Emerging Anticancer Activity of Candidal Glucoseamine-6-Phosphate Synthase Inhibitors upon Nanoparticle-Mediated Delivery

Article abstract of DOI:10.1021/acs.langmuir.8b04250

Numerous glutamine analogues have been reported as irreversible inhibitors of the glucosamine-6-phosphate (GlcN-6-P) synthase in pathogenic Candida albicans in the last 3.5 decades. Among the reported inhibitors, the most effective N³-(4-methox

Full text of DOI:10.1021/acs.langmuir.8b04250

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Biological and Environmental Phenomena at the Interface
Emerging Anticancer Activity of Candidal Glucoseamine-6-
Phosphate Synthase Inhibitors upon Nanoparticle-mediated Delivery
Ahmet Kertmen, #ucja Przysiecka, Emerson Coy, Lukasz Popenda,
Ryszard Andruszkiewicz, Stefan Jurga, and S#awomir Milewski
Langmuir, Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019
Downloaded from http://pubs.acs.org on March 26, 2019
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Emerging Anticancer Activity of Candidal Glucoseamine-6-Phosphate
Synthase Inhibitors upon Nanoparticle-mediated Delivery
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Ahmet Kertmen,§†‡* Łucja Przysiecka,‡ Emerson Coy,‡ Łukasz Popenda,‡ Ryszard Andruszkiewicz,† Stefan Jurga,‡ Sławomir
Milewski,†*
§Department of Quantum Electronics, Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan,
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Poland
†Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, G. Narutowicza 11/12,
80-233 Gdansk, Poland
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‡NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland
*Corresponding authors: ahmker@amu.edu.pl ; slamilew@pg.edu.pl
Abstract
Numerous glutamine analogs have been reported as irreversible inhibitors of Glucosamine-6-phosphate (GlcN-
6-P) synthase in pathogenic Candida albicans in last three-and-half decades. Among the reported inhibitors, the
most effective N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) has been extensively studied in
order to develop its more active analogs. A number of peptide-FMDP conjugates were tested to deliver FMDP
to its subcellularly located GlcN-6-P synthase target. However, rapid development of fungal resistance to FMDP-
peptides required development of different therapeutic approaches to tackle antifungal resistance. In the current
state of the of the global antifungal resistance, subcellular delivery of FMDP via free diffusion or endocytosis
has become crucial. In this study, we report on in vitro nanomedical applications of FMDP and one of its ketoacid
analogs, N3-trans-4-oxo-4-phenyl-2-butenoyl-L-2,3-diaminopropanoic acid (BADP). FMDP and BADP
covalently attached to polyethylene glycol coated iron oxide/silica core-shell nanoparticles, are tested against
intrinsically multidrug resistant Candida albicans. Three (3) different human cancer cell lines potentially
overexpressing GlcN-6-P synthase enzyme are tested to demonstrate the immediate inhibitory effects of
nanoparticle conjugates against mammalian cells. It is shown that nanoparticle-mediated delivery transforms
FMDP and BADP into a strong anticancer agent by inhibiting the growth of tested cancer cells, while their anti-
Candidal activity is decreased. This study discusses the emerging inhibitory effect of the FMDP/BADP –
nanoparticle conjugates based on their cellular internalization efficiency and biocompatibility.
Keywords: GlcN-6-P; GFAT; antifungal; iron oxide-silica nanoparticles; drug delivery; glutamine
Introduction
Among several promising fungal cytoplasmic enzyme targets reported in literature, targeting
glucosamine-6-phosphate (GlcN-6-P) synthase is considered advantageous because it can be inhibited by analogs
of amino acids that are relatively more accessible, low molecular weight compounds1–3. The glutamine analogs,
N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) and N3-trans-4-oxo-4-phenyl-2-butenoyl-L-2,3-
diaminopropanoic acid (BADP), particularly stand out with their irreversible inhibitory effect against Candida
albicans 4,5. However, anti-Candidal activities of BADP and FMDP are limited by poor cellular uptake efficiency.
These low molecular weight compounds, sometimes containing charged organic functionalities due to their
amino acid nature, do not diffuse through the cell membrane. On the other hand, they are poorly transported by
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fungal amino acid permeases. Since the cell membrane is impermeable, they cannot reach the target. In order to
reach their targets, these molecules must also compete with proteinogenic amino acids present in synthetic growth
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media and in mammalian extracellular body fluids, including blood serum 4,6–8
.
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To allow the internalization of the glutamine analogs and enhance their potential as antimicrobials,
“warhead delivery” or “smuggling” concept has been developed. The concept was based on the idea of
incorporating amino acid enzyme inhibitors into a short peptide (2-4 amino acid residues). Then, inhibitor-peptide
conjugates were delivered to the target by oligopeptide permeases (via active transport) that demonstrate a broad
range of substrate specificity. Eventually, the active inhibitors were expected to be released from the peptide
carrier upon the intracellular enzymatic hydrolysis9,10. Using this concept, several peptides exhibiting good
antifungal in vitro activity were designed, synthesized and characterized11–17. However, properties of these
peptide conjugates in in vitro and in vivo conditions were substantially limited by the poor serum stability and
fast development of fungal specific resistance resulting from the phenotypic repression of fungal oligopeptide
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transport systems 13,18–20
.
Since GlcN-6-P synthase requires intracellular access to function, identifying new strategies for inhibitor
translocation across cell membrane is essential. Although inhibitors can sometimes be chemically modified to
improve permeability, this often requires many interactions before translocation is achieved without sacrificing
activity. Alternatively, a “synthetic nanocarrier” can be utilized to promote cellular uptake of inhibitors without
chemical modification. Linkage of inhibitors to a nanocarrier may provide an entirely novel rationale for
antifungal chemotherapy by crossing the cell membrane by unspecific mechanism of internalization (free
diffusion or by endocytosis). Construction of such antifungal-nanocarrier conjugates may exhibit a broad
spectrum of inhibitory activity. The conjugates can remain active against the multiple drug resistant (MDR)
microbial cells as long as the intracellularly released fungal enzyme inhibitors would not be effluxed from the
cells by the MDR drug exporters. In fact, nanomedical approach is considered as a vital step towards development
of novel antifungal chemotherapies21 because the number of antifungals in use in clinics are already limited by
the emerging MDR; and the recent antifungal pipeline does not promise any new therapeutics in near-future.
From the practical point of view, developing nanomedical applications of GlcN-6-P synthase inhibitors
have number of reasons. In the era of antimicrobial resistance, it is a very well-known fact that the patients
undergoing immunosuppressive chemotherapy, such as in cancer, are especially prone to morbidity by systematic
candidiasis that eventually leads to 50-100% mortality22. Although treatment of Candidiasis is primarily
important in cancer patients, targeting fungal GlcN-6-P synthase during cancer is even more challenging than
previously estimated. According to most recent reports, GlcN-6-P synthase is not only essential to fungi but also
plays a major role in high glucose-induced progression of the multiple types of cancer23,24 including
cholangiocarcinoma25, colorectal cancer26 and adenocarcinoma27. Overexpression of GlcN-6-P synthase was
found responsible from aberrant glycolisation levels in cancer cells23,24. Thus, it is highly crucial to reveal
potential toxic effects of nanoparticle conjugates of GlcN-6-P synthase inhibitors toward human cancer cells.
Notably, from the research point of view, revealing GlcN-6-P synthase activity in cancer cells would be
advantageous. Using cancerous mammalian cells overexpressing the GlcN-6-P synthase, inhibitory effect of
nanocarrier conjugates on mammalian cells could be easily reflected with even low doses of inhibitors. Such a
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research is vital to understanding the immediate impact of nanomedical approach in GlcN-6-P synthase targeted
therapy.
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Therefore, in this work, we aim to study the nanoparticle conjugates of GlcN-6-P synthase inhibitors,
FMDP and BADP, to provide the first outlook for the GlcN-6-P synthase targeting in both fungal and human
cancer cells by a nanomedical approach. In order to prepare inhibitor-nanoparticle conjugates, we use the
superparamagnetic monodispersed iron oxide-silica core shell nanoparticles (CSNPs) synthesized by a very
recently described “nanoemulsion process” 28,29. We firstly describe a synthetic route for linking FMDP and
BADP to hydroxyl polyethylene glycol (OH-PEG) functionalized CSNPs, which forms “inhibitor-PEG-CSNP”
conjugates. Then, the inhibitory activity of inhibitor-PEG-CSNP conjugates are studied under in vitro conditions
against three (3) different human cancer cell lines and Candida albicans. The study primarily aims to demonstrate
potential of the nanomedical approach to replace “warhead delivery” in GlcN-6-P synthase targeting in Candida
albicans. The impact of the nanomedical approach on human cancer cells that overexpress the GlcN-6-P synthase
enzyme is discussed in the concept of GlcN-6-P synthase targeting. Internalization efficiency of the PEG-CSNPs
by impermeable fungal cell membrane and the human cancer cells are further discussed to understand the
“biocompatibility” criteria of Candida albicans in nanomedical applications.
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Experimental
The study consisted of a sequence of experiments that can be grouped in eight (8) steps: i) Obtaining
large amounts of CSNPs by “scaled-up” version of the previously reported “nanoemulsion process”28,29; ii)
purification of as-synthesized CSNPs by dialysis; iii) synthesis of FMDP and BADP with optically active Fmoc
protecting group; iv) functionalization of CSNPs by OH-PEG groups; v) development of synthetic procedure for
inhibitor-CSNP conjugation; vi) qualitative and quantitative detection of inhibitor-CSNP conjugation efficiency
by UV-Vis absorbance spectroscopy; vii) surface analysis of CSNPs by XPS for detection of surface purification
and conjugation of OH-PEG and inhibitors; viii) in vitro biological experiments with mammalian cells and the
Candida albicans to determine OH-PEG-CSNPs uptake and inhibitory activity of inhibitor-PEG-CSNP
conjugates.
Chemicals and Materials
Following reagents, solvents and materials were acquired from Sigma-Aldrich: CTAB (≥98%), oleic acid
(technical grade, 90%), 1-octadecene (technical grade, 90%), FeCl3·6H2O (≥98%), OH-PEG5K-NHS (hydroxyl-
PEG-NHS ester, average Mn = 5000), piperidine (ReganentPlus®, 99%), N,N’-diisopropylcarbodiimide (DIC,
99%), Nα-Fmoc-L-2,3-diaminopropionic acid (Fmoc-Dap-OH, 97%), N-hydroxysuccinimide (98%), N,N’-
dicyclohexylcarbodiimide (98%), Fmoc-Osu (Novabiochem®), mono-methyl fumarate (97%), CTAB (≥98%),
ethyl acetate (anhyd. 99.8%), chloroform, tetraethylorthosilicate (TEOS, 99%), 3-(aminopropyl)triethoxysilane
(APTES, ≥98%), n-hexane (99%), diethyl ether (99.5%), iron(II) acetate (95%), and dialysis tubing (cellulose
ester membrane with molecular weight cutoff = 14 000). Following reagents and solvents were acquired from
Avantor Performance Materials Poland S.A. (formerly POCH S.A.): sodium hydroxide (99.8%), nitric acid
(65%), hydrochloric acid (35 − 38%), ethanol (96%), acetic acid (glacial), 2-propanol (99.7%), sulfuric acid
(95%), hydrogen peroxide (30%). Epoxy resin (Polysciences Embed-it™ low viscosity epoxy kit). The following
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reagents were acquired from Alfa Aesar: ethyl isonitrosocyanoacetate (oxyma, 97%), trans-4-oxo-4-phenyl-2-
butenoate (predominantly trans, 97%).
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Characterization Methods
A Tensor 27 (Bruker Optics) spectrometer was used to record the FT-IR spectra of samples in the 4000-
200 cm-1 range with 2 cm-1 resolution (512 scans). Samples were prepared as KBr pallets. The background spectra
of water and CO2 were automatically subtracted by the OPUS operating software of the equipment.
TEM images of CSNPs and the biological TEM images of samples embedded in epoxy resin were
obtained by JEOL 1400 (60-120kV) working with a LB6 filament.
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X-ray photoelectron spectroscopy analysis was performed by SCIENTA R3000 hemispherical
photoelectron spectrometer equipped with monochromatic Al Kα source operating at 300W. The base pressure
in the analysis chamber was better than 1x10-10 mbar. All acquired spectra were calibrated to adventitious carbon
C1s at 285eV. The overall resolution of the spectrometer during measurements was 0.65eV as a full width of half
maximum (FWHM) of the Ag3d5/2 line. After subtraction of the Shirley-type background, the core-level spectra
were decomposed into main components with mixed Gaussian–Lorentzian lines (70% G + 30% L for majority
of photo-peaks) by a non-linear least squares curve-fitting procedure, using CasaXPS software.
NMR data were recorded on an Agilent DD2 800 spectrometer equipped with a 5 mm 1H{13C/15N} probe
head and operating at a resonance frequency of 799.890 and 201.151 MHz for 1H and 13C, respectively. All
measurements were carried out at 298 K. Chemical shifts were calibrated with respect to the residual solvent
peak (DMSO-d6: 2.50 ppm for 1H, 39.5 ppm for 13C). The signal assignment has been made on the basis of 1H,
13C and 2D 1H-13C gHSQC and 1H-13C gHMBC experiments.
ICP-MS analysis was carried out separately for OH-PEG-CSNPs, FMDP-PEG-CSNPs and BADP-PEG-
CSNPs samples according to PN-EN ISO 17294-2:2016-11 standards. From each aqueous stock sample, 100 µl
was transferred to a glass vial and dried at 80-90 °C till the water completely evaporated. Then the dry particles
were firstly digested in 0.2 ml piranha solution (v/v, 3:1 96% H2SO4 – 30% H2O2) under 30s sonication in a
sonication bath and cooled in liquid nitrogen till it freezes. Then, frozen piranha solutions were mixed with 0.2
ml aqua regia (3:1 HCl - HNO3) and sonicated for another 30s. Finally, the acid solutions were diluted to 10 ml
by addition of Milli-Q water. ICP-MS samples were prepared by further diluting 2 ml of the digestion solutions
to 10-15 ml in milli-Q water. For each sample 3 different dilutions were prepared, and the ICP-MS analysis
results were averaged.
Absorbance spectra were recorded by UV/Vis/NIR LAMBDA 950 spectrophotometer (Perkin Elmer) at
200 – 350 nm region (1 nm intervals) using 700 µl quartz vials with 2 mm path length. For each measurement,
background was removed by using identical solvents or solvent mixtures from respective chemical reactions.
Synthesis of antifungals and their intermediates
N-succinimidyl, methyl fumarate
Compound was synthesized according to previously described method4. In short, N-hydroxysuccinimide
(NHS) (2.54 g, 22 mmol) and dicyclohexycarbodiimide (DCC) (4.54 g, 22 mmol) were added to the solution of
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methyl hydrogen fumarate (2.6 g, 20 mmol) in 40 ml tetrahydrofuran at 0 °C under magnetic stirring. After
stirring overnight at room temperature, either of two different methods were flowed: i) THF was removed in
vacuo, resulting white powder including N,N’-dicyclohexylurea was re-dispersed in ethyl acetate, N,N’-
dicyclohexylurea filtered off and residue was crystallized from ethyl acetate/hexane or; ii) after N,N’-
dicyclohexylurea was filtered off and THF was removed in vacuo, residue was crystallized from ethyl
acetate/hexane (if the white residue is not fully dissolved in ethyl acetate, it is heated up to boiling point. Then
hexane is added when it cools down to room temperature). Crystallization process was initiated at room
temperature and continued at 4 °C. White solid, yield 70%. 1H NMR (800 MHz, DMSO-d6):  7.12 (d, J = 15.9
Hz, 1H), 7.09 (d, J = 15.9 Hz, 1H), 3.79 (s, 3H), 2.86 (bs, 4H); 13C NMR (201 MHz, DMSO-d6):  169.9, 164.0,
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160.6, 137.9, 127.0, 52.6, 25.5 (original H, 13C and 2D H-13C gHSQC data can be found in supporting
information Fig.S1-S3).
N-succinimidyl, trans-4-oxo-4-phenyl-2-butenoate
Compound was synthesized according to previously described method 30. In details, to the stirring
solution of trans-4-oxo-4-phenyl-2-butenoate (3.53 g, 20 mmol) and N-hydroxysuccinimide (NHS) (2.54 g, 22
mmol) in 50 ml THF at 0 °C, DCC (4.54 g, 22 mmol) was added. The mixture was stored at -20 °C overnight.
N,N’-dicyclohexylurea was filtered off and the solvent was removed in vacuo. Resulting residue was either
recrystallized from isopropanol (recrystallization assisted by hot sonication bath) or dissolved in hot isopropanol
to let the oily part to precipitate rapidly and then the solution was crystallized from isopropanol/hexane after
separation of oily phase by filtration. Yellow solid, yield: 70%. 1H NMR (800 MHz, DMSO-d6):  8.29 (d, J =
15.7 Hz, 1H), 8.08 (pseudo-d, 2H), 7.74 (pseudo-t, 1H), 7.60 (pseudo-t, 2H), 7.03 (d, J = 15.7 Hz, 1H), 2.88 (bs,
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4H); C NMR (201 MHz, DMSO-d6):  188.6, 170.0, 161.2, 142.3, 135.5, 134.4, 129.1, 124.8, 25.5 (original
1H, 13C, 2D 1H-13C gHSQC and 2D 1H-13C gHMBC data can be found in supporting information Fig.S4-S7).
N2-(Fmoc), N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (Fmoc-FMDP)
Compound was synthesized similarly to previously described method for its tert-butoxycarbonyl
protected analogue 4. In details, to the stirring solution of Nα-Fmoc-L-2,3-diaminopropionic acid (245 mg, 0.75
mmol) and NaHCO3 (630 mg, 7.5 mmol) in 18 ml 2:1 (v/v) water-methanol at 0 °C, N-succinimidyl ester of
methyl fumarate (170 mg, 0.75 mmol) was added. After 4h, the solvents removed in vacuo and replaced by 15
ml water. After adjusting pH with 10% citric acid, product was extracted with ethyl acetate (3x30 ml). Then the
crude organic phase was washed with saturated NaCl and dried over MgSO4. Product was crystallized by addition
of hexane. White solid, yield: 74.5%. 1H NMR (800 MHz, DMSO-d6):  12.84 (bs, 1H), 8.67 (t, J = 5.9 Hz, 1H),
7.89 (d, J = 7.5 Hz, 2H), 7.72 – 7.70 (m, 2H), 7.63 (d, J = 8.5 Hz, 1H), 7.42 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.4
Hz, 2H), 7.03 (d, J = 15.5 Hz, 1H), 6.58 (d, J = 15.5 Hz, 1H), 4.29 (d, J = 7.2 Hz, 2H), 4.23 (t, J = 7.1 Hz, 1H),
4.16 (td, J = 8.3, J = 5.2 Hz, 1H), 3.72 (s, 3H), 3.62 (dt, J = 13.5, 5.6 Hz, 1H), 3.41 (ddd, J = 13.8, 8.1, 6.0 Hz,
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1H); C NMR (201 MHz, DMSO-d6):  171.8, 165.5, 163.2, 156.0, 143.7, 140.7, 137.4, 128.2, 127.6, 127.1,
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125.2, 125.2, 120.1, 65.7, 53.4, 52.0, 46.6, 40.0 (original H, C, 2D H-13C gHSQC and 2D H-13C gHMBC
data can be found in supporting information Fig.S8-S11).
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N2-(Fmoc), N3-trans-4-oxo-4-phenyl-2-butenoyl-L-2,3-diaminopropanoic acid (Fmoc-BADP)
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Compound was synthesized similarly to previously described method for its tert-butoxycarbonyl
protected analogue 5. In details, to the stirring solution of Nα-Fmoc-L-2,3-diaminopropionic acid (326.4 mg, 1
mmol) and NaHCO3 (84 mg, 1 mmol) in 20 ml 1:1 (v/v) water-methanol at 0 °C , N-succinimidyl ester of trans-4-
oxo-4-phenyl-2-butenoate (273 mg, 1 mmol) was added. After stirring overnight, the solvent removed in vocuo
and replaced by 20 ml water. After adjusting pH with 1 M KHSO4 to pH 2, product was extracted with ethyl
acetate (3 x 30 ml). Finally, organic phase was dried over MgSO4. After Addition of minimum amount of
methanol to obtain a clear solution, product was crystallized by addition of petroleum ether and ethyl ether. White
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solid, yield 60%. H NMR (800 MHz, DMSO-d6):  12.85 (bs, 1H), 8.73 (t, J = 5.9 Hz, 1H), 7.99 (pseudo-d,
2H), 7.88 (d, J = 7.6 Hz, 2H), 7.75 (d, J = 15.3 Hz, 1H), 7.72 – 7.68 (m, 3H), 7.65 (d, J = 8.4 Hz, 1H), 7.57
(pseudo-t, 2H), 7.42 – 7.38 (m, 2H), 7.33 – 7.30 (m, 2H), 7.00 (d, J = 15.3 Hz, 1H), 4.30 (d, J = 7.2 Hz, 2H),
4.23 (t, J = 7.2 Hz, 1H), 4.20 (td, J = 8.4, J = 5.2 Hz, 1H), 3.66 (dt, J = 13.5, J = 5.5 Hz, 1H), 3.46 (ddd, J = 13.8,
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J = 8.1, J = 6.0 Hz, 1H); C NMR (201 MHz, DMSO-d6):  189.8, 171.8, 163.9, 156.0, 143.7, 140.7, 136.5,
136.1, 133.7, 132.1, 129.0, 128.6, 127.6, 127.1, 125.2, 125.2, 120.1, 65.7, 53.5, 46.6, 40.2 (original 1H, 13C, 2D
1H-13C gHSQC and 2D 1H-13C gHMBC data can be found in supporting information Fig.S12-S15).
cis-2-(9-Fluorenylmethyloxycarbonyl)amino-cyclopentanecarboxylic acid (Fmoc-cispentacin)
Compound was synthesized according to a combination of previously described methods 31,32. In details,
to the stirring solution of 2-aminocyclopentane-1-carboxylic acid (0.375 g, 2.9 mmol) and NaHCO3 (2.44 mg,
14.5 mmol) in 36 ml 2:1 (v/v) acetone-water at 0 °C, Fmoc-Osu (0.98 g, 2.9 mmol) was added. After stirring
overnight at room temperature, reaction mixture was diluted with addition of 12 ml water and acetone fraction
was removed in vacuo. Volume of aqueous residue was increased to 50 ml with addition of water and the
unreacted content was extracted with 50 ml by stirring in diethyl ether for 1h. After the removal of organic layer,
aqueous layer was acidified to pH 2 using 1N HCl and the product was extracted with ethyl acetate. Resulting
colorless oil was purified by crystallization from n-hexane/chloroform. White solid, yield: 83%. 1H NMR (800
MHz, DMSO-d6): δ 11.99 (bs, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.74 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H),
7.41 (t, J = 7.5 Hz, 2H), 7.35 – 7.31 (m, 2H), 7.31 (d, J = 8.7 Hz, 1H), 4.26 – 4.22 (m, 1H), 4.20 – 4.14 (m, 3H),
2.83 (q, J = 7.6 Hz, 1H), 1.96 – 1.90 (m, 1H), 1.87 – 1.82 (m, 1H), 1.81 – 1.72 (m, 2H), 1.66 – 1.60 (m, 1H), 1.52
– 1.46 (m, 1H); 13C NMR (201 MHz, DMSO-d6):  174.3, 155.5, 144.0, 143.7, 140.7, 140.6, 127.6, 127.6, 127.1,
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127.0, 125.5, 125.3, 120.0, 120.0, 65.5, 54.0, 47.4, 46.7, 31.3, 26.4, 21.6 (original H, C, 2D H-13C gHSQC
and 2D 1H-13C gHMBC data can be found in supporting information Fig.S16-S19).
Preparation of oleic acid capped iron oxide nanoparticles
Oleic acid capped iron oxide nanoparticles (OA-IONPs) with 12 nm average size were synthesized by
the thermal decomposition of the iron oleate complex according to a previously described method 29,33. Briefly,
ethanol/water/n-hexane (3:4:7, v/v/v) mixture was used to reflux 1.08 g of FeCl3·H2O and 3.65 g of sodium oleate
at 70 °C for 4 h to form iron oleate complex. After cooling down the iron oleate complex solution to room
temperature and separation of the aqueous phase by extraction (diethyl ether can be optionally used for
extraction), extracted phase was washed with water several times and organic solvents were removed in vacuo.
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In the next stage, 3.6 g of the resulting iron oleate complex and 0.57 g of oleic acid were refluxed in 20 g of 1-
octadecene for 30 min once the temperature reached 318-325 °C. Resulting nanoparticle suspension was collected
by precipitation in ethanol under centrifugal force (optionally, 1-part of hexane can be added to 5-parts of
ethanol). It is noteworthy to mention that the particle size can be adjusted to 8.0 ± 1.5 nm using the same
procedure as reported earlier34.
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Preparation of CSNPs
CSNPs were prepared by the slightly modified and “5-fold scaled-up” version of the previously described
“nanoemulsion process”29. The method was based on a fusion process of the clusters (colloidosomes) of oleate
capped iron oxide nanoparticles (IONPCs) to the nanoemulsions of silica precursors in acetate buffered aqueous
solution. In details, ∼150 mg of the OA-IONPs from a stock solution in hexane was dispersed in 6-8 ml
chloroform and transferred into a round flask containing 0.675 M CTAB aqueous solution. After a homogenous
emulsion is obtained by rigorous magnetic stirring, chloroform was removed by heating at 60C under N2 flow.
Volume of the resulting IONPC dispersion was increased to 250 ml by addition of water in a 500 ml two-necked
round flask. After adjusting pH to 11-12 by addition of 1.5 mL 2M NaOH solution, temperature was increased
to 60C and pH was readjusted to 7 by addition of 15 ml ethyl acetate. After a careful adjustment of temperature
to 60 C, 1.5 mL TEOS added. As an important modification to the originally reported method, amount of APTES
was increased by 15-fold and 750 μL APTES was added to solution (instead of 250 μL). Dispersion refluxed at
72− 76 °C in an oil bath for 3.15h and then allowed to cool down slowly for 1h, while keeping the flask in oil
bath under continues stirring. Reaction was quenched by addition of 7.5 ml aqueous HNO3 solution and bringing
the pH value to 3-4. Finally, resulting CSNPs were washed by precipitation from ethanol under 18000-24000
rpm centrifugal force (it is noteworthy to mention that the APTES amount was increased in order to introduce
increased amounts of amino groups in the silica shells of the particles. Minimum amount of APTES required for
original method29 is 50 uL in 50 ml solution. Therefore, for the 5-fold scaled-up version of process as reported
here, minimum 250 μL APTES in 250 ml solution would be sufficient under normal conditions. However, by
using 750 μL, we have introduced 3-fold “additional” APTES).
Dialysis of CSNPs
In order to purify CSNPs from toxic CTA+ molecules under mild conditions, as-synthesized CSNPs were
dispersed in 50 ml of acetic acid/sodium acetate buffer (pH 3.7 – 3.8) and transferred into the dialysis membrane
tubing. Nanoparticles were dialyzed against 1 L of acetic acid/sodium acetate buffer for seven (7) days. Then
solvent was replaced by EtOH-H2O (1:1, v/v) and dialysis continued for 3 days. In the last step, EtOH-H2O
dialysis solution was replaced by Milli-Q water and dialysis continued for 6-8 h. Finally, particles were stored at
4°C for further use.
Conjugation of inhibitors to CSNPs
Before the conjugation of Fmoc-FMDP and Fmoc-BADP to CSNPs, concentration of free surface amino
groups on CSNP surfaces were determined according to slightly modified version of a previously reported
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method . Briefly, a specific amount of nanoparticles from a stock solution was respectively washed with 1 x
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EtOH, 1 x H2O and 1 x 0.1 M NaB buffer (pH 8.15 - 8.50). Then the particles were dispersed in 1 ml 0.1 M NaB
buffer and added to 1 ml solution containing 1 mM Fmoc-NHS in a 2 ml Eppendorf. Reaction was carried out
for 3h under vigorous shaking in a thermoshaker at room temperature. After the removal of reaction solvents by
centrifuge, particles were thoroughly washed with 1:1 NaB – acetonitrile mixture until no absorbance was
detectable in UV-Vis spectroscopic measurements. Particles were then dispersed in 1 ml of acetonitrile and added
to 1 ml of 1% piperidine-DMF mixture in a 2 ml Eppendorf to release the covalently attached Fmoc group. After
24h of vigorous shaking in a thermoshaker at room temperature, particles were centrifuged and the supernatant
containing the piperidine-Fmoc adduct was separated into a clean vial. Concentration of the free-amino groups
of CSNPs were calculated according to absorbance calibration curve obtained from Fmoc – piperidine adduct
obtained for Fmoc-cispentacin compound in %1 piperidine solution.
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In order to prepare hydroxy polyethyleneglycol functionalized core-shell nanoparticles (OH-PEG-
CSNPs) 10-times excess OH-PEG-NHS in was dissolved in NaB buffer (pH pH 8.15 - 8.50) dispersion of CSNPs
at 0°C. After initial mixing at 0°C for 30 min, reaction was carried out for 24h at room temperature. The amount
of OH-PEG-CSNPs needed for reaction was calculated according to free-amino group density of CSNPs as
calculated above (please see the results section for discussion).
Attachment of Fmoc-FMDP and Fmoc-BADP to OH-PEG-CSNPs was carried out by their esterification
to OH-PEG chains. However, considering that OH-PEG functionalization might have not proceeded with 100%
efficiency, any remaining free-amino group on the CSNP surfaces after OH-PEG attachment was also considered
for amide formation. Therefore, DIC – oxyma crosslinking method suitable for both esterification and amidation
36
reactions in water containing environment was adopted with some modifications . In order to make sure that
maximum amount of free amino and OH groups available on the surface of CSNPs were functionalized by Fmoc-
FMDP and Fmoc-BADP, reagents was used in excess. Briefly, assuming that the concentration of amino groups
were equal to 1.5 x 10-8 mol, 60-fold excess of Fmoc-FMDP or Fmoc-BADP (9.1 x 10-7 mol); 90-fold excess of
diisopropylcarbodiimide (13.7 x 10-7 mol) and oxyma (13.7 x 10-7 mol); 360-fold excess of NaHCO3 (8.2 x 10-5
mol) were used. In other words, molar ratios of the reagent/reactant solutions used in this process were 5:5:1:5:4
(DIC : oxyma : NaHCO3 : Fmoc-FMDP/Fmoc-BADP : OH-PEG-CSNPs). Reactions were carried out in 2 ml
Eppendorf vials at room temperature for 8 h. Resulting Fmoc-FMDP-PEG-CSNPs and Fmoc-BADP-PEG-
CSNPs were used in Uv-vis, FTIR and XPS analysis.
In vitro Biological experiments
Cell lines and cell culture conditions
Human cervical cancer (HeLa), hypopharyngeal carcinoma (FaDu) cell lines and Candida albicans were
obtained from American Type Culture Collection (ATCC). Human fibroblast (MSU-1.1) cell line was obtained
from Prof. C. Kieda (CBM - CNRS, Orléans, France). Cells were cultured in a complete medium (Dulbecco’s
Modified Eagle’s Medium (DMEM) or Minimum Essential Medium (MEM- FaDu cell line) supplemented with
10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin and grown at 37 °C in humidified
atmosphere containing 5% CO2. Growth medium of human cells were supplemented by L-alanyl-L-glutamine
dipeptide (glutamax).
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Cytotoxicity and viability assays of human cells
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Human cervical cancer (HeLa), hypopharyngeal carcinoma cell line (FaDu), and fibroblasts with
malignant transformation potential (MSU-1.1)37,38 were used for in vitro cellular toxicity studies of OH-PEG-
CSNPs, FMDP-PEG-CSNPs and BADP-PEG-CSNPs. 1×103 cells/100 μl were seeded onto 96-well plates and
incubated overnight at 37 °C under a 5% CO2 atmosphere. The medium in the wells was then replaced with fresh
medium containing increasing concentrations of OH-PEG-CSNPs, FMDP-PEG-CSNPs and BADP-PEG-CSNPs
(2.5 - 50 μg/ml) and incubation continued for 24 h. The medium without any nanoparticles and with 50% DMSO
were used as a negative and positive control, respectively. The cytotoxic effect of nanoparticles on human cell
lines was determined by WST-1 assay according to manufacturer’s instructions (Takara, Clonetech). Briefly, 10
μl of WST-1 solution was added to each well and the plates were further incubated. After 2 h, the absorbance
was measured with a microplate reader (Anthos Zenyth 340rt) at 450 nm and 650 nm as reference. The
mitochondrial function and, by extension, the relative cell viability (%) related to the negative control were
calculated by test sample/negative control × 100%. Results are reported as the average ± standard deviation (SD)
of wells performed in triplicate.
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Growth kinetics and viability of Candida albicans
Candida albicans strain ATCC-10231 (wild type, intrinsically resistant to multiple drugs) was used in
this study. For routine culture, cells from stocks stored at -80 °C were propagated by streaking onto Yeast-
peptone-dextrose (YPD) agar plates, and incubated overnight at 30 °C. From these, a loopful of the overnight
Candida albicans growth was inoculated into flasks containing 8 ml of YPD liquid media in an orbital shaker at
200 rpm and 30 °C for 16 h. Candida albicans cells were diluted between 2.5 x 105 and 5x105 cells/ml in YPD
medium. 50 µl of the cell suspension were then added to each well of a 96-well plate. Appropriate concentrations
of OH-PEG-CSNPs, FMDP-PEG-CSNPs and BADP-PEG-CSNPs (2-50 µg/ml) were added and placed at 30 °C
incubator. Turbidity of the suspension as a measure of fungal growth was recorded spectrophotometrically at 570
nm (OD570 nm) with a microplate reader (Anthos Zenyth 340rt) at every 2 h interval up to 10 h and then up to 24
h. To avoid potential interference during optical measurements caused by the light scattering properties of the
nanoparticles, the same liquid medium without microorganism, but containing the same concentration of
nanoparticles were used as blank controls. The obtained data were plotted and comparative studies were
performed between control with and without nanoparticles. Results were reported as the average ± standard
deviation (SD) of wells performed in triplicate.
Cellular internalization studies
Internalization of OH-PEG-CSNPs was assessed by TEM analysis. Both Candida albicans and human
cancer cell lines (HeLa and FaDu) were exposed to 25 μg/ml of nanoparticles. After 3h and 24 h incubation
periods, cells were washed three times with PBS, fixed with a 2.5% glutaraldehyde in PBS for 2 h. Fixed cells
were post-fixed in 2% osmium tetroxide in PBS for 1 h, washed 3 x PBS, 2 x ddH2O for 10 min, dehydrated
through a graded ethanol series (50%, 70%, 80%, 90%, 2 x 100%) followed by acetone for 10 min, and embedded
in epoxy resin. After 48 h incubation at 60 °C, ultrathin sections (100 nm) were prepared using RMC
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ultramicrotome. Then, the ultrathin sections were stained with 2% uranyl acetate to improve the contrast before
observation under JEM 1400 transmission electron microscope.
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Characterization of CSNPs
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Figure 1: A,B) Low and high magnification TEM images of as-synthesized CSNPs prepared by “nanoemulsion process”; C,D) low and high magnification
TEM images of CSNPs dialyzed in acetate buffer.
As-synthesized CSNPs by “nanoemulsion process” having characteristic apple-bite-like cavities, were
already investigated in details by different TEM methods in our previous report29. Herein we show that the
nanoemulsion process could be successfully 5-fold scaled-up for the studies requiring large amounts of particles.
Here in demonstrated reproducibility of nanoemulsion process can also tolerate the use of 3-fold “additional
increase” in the amount of APTES (please see the explanatory note in experimental section). The additional
APTES was used to increase the number amino groups on the surface of CSNPs for further surface engineering
by covalent attachments. TEM images of the as-synthesized CSNPs evidences that the scaling-up the
nanoemulsion process or increasing the amount of APTES had no negative impact on the formation of
monodispersed CSNPs (Figure1A-B) in comparison to the particles obtained in the original report29.
It was previously evidenced that as-synthesized CSNPs were coated by thick carbon shell of toxic CTA+
molecule29. The CTA+ molecules may cause complications in further chemical functionalization studies by
hindering the nanoparticle surface or causing unspecific toxicity responses in biological studies. Therefore,
CSNPs were washed by dialysis against 2M acetic acid solution and the thick CTA+ layer was successfully
removed29. Herein, we demonstrate that acetate buffer (pH 3.7) could be also used for surface purification of
CSNPs, which serves as a much milder method in comparison to concentrated acetic acid solution. TEM images
of the CSNPs dialyzed in acetate buffer (dia-CSNPs) demonstrated that the particles were still intact, having
mean particle size of 46.5  1.8 nm (Figure 1C,D). Particles were observed to be slightly aggregated on
hydrophobic carbon supported TEM grid due to increased hydrophilicity after dialysis. Absence of any necking
between the silica shells indicates that agglomeration is not permanent. On the other hand, black dots observed
on particles in Figure 1D was caused by acetic acid/acetate salt contamination coming from acetate buffer, which
is difficult to remove under vacuum.
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Removal of CTA+ layer from the surface of CSNPs after dialysis process was correlated by the
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accessibility of free amino groups within the silica shell of CSNP. In order to do this, CSNPs were functionalized
by Fmoc groups by reaction of Fmoc-NHS with CSNPs to form Fmoc-CSNP conjugates. Then, the concentration
of Fmoc-piperidine adducts obtained from 1% piperidine solution of Fmoc-CSNPs was determined by
absorbance spectroscopy. The amount of Fmoc directly attached to surface of dia-CSNPs was determined to be
equal to 15.2 nmol (further details in supporting information Fig.S20). Detection of Fmoc detached from CSNP
surface simply confirmed the accessibility to amino groups on dia-CSNPs. It is noteworthy to mention that, the
reason of employing Fmoc-cispentacin was the ease of compound preparation (one-step reaction) and its amino
acid nature, although it is a non-proteogenic amino acid. The protocol developed for Fmoc-cispentacin was
further used for analysis of more complex, laborious, costly Fmoc-BADP and Fmoc-FMDP molecules.
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Figure 2: General functionalization scheme of CSNPs via amino groups. In the first step, OH-PEG chains (blue) attached to amino groups of silica shell
by amide bonding. In the next step, Fmoc-FMDP/BADP (red) is attached to OH-PEG chains by esterification or silica shell by amide bonding.
Impact of dialysis procedure on CSNPs was additionally investigated by XPS spectroscopy. Notably,
N1s XPS analysis of the samples gave a totally different impression than the XPS spectra obtained for previously
reported as-synthesized CSNPs29 (please see Fig.S21 in supporting information). In the previous report, 402.29
eV was assigned to C-N species of CTA+ molecules whereas 400.27 eV was assigned to amino groups of the
APTES molecules29. However, being differently than the previous XPS measurement where the surface of CSNPs
was cleaned/etched by ion sputtering, herein a mild chemical cleaning procedure was applied. Chemical process
could be expected to introduce additional chemical species that can alter binding energies. Interaction of acetate
species with amino groups during the dialysis process is one of the most probable scenarios. Correspondingly,
the new XPS spectra obtained after dialysis shows an improvement in the intensity of nitrogen species with a
negligible shift in binding energy (399.7 eV for as-synthesized CSNPs and 400.09 eV for dialyzed CSNPs).
Although, it was demonstrated previously 29 that dialysis removed the CTA+ molecules from the surface
of as-synthesized CSNPs, XPS results shown here evidenced similar C-C/C-O ratios for as-synthesized and
dialyzed CSNPs (Table 1). This observation was attributed to the exposed propyl groups of APTES species and
the acetate species adsorbed on the surface of CSNPs after dialysis process.
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Quantitative O1s XPS analysis of the samples demonstrated that the relative amount of oxygen content
of the samples was calculated as 50.14% for as-synthesized CSNPs and 48.38% for dia-CSNPs. It was clear that
the amount of oxygen removed with the silica layer after dialysis was replaced by other species. Oxygen rich
acetate species might have replaced the lost SiO2 oxygen atoms. This observation is also accordance with the
N1s and C1s data hinting the interaction of acetate and amino groups on the surface of CSNPs after dialysis. The
same observation was also correlated by the equal amount of C-O species found in C1s data. Presence of acetate
species must have compensated the lost oxygen and the C-C energies after the removal of CTA+ molecules by
dialysis (considering that CTA+ molecules consist of 19 carbon atoms; a large amount of acetate molecules must
have been adsorbed).
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C-C
C-O / C-O-C
samples
energy fwhm
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energy fwhm
%
as-synthesized CSNPs
284.99 2.507 80.29 286.39 3.956 19.71
284.98 2.688 80.87 286.38 19.13
285.01 1.774 41.53 286.41 2.731 58.47
dia-CSNPs
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Fmoc-FMDP-PEG-CSNPs 284.98 2.486 58.96 286.38 3.532 41.04
Fmoc-BADP-PEG-CSNPs
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2.086 57.97 286.4 3.136 42.03
Table 1: C-C and C-O, C-O-C ratios obtained from XPS analysis
Conjugation of inhibitors to CSNPs
Fmoc-FMDP (N2-Fmoc, N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid) and Fmoc-BADP (N2-
Fmoc, N3-trans-4-oxo-4-phenyl-2-butenoyl-L-2,3-diaminopropanoic acid) have been synthesized in moderate to
high yields according synthetic procedure presented in Fig.S22 of supporting information. 2D-NMR studies
confirmed purity and identity of the target compounds. Details of the synthetic procedures were given in the
Experimental Section.
In the first step of conjugation studies, CSNPs were functionalized with hydroxyl polyethylene glycol
(OH-PEG) chains to increase their water dispersibility/stability, to provide them with increased biocompatibility.
Moreover, PEG chains were used to facilitate the covalent attachment of GlcN-6-P synthase inhibitors via ester
bonds according to scheme presented in Figure 2. Conjugation of OH-PEG chains to CSNPs was confirmed by
FTIR spectroscopy. When OH-PEG functionalized CSNPs (OH-PEG-CSNPs) and acetate buffer dialyzed
CSNPs (dia-CSNPs) were compared to as-synthesized CSNPs, it was seen that all the significant vibrations of
Si-O-Si, C-O-C and C-C vibrations were concentrated at 900-1300 cm-1 band (please see the data in supporting
information Fig.S23). In order to reveal the impact of the acetic acid/acetate assisted purification of CSNPs,
previously used dialysis method against concentrated acetic acid solution29 and acetate buffer were also
compared. Comparison of acetic acid and acetate buffer dialyzed CSNPs revealed the minor differences between
C-C vibrations of CTA+ and Si-O-Si vibrations of silica shells. As shown in previous report29, the outer surface
of the CSNPs were coated by CTA+ molecules, however, acetic acid dialysis removed a thick ∼12 nm of silica
shell along with the CTA+ molecules. Therefore, upon acetic acid treatment, vibrations at 1066 cm-1 belongs Si-
O-Si bonding were revealed while a significant reduction in intensity at 1100-1200 cm-1 region observed.
Altogether, these vibrations were attributed to removal of CTA+ molecules. On the other hand, when compared
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to as-synthesized CSNPs, the band shape of the acetate buffer dialyzed (dia-CSNPs) and OH-PEG functionalized
samples were preserved while the intensity of the bands significantly changed. For these latter two samples, 1096
cm-1 bandbecame the only significant vibration. No spectral difference between the as-synthesized and OH-PEG
functionalized samples were observed. Therefore, C-C vibrations of both PEG and CTA+ molecules were
concentrated at 1096 cm-1 region. This conclusion correlated by the functionalization of CSNPs with different
amounts of OH-PEG that resulted in significant change in band intensity at 1096 cm-1 while the band shape was
retained (samples assigned as 1x or 5x, which stand for 1:1 and 1:5 molar ratios of PEG - free amino group).
In the following steps of conjugation studies, Fmoc-FMDP and Fmoc-BADP were covalently attached
to OH-PEG-CSNPs to obtain desired inhibitor – nanocarrier conjugates (Fmoc-FMDP-PEG-CSNPs and Fmoc-
BADP-PEG-CSNPs). FTIR analysis of Fmoc-FMDP-PEG-CSNPs and Fmoc-BADP-PEG-CSNPs in
comparison to OH-PEG-CSNPs did not provide any conclusive information since all the aliphatic C-H vibrations,
including Si-O-Si and C-O-C vibrations, were concentrated at 1000-1250 cm-1 band. Only the difference
observed for Fmoc-FMDP-PEG-CSNPs was the appearance of additional weak peak at 1394 cm-1, which was
attributed to do the exitance of -OCH3 vibrations from FMDP molecules39. On the other hand, apart from slightly
shifted bands of Fmoc-FMDP-PEG-CSNP sample, there were no significant difference at 2800-3000 cm-1 region
to distinguish the C-H stretching vibrations (please see the data in supporting information Fig.S24). However,
conjugation of Fmoc-FMDP and Fmoc-BADP to OH-PEG-CSNPs were qualitatively and quantitatively
confirmed by UV-Vis spectroscopy by the release of Fmoc protecting groups of conjugated FMDP and BADP
molecules in 20% piperidine-DMF solution. Both samples exhibited the characteristic absorbance peak of Fmoc-
piperidine adduct at 300-301 nm (please see the data in supporting information Fig.S25). Calibration curves
constructed from absorbance spectra of (Fmoc-BADP)-20% piperidine and (Fmoc-FMDP)-20% piperidine
solutions were used to calculate the concentration of Fmoc – piperidine solutions obtained from Fmoc-BADP-
PEG-CSNPs and Fmoc-FMDP-PEG-CSNPs samples. Correspondingly, the concentration of Fmoc-BADP and
Fmoc-FMDP were 206 and 308 nmol (173.3 and 205.3 µM), respectively (Please see the table in Fig S25 in
supporting information). Quantitative UV-vis absorbance measurements of the Fmoc-piperidine adducts obtained
from Fmoc-BADP-PEG-CSNPs and Fmoc-FMDP-PEG-CSNPs were strong indications of covalent Fmoc-
FMDP and Fmoc-BADP attachments to CSNPs.
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Despite the UV-vis absorbance measurements strongly indicative of successful Fmoc-FMDP and Fmoc-
BADP attachments, further confirmation was sought by high-resolution XPS analysis. C1s analysis of Fmoc-
BADP-PEG-CSNPs and Fmoc-FMDP-PEG-CSNPs in comparison to as-synthesized CSNPs, dia-CSNPs and
OH-PEG-CSNPs clearly evidenced that OH-PEG was successfully attached to CSNPs (please see Fig.S26 in
supporting information). This information was retrieved from the significant increase observed in the intensity
of C-O/C-O-C binding energy of OH-PEG-CSNPs at 286.4 eV relative to C-C binding energy of as-synthesized
and dialyzed CSNPs at∼285 eV (Table 1). O-C=O binding energy visible in Fmoc-FMDP-PEG-CSNPs spectrum
was regarded as the indication of Fmoc attachment. Since the same energy level was missing from Fmoc-BADP-
PEG-CSNP sample, presence of this band as regarded as a strong contribution from methyl ester groups of FMDP
molecules.
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Figure 3: A) MSU-1.1, B) FaDu and C) HeLa viability test results. FMDP-PEG-CSNPs were tested in 2.5-25 µg/ml concentration range. BADP-PEG-
CSNPs and OH-PEG-CSNPs were tested in 2.5-50 µg/ml concentration range.
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N1s XPS analysis of the samples demonstrated the attachment of OH-PEG species to CSNPs caused a
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significant shift to 401.7 eV was observed in comparison to dia-CSNPs (please see Fig.S21 in supporting
information). This binding energy can be attributed to the formation of amide bonds (N-C=O). On the other hand,
400.08 eV binding energy equally observed for both Fmoc-FMDP-PEG-CSNPs and Fmoc-BADP-PEG-CSNPs
confirmed the fact that both molecules carry the same N1s species. The N1s species of Fmoc-FMDP-PEG-CSNPs
and Fmoc-BADP-PEG-CSNPs cause 1eV shift from OH-PEG-CSNP spectrum that lacks the same N1s species.
O1s XPS analysis of the conjugates demonstrated that the relative amount of oxygen content of the
samples was 47.93% (OH-PEG-CSNPs), 48.74% (Fmoc-FMDP-PEG-CSNPs) and 48.84% (Fmoc-BADP-PEG-
CSNPs). Only 0.45% difference between the dia-CSNPs and the OH-PEG-CSNPs suggests two possibilities: i)
the adsorbed acetate species by dia-CSNPs removed / replaced by almost equal amounts of long PEG chains or;
ii) after attachment of long PEG chains on the surface of the CSNPs, XPS can no more reflect the contributions
of adsorbed oxygen species hindered below PEG coating. However, we can still see the ~1% difference in oxygen
species after the conjugation of Fmoc-FMDP/BADP to OH-PEG-CSNPs as a positive confirmation of
attachment. When the XPS spectra was analyzed further, the shift at ∼534.6 eV observed for OH-PEG-CSNPs
and as-synthesized CSNPs was attributed to the water content (please see Fig.S26 in supporting information).
Especially the shift of OH-PEG-CSNPs spectrum to the higher energy levels relative to spectra of other samples
makes the presence of water more probable. For dialyzed and Fmoc-FMDP/BADP-PEG-CSNPs samples, the
peak at 533.4 eV was attributed to the O-C=O bonds of ester species. Such observation is particularly important
since it reveals the existence of ester bonds after dialysis. Considering that the dialysis with acetate buffer
removed ∼10-12 nm of silica shell during dialysis, the breakage of the Si-O-Si bonds might have facilitated by
formation of ester bonds between Si-O and acetate species.
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Figure 4: A) viability of Candida albicans in the presence of 0.4-25 µg/ml OH-PEG-CSNPs and 0.4-25 µg/ml FMDP-PEG-CSNPs and 0.4-50 µg/ml
BADP-PEG-CSNPs; B) Candida albicans growth kinetics in the presence of 25 µg/ml OH-PEG-CSNPs, 25 µg/ml FMDP-PEG-CSNPs of CSNPs and 50
µg/ml BADP-PEG-CSNPs. The results are expressed as the mean of three independent experiments and the error bars show the standard deviation.
In vitro biological studies
Since the Glcn-6-P synthase enzyme is overexpressed in cancer cells, the cancer cells constitute a good
model to understand the toxic effect of the inhibitor-nanoparticle conjugates on mammalian GlcN-6-P synthase
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enzymes. In order to make sure that the inhibitory effect was not caused by nanoparticles, cytotoxicity of 2.5-50
µg/ml OH-PEG-CSNPs were tested on human hypopharyngeal carcinoma cells (FaDu), human cervical cancer
cells (HeLa) and fibroblasts with malignant transformation potential (MSU-1.1). Concentration of nanoparticle
conjugates were determined by ICP-MS analysis as described in Experimental Section (concentrations were
expressed as the total mass of iron and silicon elements in CSNPs). The tests revealed that OH-PEG-CSNPs did
not exhibited any cytotoxic effects against either of the cancer cell lines tested (Figure 3).
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Figure 5: TEM images of HeLa cells after incubation with 25 µg/ml OH-PEG-CSNPs. X’ and X’’ represents two different levels of magnification of
selected area indicated by red rectangles in image X. X* represents the magnified view of the selected area indicated by yellow rectangle in image X.
After confirming highly biocompatible nature of OH-PEG-CSNPs with human cancer cells in in vitro
tests, inhibitory effects of the inhibitor – CSNP conjugates were further tested. In order to do this, firstly, Fmoc
protecting groups of the Fmoc-FMDP-PEG-CSNPs and Fmoc-BADP-PEG-CSNPs were removed in 20%
piperidine to prepare FMDP-PEG-CSNPs and BADP-PEG-CSNPs. Unprotected conjugates were further purified
with successive ethanol and water washes. At 2.5-10 µg/ml concentration range, FMDP-PEG-CSNPs and BADP-
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PEG-CSNPs were as biocompatible as OH-PEG-CSNPs in in vitro tests with HeLa and FaDu cells. However, at
10 µg/ml concentration, FMDP-PEG-CSNPs started to be cytotoxic against MSU-1.1 cells by decreasing the cell
viability ∼20%. At 25 µg/ml concentration, FMDP-PEG-CSNPs caused a significant decrease in cell viability of
FaDu (up to 85%) and MSU-1.1 (90%) whereas only a moderate decrease (∼50%) in viability of HeLa cells was
observed. On the other hand, at 2.5 – 25 µg/ml concentration range, BADP-PEG-CSNPs did not exhibit any
inhibitory effect towards either of the human cancer cells tested. However, when the concentration of BADP-
PEG-CSNPs was increased to 50 µg/ml, 36% of FaDu, 63% of MSU-1.1 and ∼70% of HeLa cells were inhibited
(Figure 3).
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Figure 6: TEM images of FaDu cells after 3 h (A-C); and 24 h incubation with 25 µg/ml OH-PEG-CSNPs (D-E). X’ and X’’ represent the magnified view
of the selected area indicated by yellow rectangles in image X. X* represents the magnified view of the selected area indicated by red rectangles in image
X.
Viability and growth kinetics of Candida albicans were determined in the 0.4-50 µg/ml concentration
range for FMDP-PEG-CSNPs, BADP-PEG-CSNPs and OH-PEG-CSNPs. Results presented in Figure 4
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indicates that C. albicans as opposed to cancer cell lines were quite resistant to FMDP-PEG-CSNPs and BADP-
PEG-CSNPs. OH-PEG-CSNPs did not exhibit any cytotoxic effects within the indicated concentration range.
FMDP-PEG-CSNPs caused 17% decrease in the viability of C. albicans. Viability of C. albicans further
decreased by 28% when 50 µg/ml of BADP-PEG-CSNPs was used.
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Figure 7: TEM images of Candida albicans after 24 h incubation with 25 µg/ml OH-PEG-CSNPs showing the particles retained in the cell-wall (A-D),
internalization of particles by shifted cytoplasm of the cells (E-G) and the internalized nanoparticles (H-J).
Determination of CSNP internalization by TEM imaging
Internalization efficiency of OH-PEG-CSNPs into human cervical cancer (HeLa) and hypopharyngeal
carcinoma (FaDu) cell lines have been investigated by TEM imaging. 3h and 24h incubated HeLa cells did not
show any significant difference in terms of number of internalized nanoparticles. Figure 5A-D evidence that
large amounts of nanoparticles were encapsulated into intracellular departments of HeLa cells. However,
nanoparticle internalization efficiency of FaDu cells significantly differed between 3h and 24h incubation periods
as opposed to HeLa cells. Only couple of individual nanoparticles were internalized by FaDu cells after 3h of
incubation (Figure 6A). After 24 h incubation, both HeLa and FaDu cells were occupied with large amounts of
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OH-PEG-CSNPs (Figure 6B-C). For the 24h incubated FaDu cells, it was also possible to observe the early
stages of endocytosis process where the nanoparticles were getting engulfed by the cell membrane (Figure 6C’
and Figure 6D-F). It is noteworthy to highlight that most of the particles in HeLa cells were still encapsulated
in endosomes while the particles in FaDu cells seemed released into cytosol within 24h. This observation could
explain the higher levels of decrease in viability of FaDu cells in cytotoxicity tests with FMDP-PEG-CSNPs.
Despite the fact that 3-24 h incubation was sufficient for FaDu and HeLa cells to internalize significant
amounts of nanoparticles, Candida albicans were just in the beginning of internalization process after 24h of
incubation. Although most of the nanoparticles were aggregated around Candida albicans cell walls, images in
Figure 7A-D demonstrated that some particles were able to penetrate into the cell walls. Images in Figure 7E-
G showed that the cytoplasm of some cells were shifted towards to area where large amounts of nanoparticles
were getting internalized altogether. Nevertheless, images in Figure 7H-J demonstrated that some nanoparticles
were able to pass through the cell wall and cell membrane, which eventually reached cytosol.
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TEM imaging studies performed to observe the internalization efficiency of OH-PEG-CSNPs evidenced
that the cell membrane of the Candida albicans was indeed very efficient in their mechanism of acting as an
impermeable barrier to hydrophilic (biocompatible) nanoparticles. It is shown for the first time that nanoparticles
having 50 nm average size can pass through cell membrane of Candida albicans and reach the cytoplasm of
fungal Candida albicans cells within 24h. However, the low efficiency of the nanoparticle internalization raises
the questions whether achieving high biocompatibility by using highly biocompatible PEG chains should be the
primary concern in nanoparticle-based antifungal therapy or not. Inefficient internalization of OH-PEG-CSNPs
by Candida albicans suggests that the use of more lipophilic formulations of conjugates while slightly scarifying
the biocompatibility towards mammalian cells might be a better strategy for more efficient nanoparticle
internalization by fungal cells. Similar strategies were very recently adopted for the development of lipophilic
FMDP prodrugs that have already proven the applicability of high lipophilicity concept40,41
.
The current results obtained for the GlcN-6-P synthase targeting by nanomedical approach further
complicates the antifungal therapy expectations. Results were in conflict with the previously reported non-toxic
in vitro activity of FMDP – peptide conjugates against human cell lines16,17,42. However, it must be noted that, in
case of peptide conjugates, peptide permeases located in fungal cell membrane are responsible from the delivery
of the inhibitors. Lack of the same permeases in mammalian cell is considered as the primary reason of
insufficient delivery of the peptide conjugates to the mammalian cells, which explains their low inhibitory effect
towards mammalian cells. However, in the case of nanoparticle conjugates, unspecific cellular delivery function
(free-diffusion or endocytosis) results in delivery of elevated number of inhibitors to the mammalian cells. As it
was observed from the TEM images, large numbers of CSNPs internalized into cancer cells explains the high
cytotoxic effects of CSNP conjugates of FMDP and BADP toward cancer cells.
It is very important to highlight that increased amount of glutamine is known to be capable of reversing
the inhibitory activity of FMDP as it was demonstrated in the past 6,20,43. In fact, glutamine-induced reversibility
of enzyme function might be the explanation of why the conjugates were not cytotoxic to human cancer cells at
low 0.4-10 µg/ml concentrations because a glutamine analog (L-alanyl-L-glutamine dipeptide) was also used in
in vitro tests carried out in this study. On the other hand, it must be noted that the primary reason of using human
cancer cells was the overexpressed GlcN-6-P synthase enzyme that could more prominently reflect inhibitory
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effects of the FMDP-PEG-CSNPs and BADP-PEG-CSNPs. Thus, highly decreased viability of the human cells
by FMDP-PEG-CSNPs and BADP-PEG-CSNPs at slightly higher 25-50 µg/ml range easily overcome the
reversibility effects of the glutamine content. These results reveal the effectiveness of nanoparticle mediated
delivery of the GlcN-6-P synthase inhibitors in human cells in even glutamine rich growth environment. These
observations suggest that adverse effects of nanoparticle-mediated GlcN-6-P synthase targeting in the presence
of mammalian cells must be cautiously taken under consideration.
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Conclusions
The study evidenced that CSNPs prepared by recently reported “nanoemulsion process”29 pose no
cytotoxic effects to human and fungal cells when they were coated by the hydroxyl polyethylene glycol (OH-
PEG) chains. Despite the high level of biocompatibility exhibited by OH-PEG-CSNPs towards the human cells,
low efficiency of particle internalization by Candida albicans raises questions about the mode of biocompatibility
in antifungal therapy. The study suggests that achieving high biocompatibility towards human cells by
employment of hydrophilic PEG chains does not constitute a good nanoparticle mediated antifungal treatment
approach due to highly lipophilic nature of fungal cell membrane. Consequently, low inhibitory effects of FMDP-
PEG-CSNP and BADP-PEG-CSNP towards Candida albicans were attributed to highly hydrophilic nature of
the conjugates that are incompatible with the fungal cell membranes. However, it is still an open question whether
achieving biocompatibility towards fungal cells by increasing the lipophilicity of nanoparticles (by potentially
decreasing the uptake by human cells) would constitute a more feasible approach or not.
The study demonstrated that construction of the FMDP-PEG-CSNP and BADP-PEG-CSNP conjugates
was highly feasible from the chemistry point of view. However, FMDP-PEG-CSNP and BADP-PEG-CSNP had
entirely different fates than the previously reported FMDP – peptide conjugates. Although FMDP-dipeptides
were relatively non-cytotoxic to human cancer cells16,17,42, herein reported FMDP-PEG-CSNP and BADP-PEG-
CSNP conjugates were able to decrease the viability of the human cancer cells up to 90%. The results raise
important safety concerns about the use of nanoparticle formulations of GlcN-6-P synthase inhibitors in the
presence of mammalian cells. Considering that the fate of GlcN-6-P synthase inhibitors can dramatically change
by nanoparticle-mediated delivery, any future nanomedical application by targeting GlcN-6-P synthase enzyme
in the presence of mammalian cells must be taken cautiously.
Acknowledgements
AK acknowledges the PRELUDIUM grant provided by the National Science Centre (NCN) of Poland (UMO-
2015/17/N/NZ7/01087), European Union Human Capital Operational Programmes for the projects entitled “The
development of interdisciplinary doctoral studies at the Gdansk University of Technology in modern
technologies” (POKL.04.01.01-00-368/09) and “The Centre for Advanced Studies - the development of
interdisciplinary doctoral studies at the Gdansk University of Technology in the key areas of the Europe 2020
Strategy” (POKL.04.03.00-00-238/12). Authors thank dr. Błażej Scheibe and dr. Zuzanna Pietralik for their
collaboration. Support of dr. Grzegorz Nowaczyk regarding the TEM analysis is gratefully acknowledged.
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Author Contributions
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Original idea: AK, SM; Project conception: AK, SM, EC, RA; Synthesis of nanoparticles and antifungals:
AK; Biological studies: LPr; NMR analysis: LPo; TEM analysis: AK, EC; XPS analysis: AK; FTIR and UV-
Vis spectroscopy measurements and analysis: AK. Results Discussion: AK, EC, SM; Manuscript
Preparation: AK,
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