against a range of diseases14,15, and demonstrate anticancer activity against
different cancer types16,17,18. Among these small Se-containing molecules, Se heterocycles have demonstrated low toxicity for
healthy cells, along with promising anticancer effects. Ebselen is one of the most well studied Se heterocyclic compounds in can-
cer19,20
.
One of the limitations of most Se-based drugs however is their poor solubility and bioavailability, which can be overcome with the
use of suitable nanocarriers21. Polymeric micelles based on polyethylene oxide (PEO) and polypropylene oxide (PPO) are emerging

as promising nanocarriers, displaying a large array of architectures and properties, in addition to displaying biological inhibitory
activity towards drug efflux transporters of the ATP-binding cassette22,23. Tetronic® is the trade name of BASF for poloxamines, a
family of X-shaped amphiphilic copolymers based on PEO and PPO. Each of the four arms comprises a PEO and a PPO block,
which are connected by a central ethylene diamine group. This morphology drives self-assembly into micelles with a lipophilic core
(PPO blocks) and a hydrophilic outer shell (PEO blocks), which is highly hydrated. The number of PO and EO units forming the
arms varies, affording a range of molecular weights and hydrophilic-lipophilic balance (HLB), and consequently a rich phase be-
havior and properties24,25,26. If the configuration of the blocks is inverted, the resulting amphiphile is called a “reverse” poloxamine
(Tetronic-R). The core of the aggregates offers a non-polar environment for the encapsulation of hydrophobic, poorly water-soluble
drugs, while the protonable amino connector affords pH-responsiveness of the central moiety and thus controllable release of the
cargo.
Within this framework, we have synthesized a family of selenodiazole derivatives bearing a hydrocarbon chain of variable length
(from 3 to 12 carbon atoms, SE3-SE12). This selenodiazole skeleton was chosen based on its similarity with ebselen (also known as
PZ51, DR3305, or SPI-1005), and our previous experience on the antitumor and leishmanicidal in vitro activity of selenodiazole
derivatives substituted on 5-position with different amides27,28. The synthetic strategy to obtain selenodiazole derivatives is outlined
in Scheme 1.
These selenocompounds were formulated with two selected poloxamines, T904 (15 EO, 17 PO per arm, Mw 6,700) and T1107 (60
EO, 20 PO per arm, Mw 15,000), and their combinations with T90R4, which presents a “reverse” structure compared to T904,
bearing its PPO blocks on the outer parts of the four arms. T904 and T1107 were chosen according to their different HLB (12-18
and 18-23, respectively) and capacity of T1107 to form gels, thus widening the scope of formulations to injectable thermogels. The
combined use of the reverse poloxamine in the mixed micelles is based on the hypothesis that the incorporation of a hydrophobic
amphiphile, which, on its own does not aggregate into micelles, can lead to an enhanced hydrophobic locus within the nanoaggre-
gates and, consequently, a higher solubilization capacity of the selenocompounds. Although there are numerous routes of admin-
istration for chemotherapeutic drugs, i.e. intramuscular, intra-arterial or intrathecal among other, and ideally oral, the vast majority
of these drugs are administered intravenously (i.v.), which would be the preferred route of administration for the new derivatives
presented here. In this framework, the poloxamine micelles could help to overcome future issues in the pre-clinical development of
selenodiazoles.
2. Materials and methods
2.1. Synthesis of the selenocompounds
Compounds derived from benzo[c][1,2,5]selenodiazole were synthesized in the solid phase by cyclization of 3,4-diaminobenzoic
acid with SeO2 at high temperature and constant stirring. The product is reacted with an excess of thionyl chloride under reflux
conditions, yielding chlorination of the acid. The formation of the amide derivative follows a nucleophilic acyl substitution in the
presence of triethylamine. Final products account for amides with a linear 3- (SE3), 4- (SE4), 5- (SE5), 6- (SE6) and 12- (SE12)
carbon chain (see SI for details of the synthesis and compound characterization).
Analytical thin-layer chromatography (TLC) was conducted on E. Merck silica gel 60 F254 plates, 250 µm thickness and detection
at 254 nm. Compounds were purified by chromatographic column using the solvent mixtures specified in each case. Infrared (IR)
spectra were registered using a Thermo Nicolet FTIR Nexus Euro spectrometer with OMNIC 6.0 software. Products are obtained as
solids and their spectra were recorded using transmission KBr pellets. Organic elementary analysis (CHN) was made in a LECO
CHN-900 microprocessor. The error interval admitted for each compound is ± 0.5 %. Melting points were obtained using a Mettler
FP82 heating stage and FP80 control unit, with a 5 ºC per minute heating rate. Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker 400 UltrashieldTM spectrometer (1H, 400 MHz; 13C, 100 MHz) at room temperature (r.t.). Chemical shifts
(δ) are expressed in parts per million (ppm) and coupling constants (J) in hertz (Hz). The following abbreviations are used to de-
scribe peak patterns when appropriate: s (singlet), d (doublet), t (triplet), q (quartet), qt (quintet), dd (double doublet), dq (double
quartet) and br (broad). Fluorescence spectra were measured (Edinburgh Instruments FLS920 spectrofluorimeter) to find the char-
acteristic wavelengths of excitation and emission of the synthesized compounds in ethanol and water, so as to simulate the core and
shell environments of the poloxamine micelles. UV-Vis spectra were recorded (Agilent 8453 diode-array spectrophotometer) to
obtain the characteristic molar absorptivity by the Beer-Lambert law in ethanol and water, in the range 5·10-6 M to 3·10-5 M.
The polymeric surfactants, Tetronic 1107 (T1107) and Tetronic 904 (T904) were a gift from BASF, with reported compositions per
arm 60 EO and 20 PO for T1107 (average molecular weight 15000 gmol−1) and 15 EO and 17 PO for T904 (average molecular
weight 6700 gmol−1). The “reverse” Tetronic 90R4 (T90R4) was purchased from Sigma-Aldrich (block composition 16 EO and
18PO).
2.2. Characterization of the nanocarriers
2.2.1. Small-Angle Neutron Scattering (SANS)
Mixtures of T904 with T90R4, and T1107 with T90R4 (5, 15, 25% w/w, in ratios of 80% direct Tetronic to 20% reverse Tetronic)
were prepared at their natural pH (8.2), pH 3 (diprotonated form) and pH 6 (monoprotonated form). Experiments were carried out
on LOQ instrument at the ISIS spallation neutron source (ISIS, Rutherford-Appleton Laboratory, STFC, Didcot, Oxford). LOQ
uses incident wavelengths from 2.2 to 10.0 Å, sorted by time-of-flight, with a fixed sample-detector distance of 4.1 m, which pro-

vides a range of scattering vectors (q) from 0.009 to 0.29 Å-1. The samples were prepared in D2O (Aldrich, > 99.9% in D) to ensure
sufficient contrast between the polymer and the solvent and then placed in clean disc-shaped quartz cells (Hellma) of 1 mm path
length, controlling the temperature from 20 to 50°C with an external thermostat. All scattering data were first normalized for sam-
ple transmission and then background-corrected using a quartz cell filled with D2O, and finally corrected for the linearity and effi-
ciency of the detector response using instrument-specific software package. The data were then converted to the differential scatter-
ing cross-sections, expressed in absolute units of cm-1, using the standard procedures for LOQ. SANS curves were fitted using
SasView 3.1.0 software29. Scattering curves from the poloxamines in their unimer form were fitted with a four-arm star-shape (4SP)
polymer model, while micelles were fitted to a core-shell sphere (CSS) model combined with a hard-sphere structure factor (HS).
When coexisting in solution, a mixed model of unimers and micelles was used by combining the above two models.
2.2.2. Phase diagrams
Aqueous solutions of T904 (1-25% wt%), T1107 (1-25 wt%) and mixtures of T904 with T90R4 as well as T1107 with T90R4 (5-
25% wt% total concentration with 80% direct Tetronic/20% reverse Tetronic) were placed into a water bath at an initial temperature
of 25 ºC. The samples were gradually heated up by 5 ºC increments up to 80 ºC. All samples were prepared at their natural pH (ca.
8.2), pH 3 and pH 6, by adding the required amount of concentrated HCl.
2.2.3. Dynamic Light Scattering (DLS)
Size distributions of the poloxamine in water were obtained with a photon correlation spectrometer Malvern Zetasizer Nano, with a
laser wavelength of 633 nm. The samples were filtered prior to the measurements by 0.22 m Millex syringe PVDF filters onto
semi-micro glass cells, and the temperature of the samples controlled with 0.1˚C accuracy by the built-in Peltier in the cell com-
partment. The viscosity and refractive index of the solvent at different temperatures were taken into account to obtain the particle
size distribution from the analysis of the autocorrelation function, which was performed with the Zetasizer software in the high-
resolution mode to better distinguish overlapping distributions.
2.2.4. Solubilization studies
An excess of compound (SE3, SE6 and SE12) was introduced in vials with 15% w/w solutions of polymer (T904, T1107,
T904+T90R4 (80%/20% direct/reverse Tetronic), T1107+T90R4 (80%/20% direct/reverse Tetronic)) or in pure water. Samples
were left to stir in the water bath at 40 ºC and measured after 72 and 92 h to assure maximum solubility was reached. Drug concen-
tration was obtained from the absorbance data using each poloxamine system as a blank, and the previously established Beer-
Lambert law equation.
2.3. Cytotoxicity studies
Synthesized selenodiazoles were tested in vitro against cultured cells of leukaemia (CCRF-CEM), colon (HT-29), lung (HTB-54),
prostate (PC-3) and breast (MCF-7) cancer by means of the tetrazolium assay (MTT). All the compounds were also tested for tox-
icity in a mammary gland cell culture derived from non-malignant cells (184B5). All cells were defrosted and maintained in Dul-
becco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at
37 ºC and 5% CO2. 1 mmol of compound was diluted in 1 mL DMSO to a concentration of 10-2 M and filtered with 0.22 µm Milli-
pore filter to ensure sterility. Serial dilutions were made using non-supplemented media to achieve concentrations from 10-3 to 10-7
M. 96-well plates were seeded with 100 µL of cellular suspension (100 cells/ µL), 80 µL of media and 20 µL of compound solu-
tion, obtaining final samples of concentration 10-4-10-8 M. Each sample was evaluated in quadruplicate measurements and repeated
in three independent cell samples. Non-supplemented media was used as a blank and controls were performed for each cell line.
The same methodology was followed to test three representative compounds (SE3, SE6, SE12) encapsulated in polymer micelles
(T904, T1107 and T904+T90R4 (80%/20% direct/reverse Tetronic)). In this case, samples were prepared in 15% (w/w) poloxamine
and dilutions performed to obtain samples at 10-3 to 10-7 M, following the previously described protocol. Blanks were made with
each poloxamine system.
3. Results and discussion
3.1. Structural characterization of the nanocarriers
Analysis of phase diagrams (SI, Tables S1A and S1B) shows that the most hydrophilic T1107 forms a gel at around 35 ºC and 25%,
while the requirements for T904 to form gel are more extreme. In the mixed systems, the incorporation of T90R4 hinders the for-
mation of a gel phase for T904 and shifts the sol-gel transition by 5 ºC for T1107 at 25%T1107.
Precise structural information on the polymeric nanocarriers can be obtained from small-angle neutron scattering (SANS) and dy-
namic light scattering (DLS). As an example, Figures 1A and 1B show the DLS size distribution and SANS scattering patterns for
mixtures of T1107 with the reverse poloxamine as a function of temperature, for 2% of each Tetronic. At 20 ºC, both surfactants are
in the form of unimers, and SANS patterns under these conditions are suitably described by a four-arm-star polymer model (4SP),
with an average radius of gyration of 25 Å, matching the hydrodynamic radii observed by DLS. As the temperature increases, upon
reaching the critical temperature (CMT), unimers self-organise to form micelles, behaviour typical of PEO-PPO based surfactants24-
26. At 30ºC a new distribution in the DLS size distribution appears at 30 nm, corresponding to the mixed micelles, although its
contribution in mass is still very low. At higher temperature, unimers coexist with the micelles, and the contribution of the peak
corresponding to the unimers concomitantly reduces, while the broad peak due to the micelles becomes narrower and shifts to
smaller sizes. At 40ºC mixed micelles are fully formed and there is just one peak at ca. 15 nm in size. The solubilization of the
reverse poloxamine T90R4 in the micelles is evidenced by a shift of the phase separation, from 36 ºC for T90R4 alone, to ca. 60 ºC

when mixed with either T904 or T1107. This effect can be observed in the phase diagrams at a higher direct/reverse Tetronic mass
ratio of 4:1 (SI, Table S1A and S1B).
Micelles are appropriately described by a core-shell spherical model (CSS) combined with a hard sphere structure (HS) factor,
allowing the estimation of the geometrical parameters and hydration state of micellar core and shell (Figure 2A to C)24.
The parameters derived from the fits (Table 1) show that substituting a direct unimer by its reverse counterpart does not have a
significant impact on the overall size of the micelles. Increasing the polymer concentration, instead, induces a slight shrinkage of
the hydrophilic shell. The scattering length density (sld) of the core, which was set as a floating parameter in the fits, reaches values
close to the sld of PPO (3.4·10-7 Å-2), reflecting a dehydrated hydrophobic core (no penetration of solvent). Instead, the sld of the
shell tends to that of pure solvent (6.36·10-6 Å-2), showing a very high extent of hydration of the PEO blocks. It is worth mentioning
that the shell sld is lower in all cases than the corresponding value for the pure poloxamine, indicating more permeability to the
solvent. At high concentrations, gels are formed (Tables S1A and S1B). Thus, at 25% T1107 + T90R4 (4:1 ratio, 40 ºC), the long-
range order responsible for gelation is reflected by a sharp diffraction peak in the 0.07-0.09 Å-1 q-region (Figure 2D), not observed
for T904. The analysis of the SANS data by means of a paracrystal bcc model yields a cell parameter of 167 Å, which matches the
dimensions obtained with the same model for T1107 alone. A core size of 35 Å is obtained for the packed micelles, which is very
similar to that of the mixed micelles under dilute conditions (Table 1), and to T1107 alone at the same concentration and tempera-
ture. The behavior of T904 in combination with 90R4 follows the same trend, as shown in Table 1.
In the view of exploiting the pH to control the delivery of the cargo from the micellar core, the effect of pH on the morphology of
the aggregates was studied next. Upon a reduction of the pH below the second pKa (pH 6), the partial protonation of the central
ethylene diamine connector leads to a decrease of the scattering. A further reduction below the first pKa (pH 3) yields curves remi-
niscent of the unimer form, with radii of gyration similar to those of the respective unimers forming the micelles at its natural pH
(pH 8.2), as obtained from fits to a 4SP model. Overall, this means that the protonation of the amino groups causes strong electro-
static repulsion between the direct Tetronic unimers in the core, and the reverse Tetronic in the shell. This destabilizes the mixed
micelles, leading first to a mixture of free unimers and aggregates (pH 6), and then to a full rupture of the structures where only
unimers are present (pH 3) (SI, Figure S1).
Overall, the structural analysis on the mixed micelles shows that the copolymer with the reverse configuration, largely insoluble on
its own, can be incorporated inside the direct micelles. The dimensions of the micelles are not impacted by substituting up to 20%
in weight by the reverse structure. The hydrophobic core presents a high degree of packing of the PPO blocks, while the main im-
pact of incorporating the reverse poloxamine is a slight increase of the hydration of the PEO shell. Hence, from a structural view-
point, any of the combinations is suitable for the solubilization of a hydrophobic cargo.
3.2. Drug Encapsulation
The solubilization of selected organoselenium constructs (SE3, SE6, SE12) was studied in direct poloxamines (T904, T1107) and in
their mixtures with the reverse copolymer (T904+T90R4, T1107+T90R4) (Figure 3). As expected, the construct with the shorter
alkyl chain, SE3, displays the highest solubility in water at 4.92·10-4 M. Increasing the length of the hydrocarbon tail drastically
reduces solubility, with values of 3.5·10-5 M and 9·10-6 M obtained for SE6 and SE12. Incorporation into the polymeric nanocarri-
ers enhances the solubility (Figure 3), with the best solubilization observed with the smaller T904. The addition of the reverse
Tetronic T90R4, hypothesized to expand the hydrophobic locus of solubilization, instead slightly reduces the total drug loading.
The effect is more pronounced with the long chain organoselenium constructs (SE6 and SE12), whereas for the less hydrophobic
compounds (SE3), a slight improvement is noted in the mixed T1107 micelles compared to T1107 alone.
Overall, while the incorporation of the reverse polymer leaves micellar dimensions virtually unchanged, a possible rearrangement
of the polymer chains (in particular a more open and hydrated corona) may lead to a different environment, less favorable to drug
solubilization, at least for the compounds synthesized here.
3.3. Anticancer activity and selectivity
The drug constructs were evaluated for their cytotoxic activity using MTT assays against a panel of five human tumor cell lines as a
representative selection of liquid, solid and hormone-dependent tumors, including lymphocytic leukemia (CCRF-CEM), lung carci-
noma (HTB-54), colon carcinoma (HT-29), prostate adenocarcinoma (PC-3) and breast adenocarcinoma (MCF-7) (SI, Figure S2).
In order to evaluate the selectivity of the drugs, their toxicity was also assessed against a non-malignant cell line derived from nor-
mal breast tissue (184B5). The results are expressed in terms of IC50, the concentration that reduces by 50% the growth of treated
cells with respect to untreated controls.
SE12 shows a remarkable cytotoxic profile, together with selectivity, under our experimental conditions, with IC50 values lower
than 1 µM in MCF-7 and PC-3 and higher than 100 µM for 184B5 (Table 2). This represents a 65 and 100-fold increase (for MCF-
7 and PC-3, respectively) compared to the reference drug ebselen. These results suggest that the long alkyl lateral chain plays a key
role in the activity of the drug, with 12 atoms of carbon being the optimal length. In addition, SE4 and SE12 combine potency and
selectivity, a highly attractive feature for anticancer drugs, whose toxic side effects are a limitation to their use.

In a further step, and since the poor solubility of Se compounds is a limitation to their clinical use, the activity of the constructs was
assessed in formulations with the different Tetronics and combinations with the reverse one. MTT assays on the most responsive
cell line (MCF-7) were repeated on representative compounds (SE3, SE6 and SE12) encapsulated in T904, T904+T90R4 and
T1107 micelles (Figure 4). Table 3 reveals a remarkable decrease of the IC50 values for the compounds loaded in T904 and
T904+T90R4 mixed micelles in comparison to the non-formulated constructs, particularly noticeable for SE12 (Figure 4), (SI,
Figures S3, S4 and S5). While T1107 and T90R4 show some cytotoxicity on their own, T904 has minimum effect on the cells,
making it the ideal candidate for the development of delivery systems (SI, Figures S3, S4 and S5).
Considering together the cell growth inhibition, it can be stated that the substitution of the selenodiazole framework in position 5
with aliphatic chains result in an outstanding increase of the anticancer activity for this class of compounds. A comparison of the
reported cytotoxic activity for the unsubstituted carboxylic acid ([1] in Scheme 1) and carboxamide27 with the derivatives presented
herein reinforce this molecular design, making it a very promising approach to develop more potent and selective antitumor agents.
To our knowledge, the only Se-containing compound to date modified to form nanocapsules is selol incorporated in a polymeric
shell composed of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA). Nonetheless, this modification yielded only a slight
increase in the cytotoxicity in lung cells after 72 h of treatment30. Accordingly, the use of poloxamines in selenodiazole derivatives
achieved a much higher increase in the antitumor activity, making this construct very promising.
4. Conclusions
In summary, based on the importance of Se for human health and positive evidence from the literature on Se-heterocycles and in
particular the commercial drug ebselen, we have created a small library of selenocompounds, 5-substituted amide selenodiazoles
with increasing hydrophobicity, derived from alkyl lateral chains ranging from 3 to 12 carbon atoms. Given the low solubility and
poor bioavailability of Se-based compounds being a barrier towards their clinical use, our constructs were encapsulated in PEO-
PPO tetrablock copolymer micelles, namely, Tetronics T904 and T1107, and their combination with the reverse copolymer T90R4,
which has hydrophobic blocks as end blocks, and is poorly soluble. The incorporation of these new constructs within the micelles
largely enhanced their solubility, thus offering a route towards higher bioavailability. Combined SANS and DLS studies allowed us
to characterize the nanoaggregates as presenting a core-shell morphology, with a dense PPO core and a largely hydrated PEO coro-
na. The largely insoluble reverse Tetronic T90R4 is incorporated within the micelles (up to 20% of the total weight), and, aside
from a higher hydration of the corona, does not impact the structural parameters of the micelles, albeit slightly reducing the drug
loading within the aggregates. Biological evaluation highlights the selectivity of the constructs towards cancer cells, MCF-7 being
the most responsive line. The construct with the longest alkyl side chain, SE12, shows the best activity by far, with up to a 100-fold
increase with respect to ebselen. A further improvement of the activity is achieved by encapsulation within the polymeric micelles.
The smaller and more hydrophobic T904 displays the highest solubilization capacity, combined to the highest activity enhance-
ment, pointing to the combination of this nanocarrier with SE12 as a promising candidate for further optimization.
5. Acknowledgements
The authors thank ISIS for the provision of beam time and Richard Heenan and Steve King (Rutherford Appleton Laboratory) for
their assistance with the SANS experiments. Financial support from projects MAT2014-59116-C2-2-R of the Spanish MINECO,
Plan de Investigación de la Universidad de Navarra, PIUNA (Ref 2014–26), and Caixa Foundation-UNED is acknowledged. A.U.
is grateful to the School of Science of the UN for the SIP and Erasmus grants for her internship at KCL.

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Figure captions
Scheme 1. Synthesis pathway for compounds SE3, SE4, SE5, SE6 and SE12. a) SeO2, 260 °C, solid phase, 1h. b) SOCl2, reflux, 2h. c) R-
NH2 (R: propyl (SE3), butyl (SE4), pentyl (SE5), hexyl (SE6), dodecyl (SE12)), CHCl3, TEA, r.t., 48h.
A
100
B
60 ºC
55 ºC
10
50 ºC
45 ºC
1
40 ºC
35 ºC
30 ºC
T1107 (20 ºC)
T1107 + T90R4 (20 ºC)
0,1
25 ºC
20 ºC
T1107 (50 ºC)
T1107 + T90R4 (50 ºC)
0,01
0,01
0,1
1
10
100
1000
q (Å-1)
hydrodynamic diameter / nm
Figure 1. (A) DLS Intensity size distribution for T1107+T90R4 2% (1:1 mass ratio) as a function of temperature. Only unimers are present
up to 25 ºC, a mixture of unimers and micelles at intermediate temperatures (30-40 ºC) and only micelles over 40 ºC. (B) SANS curves for
the T1107+T90R4 mixture and T1107 alone at 20 and 50 ºC (solid lines are fits to the 4SP and CSS models described in the text)

100
10
1
100
10
1
A
B
40 ºC
40 ºC
30 ºC
30 ºC
20 ºC
20 ºC
0.1
0.1
T904 + reverse T90R4 5%
T1107 + reverse T90R4 5%
0.01
0.1
0.01
0.1
C
D
100
10
1
100
10
1
T904 + T90R4 5%
T904 + T90R4 15%
T904 + T90R4 25%
T1107 + T90R4 5%
T1107 + T90R4 15%
T1107 + T90R4 25%
0.1
0.1
0.01
0.1
0.01
0.1
q (Å-1)
q (Å-1)
Figure 2. SANS patterns for direct and reverse poloxamine solutions at varying temperatures and concentrations. (A) T904+T90R4 5% as
a function of temperature; models used are 4SP (green), 4SP+CSS (blue) and CSS-HS (red). (B) T1107+T90R4 5% as a function of tem-
perature. Models used are: 4SP (green), 4SP+CSS (blue) and CSS-HS (red). (C) T904+T90R4 at 40 ºC, concentrations 5, 15, and 25%,
model used is CSS-HS. (D) T1107+T90R4 at 40 ºC, concentrations 5, 15, and 25%; models used are CSS+HS (green, blue) and a body
centre cube (bcc) paracrystal (red).
Figure 3. Solubility studies. Final concentration (M) of SE3, SE6 and SE12 after 3 days at 40 ºC in water, T904, T904+T90R4, T1107 and
T1107+T90R4. Tetronic concentrations used are 15 % w/w, as for the biological assays. Concentrations of SE6 and SE12 are multiplied by
a factor of ten.

120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Figure 4. Cytotoxicity results on SE12 encapsulated in Tetronic systems (15% w/w) and tested against breast cancer MCF-7 cell line.
From left to right SE12 in T904, in T904+T90R4 and in T1107.
11

Table 1. Micellar structural parameters at 40 oC in D2O obtained from SANS data analysis: Rc (core radius), t (shell thickness),  (volume
fraction from the hard-sphere potential), s (scattering length density of the shell). In the mixed systems, the concentration is given as the
total wt %, at a 4:1 weight ratio.
s 106 / Å-2
5.87
Polymeric micelles
T904 8%
Rc / Å
35
t / Å
21
26
21
19
45
44
37

0.18
0.09
0.27
0.40
0.18
0.11
0.39
T904-T90R4 5%
T904-T90R4 15%
T904-T90R4 25%
T1107 5%
35
6.22
35
6.23
34
6.20
34
5.94
T1107-T90R4 5%
T1107-T90R4 15%
34
6.08
33
6.02
Table 2. MTT results for leukaemia (CCRF-CEM), colon (HT-29), lung (HTB-54), prostate (PC-3) and breast (MCF-7) cancer cells as
well as non-cancer breast tissue cells (184B5).
IC50 / µM
CCRF-CEM
91.9
HT-29
>100
>100
>100
81.2
HTB-54
>100
>100
>100
>100
>100
68.8
PC-3
>100
>100
>100
84.4
MCF-7
>100
11.1
184B5
>100
>100
>100
>100
>100
30.1
SE3
SE4
>100
SE5
68.4
84.3
SE6
45.7
>100
0.90
SE12
Ebselen
>100
11.5
0.20
57.1
>100
>100
58.8
Table 3. IC50 values for encapsulated and non-encapsulated selenocompounds SE3, SE6 and SE12. Results obtained by MTT assay per-
formed on MCF-7 cells.
IC50 / µM
No encapsulation
T904
6.20
1.38
0.05
T904+T90R4
1.33
T1107
46.85
>10
SE3
SE6
SE12
>100
>100
0.9
0.52
0.03
>1
12