Towards potent but less toxic nanopharmaceuticals-lipoic acid bioconjugates of ultrasmall gold nanoparticles with an anticancer drug and addressing unit

Article abstract of DOI:10.1039/c8ra01107a

Modification of ultrasmall gold nanoparticles (AuNPs) with the lipoic acid derivative of folic acid was found to enhance their accumulation in the cancer cell, as compared to AuNPs without addressing units. The application of lipoic acid enabled the control of the gold nanoparticle functionalities leading to enhanced solubility and allowing for attachment of both the folic acid and the cytotoxic drug, doxorubicin. More robust attachment of doxorubicin to the nanoparticle through the amide bond resulted in toxicity comparable with that of the drug alone, opening a new perspective for designing more potent, but less toxic nanopharmaceuticals. The increased uptake was accompanied by pronounced nuclear accumulation and observable cytotoxicity. Doxorubicin binding via covalent amide bonds enhanced stability of the whole drug vehicle and provided much better control over doxorubicin release in the cell environment, as compared to physical adsorption or pH sensitive bonding commonly used for anthracycline carriers. Confocal microscopy revealed that the bond was stable in the cytoplasm for 22 h. The ability to slow down the rate of drug release may be crucial for the application in sustained anticancer drug delivery. Biological analyses performed using MTT assay and confocal microscopy confirmed that the ultrasmall AuNPs with the lipoic acid derivative of folic acid exhibit relatively low cytotoxicity, however when loaded with a chemotherapeutic, they cause a significant reduction in the cell viability.

Full text of DOI:10.1039/c8ra01107a

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Towards potent but less toxic
nanopharmaceuticals – lipoic acid bioconjugates
of ultrasmall gold nanoparticles with an anticancer
drug and addressing unit†
Cite this: RSC Adv., 2018, 8, 14947
a
Maciej Dzwonek,a Dominika Załubiniak,a Piotr Piatek,
Grzegorz Cichowicz,b
˛
Sylwia Meczynska-Wielgosz,c Tomasz Stepkowski,c Marcin Kruszewski,cd
˛
˛
a
Agnieszka Wieckowskaa and Renata Bilewicz
*
˛
Modification of ultrasmall gold nanoparticles (AuNPs) with the lipoic acid derivative of folic acid was found
to enhance their accumulation in the cancer cell, as compared to AuNPs without addressing units. The
application of lipoic acid enabled the control of the gold nanoparticle functionalities leading to
enhanced solubility and allowing for attachment of both the folic acid and the cytotoxic drug,
doxorubicin. More robust attachment of doxorubicin to the nanoparticle through the amide bond
resulted in toxicity comparable with that of the drug alone, opening a new perspective for designing
more potent, but less toxic nanopharmaceuticals. The increased uptake was accompanied by
pronounced nuclear accumulation and observable cytotoxicity. Doxorubicin binding via covalent amide
bonds enhanced stability of the whole drug vehicle and provided much better control over doxorubicin
release in the cell environment, as compared to physical adsorption or pH sensitive bonding commonly
used for anthracycline carriers. Confocal microscopy revealed that the bond was stable in the cytoplasm
for 22 h. The ability to slow down the rate of drug release may be crucial for the application in sustained
anticancer drug delivery. Biological analyses performed using MTT assay and confocal microscopy
confirmed that the ultrasmall AuNPs with the lipoic acid derivative of folic acid exhibit relatively low
cytotoxicity, however when loaded with a chemotherapeutic, they cause a significant reduction in the
cell viability.
Received 5th February 2018
Accepted 15th April 2018
DOI: 10.1039/c8ra01107a
rsc.li/rsc-advances
small, solid tumors at an early stage of development and small
metastases. Targeted therapy requires nding an appropriate
Introduction
target on the tumor cells that can be targeted by a biologically
active molecule that has an affinity for this target.
A modern targeted anticancer therapy utilizes antineoplastic
drugs attached to biologically active molecules, which can
selectively access the malformed tissue. A unique advantage of
targeted therapy is the ability to destroy diseased tissues
without damaging the healthy ones. Targeted delivery of cyto-
toxic drugs is particularly important for non-solid tumors or
Gold nanoparticles possess some advantages such as a small
size and ligand versatility due to the ease of surface function-
alization and are therefore promising candidates for medical
use.1 The acceptable toxicity of AuNPs enables their use for drug
delivery to living organisms. The sizes of cells and organelles are
in the micrometer range; therefore, nanoparticles are excellent
candidates for transferring drugs into these biological struc-
tures.2–4 One-pot synthesis of gold nanoparticles gives the
possibility of producing functional nanoparticles with a high
monodispersity index and well characterized core and surface
properties without additional steps of exchanging ligands,5 and
appropriately chosen monolayers protecting the gold nano-
particles can provide water solubility and appropriate stability
under physiological conditions.6,7
aFaculty of Chemistry, University of Warsaw, Pasteura 1, 02-093, Warsaw, Poland.
E-mail: bilewicz@chem.uw.edu.pl; Tel: +48 22 55 26 357
bCzochralski Laboratory of Advanced Crystal Engineering, Biological and Chemical
˙
Research Centre, Faculty of Chemistry, University of Warsaw, Zwirki i Wigury 101,
02-089 Warsaw, Poland
cCenter for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and
Technology, Dorodna 16, 03-195 Warszawa, Poland
dDepartment of Molecular Biology and Translational Research, Institute of Rural
Health, Jaczewskiego 2, 20-090 Lublin, Poland
† Electronic supplementary information (ESI) available: NMR spectra, evaluation
of the AuNPs concentration and the concentration of the ligands on the AuNPs
surfaces that is based on UV-Vis spectroscopy results, thermogravimetric
analysis, cyclic voltamogramms and uorescence microscopy of live cells
treated with AuNPs-LA–Dox for 22 h. See DOI: 10.1039/c8ra01107a
In the present study, folate was used as the AuNPs address-
ing unit. The folate receptors (FR) are overexpressed on the
surface of many types of cancerous cells.8 The folate receptor
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binds folic acid (FA), as well as its derivatives, with a high
affinity (Kd ¼ 10ꢀ10 M)9 to maintain its steady-state level in fast
growing cells. The drug and folic acid attached to the carrier are
delivered directly to the cancerous cells and enter the cell via
endocytosis. Folic acid consists of pteroic acid together with
glutamic acid; however, the amino acid is not involved in the
recognition process by the FR, which makes it suitable for
modication.10
Solubility in aqueous solutions and a small size are crucial
for a promising drug delivery vehicle. Usually, AuNPs that are
modied with citrates (Turkevich-like method11) or covered with
thiol derivatives of poly(ethylene)glycol (PEG) are used.12,13 The
still limited practical usage of these AuNPs is connected with
their relatively large diameters, leading to a very slow diffusion
through the human tissue and thus a low efficiency in reaching
the cancer cell environment. Another difficulty encountered is
their slow removal from the organism. On the other hand,
PEGylated nanocarriers as liposomes, micelles, solid lipid
nanoparticles are found to induce the Accelerated Blood
Clearance (ABC) phenomenon or have impact on the func-
tioning of immune system.14
Among the various approaches to bind folic acid to gold
nanoparticles, the most common is to prepare AuNPs and then
to modify the ligands that are present at their surface using folic
acid.15,16 The difficulty in binding nonmodied folic acid
involves its low solubility in aqueous media. Moreover, the
presence of 2 carboxylic groups in its structure does not facili-
tate conjugation following AuNPs formation, even though the
gamma-COOH group is more active. The AuNPs structure may
also be affected by the harsh changes of the conditions needed
for deblocking, purifying and separation of the product.
Our approach differs from the common approach in that,
rst, the lipoic acid derivative of folic acid is synthesized, and
Experimental section
Materials and methods
Chemical and cell culture reagents. Tetrachloroauric acid
trihydrate (HAuCl4$3H2O), tetraoctylammonium bromide
(TOAB), lipoic acid (LA), folic acid (FA), sodium borohydride
(NaBH4),
N-hydroxysuccinimide
(NHS),
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), ethylenediamine
(NH2)2(CH2)2, t-butyloxycarbonyl anhydride (Boc2O), dime-
thylsulfoxide (DMSO), N,N0-dicyclohexylcarbodiimide (DCC),
triethylamine (Et3N), N-disuccinimydyl carbonate, N,N-dii-
sopropylethylamine (DIPEA), doxorubicin (Dox) were
purchased from Sigma Aldrich and used without further
purication.
Tetrahydrofuran (THF), dimethylformamide (DMF),
dichloromethane (DCM), diethyl ether (Et2O), ethyl acetate
(EtOAc), chloroform (CHCl3), sodium carbonate (Na2CO3),
anhydrous magnesium sulfate (MgSO4), acetone and any
other solvents were obtained from Avantor, Poland. All
glassware was carefully cleaned with an acidic solution of
KMnO4 (overnight), piranha solution (H2SO4 : H2O2 : H2O,
1 : 1 : 3) (2 h), rinsed with large amounts of Milli-Q water and
dried.
EMEM media was a product of the American Type Tissue
Culture Collection (ATCC, Rockville, MD). RPMI-1640
without folic acid was purchased from Thermo Fisher
Scientic (USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) were obtained from Sigma
Aldrich. Fetal calf serum was a product of Biological Indus-
tries (Israel).
Instrumentation
then it is used directly during the synthesis of gold nano- Transmission electron microscopy (TEM) images were carried
particles to form the AuNPs protective layer.16 Using a previously out using a FE-TEM Libra 120 Carl Zeiss microscope at a 120 kV
synthesized folic acid derivative that is conjugated with lipoic acceleration voltage. The samples were placed onto a carbon
acid through the diamine linker removes all of the mentioned coated copper microgrid (400 mesh) that was dried overnight. At
difficulties, allows better control of the whole procedure and least 5 different sample of nanoparticles were prepared, and
increases the population of folic acid molecules present at the from each grid at least 3 pictures were taken. The diameters of
AuNPs surface. A good solubility of the AuNPs is achieved here the cores of the nanoparticles were determined for at least 300
by covering them with lipoic acid (LA) that is naturally present nanoparticles in each image.
in both prokaryotic and eukaryotic cells.
The UV-Vis spectra were recorded using a Perkin Elmer
Doxorubicin (Dox, trade names Adriamycin®, Rubex®, Lambda 25 UV-Vis spectrophotometer.
Scheme S1†) was covalently bound to the modied AuNPs. Dox
Thermogravimetric measurements (TG) were carried out
is oen used for the treatment of various types of cancers, e.g., using a Netzsch STA 449 F1 Jupiter thermobalance. All samples
leukemia, Hodgkin's lymphoma, bladder, breast, stomach, were heated at a rate of 5ꢁC minꢀ1 up to 760 ꢁC in an argon
lung, ovarian, thyroid, so tissue sarcoma, and multiple atmosphere. Argon (99.5%) was obtained from Air Products
myeloma.17–19 Dox is a potent drug; however, it possesses several (Poland).
disadvantages, including rapid excretion and numerous side
The electrochemical experiments were performed using
effects, with the main and the most important being cardiac a CHI 750D electrochemical workstation (CHI Instruments Inc.,
toxicity attributed to highly reactive oxygen species (ROS) Austin, Texas) in the three-electrode arrangement employing
production.19,20 Therefore, we propose a novel way of preparing a platinum foil counter electrode, an Ag/AgCl (KCl sat.) refer-
targeted AuNPs carriers of Dox with a protective layer consisting ence electrode and a glassy carbon disc electrode (BASI) as
of the lipoic acid (LA) and the lipoic acid derivative of the folic a working electrode.
acid (LAFA); using microscopic imaging and cytotoxicity anal-
ysis, we demonstrate the successful uptake of the drug by cancer AVANCE 300 MHz spectrometer and the mass spectra were
All 1H and 13C NMR spectra were performed using a Bruker
cells, its nuclear accumulation, and cellular apoptosis.
recorded with a Quattro LC mass spectrometer.
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Scheme 1 General scheme of lipoic–folic (LAFA) derivative synthesis.
Ligands synthesis – lipoic derivative of folic acid (LAFA)
308 mg (2 eq.) of N-hydroxysuccinimide and 552 mg (2 eq.) of
DCC were successively added. The reaction mixture was stirred
for 16 h at room temperature, aer which the urea precipitate
was ltered through the pad of Celite. To the ltrate, 0.376 mL
(2 eq.) of triethylamine and 429 mg (2 eq.) of N-Boc-
ethylenediamine dissolved in 5 mL of DMSO were added. The
mixture was stirred overnight, and then it was added to the
mixture of 20% acetone in diethyl ether. The thin yellow
precipitate was centrifuged, washed four times with acetone
and four times with diethyl ether and was nally dried under
vacuum (690 mg, 82% yield) (Scheme 3).
Lipoic acid (LA) provides both a thiol function for covalent
attachment to AuNPs and a carboxylic function that assures
water solubility of LA functionalized nanoparticles. Folic acid
(FA), as well as most of its derivatives, is poorly soluble in most
organic solvents, which causes synthetic difficulties related
mostly to product purication. FA contains two carboxylic
groups that can undergo a nucleophilic substitution reaction.
Therefore, the separation and purication of the main g-
substituted product from a-conjugates, bi-conjugates or
unreacted FA are laborious.
The preparation of LAFA (Scheme 1) starts from regiose-
lective coupling of g-carboxylic function of FA with N-Boc-
ethylenediamine in conditions described by Trindade and co-
workers.21 Next, Boc-group deprotection followed by the
coupling of the resulting amine with N-hydroxysuccinimidyl
liponate (LA-NHS) leads to a good yield of LAFA. The LA-NHS
was obtained by the reaction of LA with N,N-disuccinimidyl
carbonate (NHS) and N,N-diisopropylethylamine in deoxygen-
ated acetone.22 The whole procedure of obtaining LAFA is
depicted in Scheme 1, and each step is described in detail
below. The respective NMR spectra are provided in ESI, S2†
(Scheme 2).
1H NMR (300 MHz, DMSO) d: 11.46 (s, 1H), 8.64 (s, 1H), 8.20–
7.86 (m, 2H), 7.68–7.62 (m, 2H), 7.30–6.72 (m, 3H), 6.66–6.63 (d,
2H), 4.48 (s, 2H), 4.27 (m, 1H), 3.09–2.85 (m, 4H), 2.28–1.85 (m,
4H), 1.35 (s, 9H).
13C NMR (300 MHz, DMSO) d: 172.72, 166.68, 161.64, 156.08,
154.18, 151.23, 151.09, 148.57, 129.66, 129.32, 128.31, 121.59,
111.87, 78.27, 52.84, 46.43, 46.02, 40.94, 39.27, 32.88, 31.51,
28.68, 9.86.
MS (Eꢀ): 582.40.
The a-lipoic acid (2.0 g, 9.7 mmole) was dissolved in deoxy-
genated acetone (50 mL). The solution was protected from
direct light by covering the reaction ask with aluminum foil.
N,N-Disuccinimidyl carbonate (3.1 g, 1.25 eq.) and N,N-diiso-
propylethylamine (DIPEA, 2 mL, 1.25 eq.) were sequentially
added and the reaction was stirred overnight at room temper-
ature in an argon atmosphere. Then, solvent was evaporated,
and the solid residue was re-dissolved in a mixture of water and
DCM. The layers were separated, and the organic layer was
washed with water (10 mL). The organic phase was dried over
MgSO4 and was ltered. The solvent was removed in vacuo, and
2.65 g of LA-NHS in the form of a white solid was obtained (90%
yield) (Scheme 4).
In a round-bottomed ask 20 mL (0.3 mole, 10 eq.) of eth-
ylenediamine in 200 mL of dry chloroform was dissolved and
stirred at 0 ꢁC. Then, a solution of Boc2O (6.5 g, 1 eq.) in 150 mL
of chloroform was added dropwise. The reaction mixture was
stirred for 24 hours at room temperature, and then the solvent
was evaporated; the residue was re-dissolved in a solution of 2 M
Na2CO3 (80 mL) and extracted four times with 60 mL of chlo-
roform. The organic phases were combined, dried over MgSO4
and ltered, and the solvent was evaporated. N-Boc-
Ethylenediamine (4.27 g, 90% yield) was isolated as a color-
less oil (Scheme 2).
1H NMR (300 MHz, CDCl3) d: 3.62–3.53 (m, 1H), 3.19–3.09
(m, 2H), 2.83 (s, 4H), 2.66–2.60 (m, 2H), 2.5–2.43 (m, 1H), 1.96–
1.89 (m, 1H), 1.81–1.54 (m, 6H) (Scheme 5).
1H NMR (300 MHz, CDCl3) d: 4.94 (s, 1H), 3.17–3.11 (q, 2H),
2.79–2.72 (t, 2H), 1.42 (s, 9H), 1.16 (s, 2H).
First, 640 mg of folic acid (1.45 mmole, 1 eq.) was dissolved
in 25 mL of DMSO (30 minutes with mild heating). Then,
Diisopropylethylamine (270 mL,
ysuccinimidyl liponate were added to
4
eq.) and N-hydrox-
solution of
a
ethylenediamine-folate (200 mg, 0.41 mmole) in 5 mL of DMSO
and stirred until a clear solution was obtained (ꢂ3 h). The
solution was protected from direct light by covering the reaction
ask with foil. The reaction mixture was poured into a mixture
of 20% acetone in diethyl ether. The thin yellow precipitate was
carefully centrifuged, washed six times with acetone and four
Scheme 2 Protection of ethylenediamine using t-butyloxycarbonyl
anhydride.
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Scheme 3 Modification of folic acid by protected ethylenediamine.
Scheme 4 Activation of lipoic acid by N-disuccinimydylcarbonate.
Scheme 5 Conjugation of lipoic and folic acid derivatives.
times with diethyl ether and nally dried under vacuum dimethylformamide, respectively. During the formation of the
polymeric structures, strict conditions as an argon atmosphere,
(202 mg, 73% yield) (Scheme 5).
1H NMR (300 MHz, DMSO) d: 8.63 (s, 1H), 8.14–7.88 (m, 2H), low temperature and a dark environment were ensured. Aer
7.68–7.62 (m, 2H), 7.17 (bs, 2H), 6,91 (bs, 2H), 6.65–6.63 (m, the reduction of the gold precursor (HAuCl4 in the corre-
2H), 4.48 (bs, 2H), 4.26 (bs, 1H), 3.59–3.49 (m, 1H), 3.41–3.34 sponding solvent) with sodium borohydride, the crude product
(m, 1H) 3.36–2.92 (m, 6H), 2.89–2.82 (m, 2H), 2.26–1.80 (m, 8H), was puried. A detailed description of the synthetic procedure,
1.69–1.39 (m, 6H), 1.36–1.23 (m, 3H).
shown in Scheme 6, is provided below.
13C NMR (300 MHz, DMSO) d: 172.53, 167.10, 161.88, 160.00,
Synthesis of AuNPs-LA. First, 197 mg of HAuCl4$3H2O (0.5
156.89, 154.66, 151.30, 148.96, 129.55, 129.19, 128.42, 121.83, mmole) was dissolved in 20 mL of anhydrous tetrahydrofuran,
111.73, 65.38, 56.57, 46.39, 45.99, 35.71, 34.56, 28.79, 25.42, and then 328 mg of tetraoctylammonium bromide (TOAB, 0.6
19.20, 15.63, 9.97.
MS (ESꢀ): 670.22.
mmole, 1.2 eq.) was added. Then, the reaction mixture was
deaerated with argon and cooled in a water/ice bath for 30 min.
Then, 413 mg of lipoic acid (LA, 2 mmole, 4 eq.) was dissolved in
15 mL of anhydrous THF and dropped into the reaction mixture
and stirred for 12 hours in the dark and an argon atmosphere.
Gold nanoparticles synthesis
Synthesis of ultrasmall gold nanoparticles (AuNPs-LA and Next, 189 mg (5 mmole, 10 eq.) of NaBH4 was dissolved in 10 mL
AuNPs-LA/LAFA) was performed in tetrahydrofuran and
Scheme 6 Synthesis of AuNPs modified with lipoic acid (AuNPs-LA) and lipoic acid/folic acid derivative (AuNPs-LA/LAFA).
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of water and added carefully to the reaction mixture. The color and three times with diethyl ether. Then, the reaction mixture
of the mixture changed to dark brown, and the mixture was was pre-concentrated under vacuum and the pH of the solution
stirred for 3 hours. The dichloromethane/water mixture was was adjusted to 7.0 with 0.1 M HCl. Then, the reaction was
added to facilitate phase separation. The organic phase was processed and concentrated with Vivaspin (MWCO 50 000 and
discarded, and the water phase was washed three times with MWCO 10 000) to remove any excess of the unreacted
dichloromethane, three times with ethyl acetate and two times substrates. The purication process was continued until the
with diethyl ether. The reaction mixture was preconcentrated ltrate showed a clear spectrum (only water) in UV-Vis. Then,
under vacuum and was processed and concentrated using the concentrate was ltered using a syringe lter (0.2 mm) and
ꢁ
Vivaspin (MWCO 50 000 and MWCO 10 000) to remove any stored at 4 C in the dark.
excess unbound lipoic acid, unreacted substrates and water-
Conjugation of doxorubicin to AuNPs-LA/LAFA. Gold nano-
soluble products of NaBH4. The purication process was particles modied with lipoic acid and the folic acid derivative
continued until the ltrate showed clear spectrum (only water) (AuNPs-LA/LAFA) were coupled with the drug doxorubicin.
in UV-Vis. Then, the concentrate was ltered through a syringe Scheme 7 presents the procedure of binding the drug, Dox, to
ꢁ
lter (0.2 mm) and stored at 4 C in the dark.
the free LA-moieties of the AuNPs modied with LAFA.
First, 20 mL of AuNPs-LA/LAFA (ꢂ0.4 mg mLꢀ1) solution was
AuNPs-LA/LAFA. First, 98.5 mg of HAuCl4 (0.25 mmole) was
dissolved in 20 mL of anhydrous dimethylformamide, and then adjusted to a pH of 8.0 with 0.01 M NaOH, and then the solution
164 mg of tetraoctylammonium bromide (TOAB, 0.3 mmole, 1.2 of coupling agents was added to 5 mL of water (15 mmole (1.7
eq.) was added. Then, the reaction mixture was degassed with mg) NHS/15 mmole (2.8 mg) EDC, 1.5 eq. according to Dox) and
argon and cooled in a water/ice bath for 1 hour. Then, 88.4 mg stirred for 2.5 h in the dark (aluminum foil). Then, the solution
of folic derivative (LA-FA, ꢂ0.125 mmole, 0.5 eq.) was dissolved of doxorubicin (10 mmole (5.8 mg) Dox) was added to 5 mL of
in 10 mL anhydrous DMF and was dropped into the reaction water. The mixture was stirred for another 5 hours and then was
mixture and stirred for 18 hours in the dark and in an argon puried and concentrated using Vivaspin (10 000 MWCO).
atmosphere. Then, the reaction mixture was again cooled in AuNPs-LA/LAFA–Dox were washed with water until the ltrate
a water/ice bath, and 180 mg of lipoic acid (LA, 0.875 mmole, 3.5 showed a clear spectrum (only water) in UV-Vis. Finally, the
eq.) was dissolved in 10 mL of DMF and dropped into the nanoparticles were collected and adjusted to a pH of 7.0 with
reaction mixture and stirred for 12 hours in the dark and in an 0.01 M HCl. Then, the product was ltered using a syringe lter
ꢁ
argon atmosphere. Then, 94.5 mg of NaBH4 (2.5 mmole, 10 eq.) (0.2 mm) and stored at 4 C in the dark.
was dissolved in 5 mL of water, added carefully to the reaction
mixture and stirred for
3 additional hours. Then, the
Cell culture
dichloromethane/water mixture was added to facilitate the
phase separation. When the phase was not separate, a few drops
of 0.5 M NaOH was added. Then, the water phase was washed
three times with dichloromethane, four time with ethyl acetate
Human cervical carcinoma KB cells (CCL-17), a derivative of
HeLa cells, were purchased from the American Type Tissue
Culture Collection (ATCC, Rockville, MD) and maintained
Scheme 7 Conjugation of Dox with AuNPs-LA/LAFA.
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according to the ATCC protocol in EMEM medium. When humidied atmosphere. Aer incubation, 20 mL of MTT [3-(4,5-
required, the EMEM medium was exchanged for a RPMI dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide] of
medium without folic acid to unblock the folic acid receptors. 5 mg mLꢀ1 stock solution in PBS (pH 7) was added to each well
All media were supplemented with 10% fetal calf serum (FCS). and incubated for 3 h at 37 ꢁC. Then, the MTT solution was
ꢁ
The cells were incubated in a 5% CO2 atmosphere at 37 C.
removed and remaining insoluble formazan crystals were dis-
solved in 100 mL DMSO. The absorbance was measured in the
plate reader spectrophotometer (Innite M200, Tecan, Austria)
at a wavelength of 570 nm. The cell metabolic activity, which
roughly relates to cell viability, was expressed as ratio of
absorbance of the treated cells to the absorbance of the cells
treated with vehicle, both aer subtraction of the reagent
control and multiplied by 100%. At least three independent
experiments in six replicate wells were conducted.
Confocal microscopy experiments
KB cells were grown on Nunc™ Lab-Tek™ II Chamber Slide™
System (Thermo Scientic) at semi conuent concentration.
One hour before treatment, EMEM culture medium was
changed to RPMI medium without folic acid. Then, the cells
were incubated for 4 h with 5 mM Dox or appropriate concen-
tration of AuNPs to provide an equimolar concentration of Dox.
Aer treatment, the cells were xed in 1.5% formaldehyde and
VectaShield DAPI stain containing anti-fade reagent was
immediately added. Samples were refrigerated overnight and
microscopic images were recorded. Confocal imaging was per-
formed using a NIKON Eclipse Ti-1 microscope equipped in
ꢃ100 oil-immersed Plan Fluor objective (Nikon) and a Nikon A1
confocal system.
Statistical analysis
The differences between samples and the control were evalu-
ated using GraphPad Prism 5.0 soware (GraphPad Soware
Inc., USA). The toxicological data were evaluated by Kruskal–
Wallis one way analysis of variance on ranks (ANOVA) followed
by the post hoc Dunnet's method. The sensitivities of the cell
lines were tested using a two-ways analysis of the variance fol-
lowed by the post hoc Tukey's test. Differences were considered
statistically signicant when the p-value was <0.05.
Cytotoxicity analysis
An MTT test, which measures the activity of mitochondrial
dehydrogenases and roughly corresponds to cellular viability,
was used to evaluate the toxicity of the synthesized compounds.
The assay was performed as described previously.23 In brief, KB
Results and discussion
Characterization of the gold nanoparticles
cells were seeded a day before the experiment in 96-well The modied AuNPs were characterized by transmission elec-
microplates (TPP, Austria) at a density of 1 ꢃ 104 cells per well tron microscopy (TEM), UV-Vis spectroscopy, thermogravi-
in 100 mL of the EMEM culture medium. One hour before the metric analysis (TGA) and voltammetry. The metallic core size of
treatment, the EMEM culture medium was changed to the RPMI gold nanoparticles was evaluated from the transmission elec-
medium without folic acid. Next, the cells were treated with tron microscopy images. The average diameter of the gold cores
AuNPs or Dox in the concentrations of 1 ꢃ 10ꢀ6 M and 5 ꢃ obtained for AuNPs-LA is 2.2 ꢄ 0.4 nm and for AuNPs-LA/LAFA
10ꢀ6 M or vehicle (control) for 24 h or 48 h at 37 ꢁC in a 5% CO2 is 2.5 ꢄ 0.5 nm (Fig. 1).
Fig. 1 TEM images of (A) AuNPs-LA and (B) AuNPs-LA/LAFA. The scale bar is 20 nm. The histograms show the size distributions of the AuNPs
evaluated from the TEM images.
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The evaluation of the AuNPs concentration and the
concentration of the ligands on the AuNPs surfaces that is
based on UV-Vis spectroscopy results (Fig. 3) is described in the
ESI, S3.†
Thermogravimetric analysis conrmed the presence of LA
and LAFA ligands on the nanoparticles surfaces (ESI, S4†).
Additionally, on the basis of the subsequent inection on the
TGA curves, we can estimate the approximate content of ligands
present on the AuNPs.
The presence of Dox on the LA and LA/LAFA modied AuNPs
was conrmed using an electrochemical method since Dox is an
electroactive molecule.24 The electrode processes of Dox are
shown in Scheme S1-B (in ESI).†
The quinone moiety of Dox is reduced at negative potentials
to hydroquinone, and electrochemical studies were then used
for the detection of Dox in the coating of AuNPs, AuNPs-LA–Dox
and AuNPs-LA/LAFA–Dox. Results were compared with the
signals obtained for free Dox in a 0.15 M McIlvaine buffer
solution, pH ¼ 5.5. Gold nanoparticles coupled with Doxoru-
bicin (ca. 1 mM) were added to the electrochemical cell and
preconcentrated at the GC electrode surface by cycling the
potential in the potential window of 0 to ꢀ0.7 V. Cyclic vol-
tammograms (ESI, S5†) show the characteristic Dox reduction/
oxidation peak couple at ca. ꢀ0.5 V vs. Ag/AgCl, which corre-
sponds to the reduction of the quinone moiety to hydroquinone
and its reoxidation in the reverse half-cycle. The Dox reduction
peak in case of AuNPs-LA/LAFA–Dox appears at a slightly less
Fig. 2 UV-Vis spectra of AuNPs-LA (black curve), AuNPs-LA/LAFA
(red), AuNPs-LA/LAFA–Dox (violet), free FA (blue), free Dox (pink). The
spectra are vertically shifted for better a visualization of the charac-
teristic bands and regions of absorption.
The AuNPs-LA and AuNPs-LA/LAFA were characterized using
optical and electrochemical methods. UV-Vis spectra of the
obtained modied gold nanoparticles in solution show the
continuous spectrum of gold nanoparticles in the visible region
and signals corresponding to the free folic acid derivative and to
the doxorubicin derivative (Fig. 2).
Fig. 3 (A) UV-Vis spectra of AuNPs-LA with increasing concentrations and (B) calibration curves for various wavelengths. (C) Spectra for
increasing concentrations of AuNPs-LA–Dox.
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negative potential than free Dox and AuNPs-LA–Dox due to the treated cells was observed 4–5 h aer treatment (data not
p–p interactions between the drug and FA groups. The revers- shown). Therefore, we decided to use a 4 h treatment time to
ible and stable pattern in the time signals of the Dox moiety perform high quality confocal images of xed cells with stained
attached to gold nanoparticles indicates an efficient coupling of nuclei. The results revealed large difference in the accumulation
Dox to the nanoparticles by the covalent bond. A non- of Dox in the cytoplasm and nuclei (Fig. 4). Dox uorescence
decreasing doxorubicin activity was observed even aer one (red) was the brightest in cells treated with Dox alone; then in
month.
AuNPs-LA/LAFA–Dox treated cells. Whereas, in AuNPs-LA–Dox
treated cells it was only just visible. However, a duly analysis of
red uorescence on the AuNP-LA–Dox image indicated the
presence of weak Dox signal in the cytoplasm, but not in the cell
nucleus (Fig. 4, row B, 3rd column), that was in line with our
observation using 22 h life cells imaging. No Dox uorescence
was seen in the control cells (not shown).
Confocal imaging revealed that folic acid enriched AuNPs
were able to deliver Dox to the nucleus of the cell. Since we did
not nd Dox accumulation in cells treated with similar particles
containing LA–Dox conjugate without folic acid addressing
molecule, we suggest that the observed difference results from
AuNPs-LA/LAFA–Dox binding to the folic acid receptor that
enhances the transfer of those particles through the outer cell
membrane and facilitates their incorporation into the nucleus.
Boshnjaku et al. found that the folic acid receptor alpha (FRa)
may be transferred through the nuclear envelope to reach DNA
and act as a transcription factor. Additionally, an unbound free
Cell studies of the AuNPs based carriers
Fluorescence and confocal microscopy imaging of AuNPs
treated KB (CCL-17) cells. A continuous observation by uo-
rescence microscopy of live cells treated with AuNPs-LA–Dox for
22 h revealed that AuNPs-LA–Dox particles were hardly uptaken
and accumulated in the cytoplasm (ESI, S6†). However, this
results proved also the stability of amide bonding used to attach
Dox to AuNPs-LA. Whereas, Dox alone freely migrated through
the cytoplasm to the cell nucleus (Fig. 4, row A, 3rd column), the
Dox uorescence from AuNPs-LA–Dox was present predomi-
nantly in the cytoplasm (Fig. 4, row B, 3rd column) conrming
that Dox was still attached to the AuNPs even 22 h aer entering
the cytoplasm (ESI, S6†).
This preliminary experiment showed that the most
pronounced difference in intracellular Dox uorescence of
Fig. 4 Confocal images showing how the presence of folic acid on an NP surface affects the internalization and nuclear accumulation of Dox. KB
cells were fixed after a 4 h incubation with 5 mM Dox (1st row, A), AuNPs-LA–Dox (2nd row, B) and AuNPs-LA/LAFA–Dox (3rd row, C). The
calculated Dox concentration in the AuNPs preparation equaled 5 mM. Cells nuclei of fixed cells were stained with DAPI dye (blue, 2nd column).
Dox fluorescence (red, 3rd column). Cell bodies were imaged using transmitted light and Nomarski DIC contrast (gray, 4th column).
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Fig. 5 Toxicity of the AuNPs conjugates and free doxorubicin to KB (CCL-17) cells treated for 24 h (A) and 48 h (B). Data are expressed as
a percent of the control and the mean ꢄ SD from three independent experiments. P < 0.05 (‘a’ denotes a statistically significant difference
between the 1 ꢃ 10ꢀ6 M and 5 ꢃ 10ꢀ6 M concentrations; ‘b’ denotes a statistically significant difference between the 24 h and 48 h treatment, ‘c’
denotes a statistically significant difference versus the control group; and ‘d’ denotes a statistically significant difference between the AuNPs–Dox
complexes and Dox alone).
FRa was found in the nuclear envelope.25 Recently, Chaumet Although Chaumet et al. did not study the FRa receptor, the
et al. showed that a substantial fraction of early endosomes may proposed mechanism seems more universal and the direct
deliver particular plasma membrane receptors to the nucleus.26 delivery of FRa to the nucleus through the endosome-mediated
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transfer seems highly probable. Thus, it is plausible that the (FA) did not show any signicant internalization or cytotoxicity.
whole complex of AuNPs-LA/LAFA–Dox, once bound to the All together it indicates that folic acid derivative was necessary
plasma membrane FRa receptor, is trapped inside the endo- for the proper up-take of the synthesized nanoparticles by the
some, which is in turn transported to nucleus. This would cells while the amide bonding provided sustained release of the
explain why we have not seen the nuclear accumulation of drug limiting it mainly to the nucleus. Results showed also that
AuNPs-LA–Dox even aer 22 h of incubation. The AuNPs-LA– AuNPs with a protective layer made of lipoic acid and/or lipoic
Dox nanoparticles were possibly uptaken by a non-specic acid derivative of an “addressing molecule” are transferred
mechanism that resulted in a much slower AuNPs accumula- through the nuclear envelope to reach DNA, likely via receptor
tion in the cytoplasm and apparent lack of cytotoxicity. The mediated mechanism.
proposed above folate receptor dependent mechanism of
The application of these targeted ultrasmall nanoparticles
nucleus transport prevails in case of AuNPs-LA/LAFA–Dox bio- opens a new route for the delivery of toxic drugs. A tight covalent
conjugates and results in better nucleus penetration and amide bond used to attach Dox to LA moiety resulted in a stable
enhanced toxicity (see below).
bioconjugate that persist at least 22 h in the cytoplasm. Thus, it
is anticipated that once cell dies, the drug is released in
unchanged form, ready to interact with another cell. This
reciprocal mechanism of action should increase the drug effi-
cacy and limit its cytotoxicity to target receptor overexpressing
cells rather than all cells present in vicinity, as in case of free
Dox released from non-covalent bound bioconjugates. In addi-
tion, Dox tightly attached to AuNPs is expected to has less
systemic toxicity than its free form, readily penetrating all
organs of the body. Finally AuNPs with attached Dox are ex-
pected to have slower body clearance than Dox alone, thus
should be more efficient in the cancer treatment. In vitro studies
on cancer cells culture proved that such nanoparticles are
promising drug carriers and they are worth to be further
investigated in in vivo experiments.
Cytotoxicity
The cytotoxic activity of tested compounds was investigated on
KB cells using the MTT assay (Fig. 5). AuNPs-LA and AuNPs-LA/
LAFA were not cytotoxic in the concentrations used. AuNPs-LA–
Dox complex expressed small, but statistically signicant
toxicity in concentration 5 ꢃ 10ꢀ6 M aer 48 h incubation. This
concentration reduced metabolic activity of KB cells to 76% of
control value. On the contrary, treatment of the cells with
AuNPs-LA/LAFA–Dox and Dox alone resulted in a marked
reduction of metabolic activity in a dose- and time-dependent
manner. The highest used concentration aer 48 h incubation
reduced the metabolic activity of KB cells to 25 and 13% for
AuNPs-LA/LAFA–Dox and Dox, respectively. However, the
further studies of toxicity are needed to obtain additional
information on mutagenicity or genotoxicity of the developed
drug carriers in view of several literature reports.27–29
Conflicts of interest
There are no conicts to declare.
Conclusions
Acknowledgements
We described a successful procedure for the preparation of This project was nanced by the National Science Centre,
bifunctional gold nanoparticles for drug delivery. The prepara- conferred on the basis of decision number DEC-2014/13/B/ST5/
tion of a lipoic acid derivative of folic acid soluble in a synthetic 04117. AW thanks the National Science Centre No. 2016/22/E/
medium allowed the attachment of the target molecule to the ST4/00572 for support.
AuNPs and increased the carrier solubility in aqueous solutions.
The carrier was made more hydrophilic by the subsequent step
of self-assembly of free lipoic acids in the voids not covered by
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