Biodegradable and pH-Responsive Acetalated Dextran (Ac-Dex) Nanoparticles for NIR Imaging and Controlled Delivery of a Platinum-Based Prodrug into Cancer Cells

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00058

Nanoparticles (NPs) based on the biodegradable acetalated dextran polymer (Ac-Dex) were used for near-infrared (NIR) imaging and controlled delivery of a Pt^IV prodrug into cancer cells. The Ac-Dex NPs loaded with the hydrophobic Pt^IV prodrug 3 (Pt^IV/Ac-Dex NPs) and with the novel hydrophobic NIR-fluorescent dye 9 (NIR-dye 9/Ac-Dex NPs), as well as Ac-Dex NPs coloaded with both compounds (coloaded Ac-Dex NPs), were assembled using a single oil-in-water nanoemulsion method. Dynamic light scattering measurements and scanning electron microscopy images showed that the resulting Ac-Dex NPs are spherical with an average diameter of 100 nm, which is suitable for accumulation in tumors via the enhanced permeation and retention effect. The new nanosystems exhibited high drug-loading capability, high encapsulation efficiency, high stability in physiological conditions, and pH responsiveness. Drug-release studies clearly showed that the Pt^IV prodrug 3 release from Ac-Dex NPs was negligible at pH 7.4, whereas at pH 5.5, this compound was completely released with a controlled rate. Confocal laser scanning microscopy unambiguously showed that the NIR-dye 9/Ac-Dex NPs were efficiently taken up by MCF-7 cells, and cytotoxicity assays against several cell lines showed no significant toxicity of blank Ac-Dex NPs up to 1 mg mL^-1. The IC₅₀ values obtained for the Pt^IV prodrug encapsulated in Ac-Dex NPs were much lower when compared with the IC₅₀ values obtained for the free Pt^IV complex and cisplatin in all cell lines tested. Overall, our results demonstrate, for the first time, that Ac-Dex NPs constitute a promising drug delivery platform for cancer therapy.

Full text of DOI:10.1021/acs.molpharmaceut.9b00058

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Article
Biodegradable and pH-Responsive Acetalated Dextran
(Ac-Dex) Nanoparticles for NIR-Imaging and Controlled
Delivery of a Platinum-based Prodrug into Cancer Cells
Carolyne B. Braga, Gabriel Perli, Tiago B. Becher, and Catia Ornelas
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00058 • Publication Date (Web): 22 Mar 2019
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Biodegradable and pH-Responsive Acetalated Dextran (Ac-Dex)
Nanoparticles for NIR-Imaging and Controlled Delivery of a
Platinum-based Prodrug into Cancer Cells
Carolyne B. Braga, Gabriel Perli, Tiago B. Becher, Catia Ornelas*
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Institute of Chemistry, University of Campinas - UNICAMP, 13083-970, Campinas, SP, Brazil
ABSTRACT: Nanoparticles based on the
biodegradable acetalated dextran polymer (Ac-
Dex) were used for NIR-imaging and controlled
delivery of a PtIV prodrug into cancer cells. The
Ac-Dex NPs loaded with the hydrophobic PtIV
prodrug 3 (PtIV/Ac‒Dex NPs) and with the novel
hydrophobic NIR-fluorescent dye
9
(NIR-
dye9/Ac-Dex NPs), as well as Ac-Dex NPs co-
loaded with both compounds (co-loaded Ac-Dex
NPs), were assembled using a single oil-in-water
nanoemulsion method. Dynamic light scattering
(DLS) measurements and scanning electron
microscopy (SEM) images showed that the
resulting Ac-Dex NPs are spherical with an average diameter of 100 nm, which is suitable for accumulation in tumors via the
EPR effect. The new nanosystems exhibited high drug loading capability, high encapsulation efficiency, high stability in
physiological conditions, and pH-responsiveness. Drug release studies clearly showed that the PtIV prodrug 3 release from
Ac-Dex NPs was negligible at pH 7.4, whereas at pH 5.5 this compound was completely released with a controlled rate.
Confocal laser scanning microscopy (CLSM) unambiguously showed that the NIR-dye 9/Ac-Dex NPs were efficiently
uptaken by MCF-7 cells, and cytotoxicity assays against several cell lines showed no significant toxicity of blank Ac-Dex
NPs up to 1 mg mL-1. The IC50 values obtained for the PtIV prodrug encapsulated in Ac-Dex NPs was much lower when
comparing with the IC50 values obtained for the free PtIV complex and cisplatin, in all cell lines tested. Overall, our results
demonstrate, for the first time, that Ac-Dex NPs are a promising drug delivery platform for cancer therapy.
KEYWORDS: acetalated dextran, polymeric nanoparticles, drug delivery, NIR-imaging, prodrug, nanomedicine
1. INTRODUCTION
increase the selective drug accumulation in tumor tissues
via the enhanced permeation and retention effect (EPR
effect) or active targeting, thereby reducing the adverse
effects while increasing the therapeutic efficacy. Other
significant advantages of nanosystems over their free drug
counterparts include their ability to increase the drug
bioavailability, stability, biodistribution and circulating
half-life, resulting in improvements on the drug therapeutic
efficacy.5, 6
In the last decades, there have been remarkable
advances in modern medical sciences, in particular with the
development of nanomedicine.1-3 Despite of the significant
improvements in cancer treatments, the number of cancer-
related deaths has continued to rise.4 Current chemotherapy
uses nonselective anticancer agents that are not able to
differentiate between healthy and cancer cells, causing
harmful side effects in patients. These undesirable side
effects often limit the doses of chemotherapeutic agents
administered to patients, compromising the treatment
effectiveness, and contributing to the development of drug
resistance, which is also responsible for failure in cancer
therapy. Recent research has focused on the development of
innovative nanomaterials that aim at selective delivery of
chemotherapeutic agents to tumor cells to avoid or decrease
the harmful side effects of conventional drugs. The driving
force behind this paradigm shift in cancer research is that
nanomaterial-based drug delivery systems can dramatically
A variety of nanomaterials with potential applications in
nanomedicine can be found in the literature, including
8
16
liposomes,7,
dendrimers,9-14 hydrogels,15,
polymeric
quantum
nanoparticles,17,
metallic nanoparticles,19,
18
20
dots,21 mechanized nanoparticles,22, 23 and polyrotaxanes.24
Despite the significant progress in the application of
nanomaterials in drug delivery applications, numerous
opportunities remain for the development of innovative
nanotherapeutic systems.
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Polymeric nanoparticles are able to carry drugs either by
longer reaction times produce the thermodynamically more
stimuli-responsive covalent attachment or encapsulation
within its hydrophobic nanoenvironments or cavity.17, 25-28
Nanoparticles (NPs) based on biodegradable polymers,
such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA),
poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone)
(PCL), chitosan or hyaluronic acid have been widely
explored for the targeted delivery of chemotherapeutics in
cancer research. These materials are very attractive
especially due to their biodegradability, versatility,
biocompatibility, and capability of sustained release, which
are crucial properties of drug delivery systems to improve
the pharmokinetics of anticancer drugs and to minimize
their negative side effects.
stable cyclic acetals.31 Thus, Ac-Dex particles are very
attractive for drug delivery applications because the release
rate of the encapsulated therapeutic agent from Ac-Dex
particles can be tailored according to the desired
application.
Several studies have explored the pH-responsive Ac-
Dex micro- or nanoparticles as a delivery vehicle for
therapeutic agents including proteins,29, 31, 33 peptides,34, 35
small molecules,36, 37 antibiotics,38 and nucleotides.39, 40 For
example, Ac-Dex NPs were able to encapsulate
hydrophobic silver carbenes in relatively high loadings, and
showed significant antimicrobial activity against both gram-
positive and gram-negative bacteria.38 More recently,
Bachelder et al. have shown that Ac-Dex microparticles
facilitated delivery of the host-directed therapeutic AR-12
to clear intracellular infections within phagocytes.41
However, there are no studies on the use of Ac-Dex
nanoparticles as drug delivery systems for anticancer drugs.
The lability of the acetal groups on Ac-Dex at pH 5.5 make
it an attractive candidate for the selective delivery of
anticancer agents to tumor cells.
Here we describe, for the first time, the use of Ac-Dex
NPs for both imaging and delivery of an anticancer prodrug
to cancer cells. The Ac-Dex NPs were tailored with an
average size of 100 nm in order to enhance the drug
pharmokinetics and to enable accumulation in tumor cells
via the EPR effect. The hydrophobic PtIV prodrug
cis,cis,trans-[Pt(NH3)2Cl2(CO2(CH2)4CH3)2] was chosen as
the anticancer agent due to its low cytotoxicity and known
ability to be reduced intracellularly into highly cytotoxic
PtII species (cisplatin).42-44 This strategy of using an inert
cisplatin prodrug improves cisplatin tolerability and
efficacy in vivo. The single nanoemulsion method used to
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Acetalated dextran (Ac-Dex) is
a biodegradable
polymer that has emerged as a potential candidate for drug
delivery applications.29 In addition to biodegradability, this
polymer presents a variety of interesting features, including
facile synthesis, pH-responsiveness, controlled release, high
loadings, high biocompatibility and versatility, and
functionalization capability.30 Ac-Dex is prepared through a
one-step synthesis from the clinically approved biopolymer
dextran, a homopolysaccharide of glucose. Protection of the
pendant hydroxyl groups of water-soluble dextran with
acetal groups affords the hydrophobic Ac-Dex polymer
containing both cyclic and acyclic acetal groups (Scheme
1).29, 31 This acetalated polymer exhibits a pH-responsive
degradation, since acetal groups undergo hydrolysis under
mildly acidic conditions (pH 5.5), converting back to its
hydrophilic and biocompatible precursor dextran.29, 32
encapsulate
hydrophobic
molecules
in
Ac-Dex
nanoparticles was further explored to encapsulate the new
hydrophobic NIR-dye to provide nanoparticles suitable for
NIR-imaging, and to co-encapsulate the PtIV prodrug and
the NIR-dye to provide nanoparticles with the dual role of
imaging and treating, providing a system suitable for
theranostic applications.45, 46
Scheme 1. Synthesis of hydrophobic acetalated dextran (Ac-Dex)
polymer from natural polyssacharide dextran: (a) 2-
methoxypropene, pyridinium p-toluenesulfonate, DMSO.29
2. EXPERIMENTAL SECTION
2.1. General Considerations. Cell counting kit 8 (CCK-
8), dextran (from Leuconostoc mesenteroides; average MW
9000-1100) and all chemical reagents were purchased from
Sigma-Aldrich and used without further purification unless
otherwise noted. DMSO was purchased anhydrous (>
99.9%) and used under nitrogen atmosphere; all other
solvents were analytical grade and were used as supplied.
DMEM, RPMI-1640 and 0.25% trypsin-EDTA solution
were purchased from Gibco®. Fetal bovine serum (FBS)
and Dulbecco modified phosphate buffered saline
(DMPBS, pH 7.4) were purchased from Nutricell
Nutrientes Celulares (Campinas, SP, Brazil). Hoechst
33342 and Alexa Fluor® 555 Phalloidin were obtained
from Life Technologies. dd-H2O (pH 8.0) was prepared
Ac-Dex polymer has been tailored into micro- and
nanoparticles by using standard emulsion methods.
Depending on the emulsion approach employed, oil-in-
water (o/w) or water-in-oil-in-water (w/o/w), the resulting
particles have been used to encapsulate hydrophobic or
hydrophilic compounds, respectively.
The uniqueness of this modified-dextran polymer over
other pH-sensitive polymers comes from its highly tunable
degradation rate, which is dependent on the ratio between
the two different types of acid-labile acetal groups (cyclic
and acyclic) on its structure. The ratio of cyclic and acyclic
acetals is tailored by the acetalation reaction kinetics, since
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using NaOH and/or HCl to adjust pH, and pH was always
checked prior to each use.
196.9 (+NCCH3), 142.3 (arom. +NCq), 141.5 (arom.
+NCqCq), 129.9 (arom. CH), 129.5 (arom. CH), 124.0
(arom. CH), 116.0 (arom. CH), 54.7 (+NCH2(CH2)4CH3),
48.2 (+NCC(CH3)2), 31.2 (+NCH2CH2(CH2)3CH3), 27.7
(+N(CH2)2CH2(CH2)2CH3), 26.0 (+N(CH2)3CH2CH2CH3),
22.5 (+NCC(CH3)2), 22.3 (+N(CH2)4CH2CH3), 14.7
(+N(CH2)5CH3), 14.3 (+NCCH3). HRMS (ESI+) m/z [M+]
calcd for C17H26N+: 244.2060, found: 244.1987.
2.2. Synthesis of Acetalated Dextran Polymer (Ac-
Dex). The Ac-Dex polymer was synthesized in one step
from dextran following a previously published procedure.29
The detailed procedure is described in the Supporting
Information.31
2.3. Synthesis of PtIV complex and its percursors.
Cisplatin was purchased from Sigma-Aldrich, and was
oxidized using hydrogen peroxide to afford the compound
2.47, 48 The resulting PtIV complex 2 reacted with hexanoic
anhydride to yield the hydrophobic PtIV prodrug 3.49 The
detailed synthetic procedures are described in the
Supporting Information.
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2.4.3. Synthesis of chlorocyanine dye 8. To a solution of
iminium salt 5 (0.6 g, 1.71 mmol) and indolium derivative 7
(1.3 g, 3.40 mmol) in absolute ethanol (50 mL), anhydrous
sodium acetate (320 mg, 3.97 mmol) was added. The
resulting mixture was refluxed for 12 h under nitrogen
atmosphere. The ethanol was removed under reduced
pressure after completion of reaction. The crude product
was purified by column chromatography over silica gel
with acetone/methanol (95:5) as eluent to afford 1.1 g of 8
2.4. Synthesis of NIR-dye 9 and its precursors.
2.4.1. Synthesis of N-[5-anilino-3-chloro-2,4-(propane-
1,3-diyil)-2,4-pentadiene-1-ylidene]anilinium chloride (5).
Phosphorus oxychloride (POCl3, 1.1 mL, 11.80 mmol) was
added dropwise to anhydrous DMF (1.3 mL, 16.79 mmol)
in an ice bath at 0 ºC. After 30 min, cyclohexanone (4) (0.6
mL, 5.79 mmol) was added dropwise, and the reaction
mixture was refluxed for 1 h under nitrogen atmosphere.
The reaction mixture was cooled to room temperature. A
solution of aniline (0.9 mL) in ethanol (100 mL) was added
dropwise, and the reaction mixture was stirred for further 1
h. The resulting deep purple mixture was poured into 1 L of
ice cold HCl aqueous solution (10%), and the product was
allowed to crystallize overnight at 4 ºC. The crystals were
filtered off, washed twice with ice-cold water, and once
with diethyl ether, and then dried under vacuum.
Compound 5 was obtained as purple crystals in 63% yield
1
(1.67 mmol, 98%) as a deep green solid. H NMR (400
MHz, DMSO-d6), δ (ppm): 8.26 (d, J = 14.0 Hz, 2H,
+NCCH), 7.64 (d, 2H, arom. H), 7.45 (m, 4H, arom. H),
7.29 (m, 2H, arom. H), 6.34 (d, J = 14.2 Hz, 2H,
+NCCHCH), 4.23 (m, 4H, +NCH2(CH2)4CH3), 2.72 (m, 4H,
ClCCCH2), 1.88 (m, 2H, ClCCCH2CH2), 1.75 (m, 4H,
+
+NCH2CH2(CH2)3CH3), 1.68 (s, 12 H, NCC(CH3)2), 1.41-
1.27 (m, 12H, +N(CH2)2CH2CH2CH2CH3), 0.86 (t, 6H,
+N(CH2)5CH3). 13C NMR (100 MHz, DMSO-d6), δ (ppm):
172.7 (+NCC(CH3)2), 143.4 (arom. +NCq), 142.5 (arom.
+NCqCq),
141.5
(+NCCHCHCCCl),
132.0
(+NCCHCHCCCl), 129.1 (arom. CH), 126.6 (ClC), 125.7
(arom. CH), 123.0 (arom. CH), 112.0 (arom. CH), 102.1
(+NCCHCHCCCl),
49.5
(+NCC(CH3)2),
44.3
(+NCH2(CH2)4CH3), 31.3 (+NCH2CH2(CH2)3CH3), 27.9
1
(1.3 g, 3.62 mmol). H NMR (400 MHz, DMSO‒d6), δ
(ClCCCH2CH2),
27.7
(ClCCCH2CH2),
27.4
(ppm): 8.54 (s, 2H, +NCH), 7.56 (m, 4H, arom. H), 7.47 (m,
4H, arom. H), 7.28 (m, 2H, arom. H), 2.73 (t, J = 6.0 Hz,
(+N(CH2)2CH2(CH2)2CH3), 26.2 (+N(CH2)3CH2CH2CH3),
22.4 (+NCC(CH3)2), 19.3 (+N(CH2)4CH2CH3), 14.3
(+N(CH2)5CH3). HRMS (ESI+) m/z [M]+ calcd for
+
+
4H, NCCCH2), 1.87 (quint, J = 6.0 Hz, 2H, NCCCCH2).
HRMS (ESI+) m/z [M]+ calcd for C20H20ClN2 : 323.1309,
C42H56ClN2 : 623.4127, found: 623.4069.
+
+
found: 323.1252.
2.4.4. Synthesis of aminocyanine dye 9. Dye 8 (1.5 g, 2.27
mmol) and hexylamine (394.6 mg, 3.90 mmol) were
dissolved in anhydrous DMF (25 mL), and the solution was
heated at 80 ºC for, 16 h. The solution color gradually
changed from green to deep blue. The solvent was removed
under vacuum and the product was purified by column
chromatography using silica gel and acetone/methanol
(99:1) as eluent. Dye 9 was obtained as a deep blue solid in
67% yield (1.1 g, 1.52 mmol). 1H NMR (400 MHz,
2.4.2. Synthesis of 1-hexyl-2,3,3-trimethyl-3H-indol-1-
ium iodide (7).50 A mixture of 1-iodohexane (3.2 g, 15.09
mmol) and 2,3,3-trimethylindolinine (2.0 g, 12.56 mmol) in
acetonitrile was refluxed with continuous stirring for 16 h
to produce a pink solution. The reaction mixture was cooled
to room temperature and the solvent was evaporated. The
crude product was dissolved in a minimum amount of
dichloromethane (1.5 mL) and poured over 400 mL of
diethyl ether under vigorous stirring. The precipitate
obtained was washed with diethyl ether (20 mL x 3) and
dried under vacuum to give 4.1 g of pure compound 7
+
DMSO‒d6), δ (ppm): 7.72 (d, J = 12.7 Hz, 2H, NCCH),
7.27 (m, 4H, arom. H), 7.05 (m, 2H, arom. H), 6.83 (m, 2H,
+
arom. H), 5.59 (d, J = 12.7 Hz, 2H, NCCHCH), 3.88 (m,
1
+
(11.04 mmol, 88%) as a purple solid. H NMR (400 MHz,
2H, NHCH2(CH2)4CH3), 3.78 (m, 4H, NCH2(CH2)4CH3),
DMSO‒d6), δ (ppm): 7.98 (m, 1H, arom. H), 7.84 (m, 1H,
arom. H), 7.62 (m, 2H, arom. H), 4.46 (t, J = 7.8 Hz, 2H,
2.47
(t,
4H,
NHCCCH2),
1.97
(quint,
2H,
NHCH2CH2(CH2)3CH3), 1.82 (m, 2H, NHCCCH2CH2),
+
+NCH2(CH2)4CH3), 2.86 (s, 3H, NCCH3), 1.83 (quint, J =
1.75 (m, 4H, +NCH2CH2(CH2)3CH3), 1.70 (s, 12 H,
7.8 Hz, 2H, +NCH2CH2(CH2)3CH3), 1.54 (s, 6H,
+NCC(CH3)2), 1.47-1.26 (m, 18H, N(CH2)2(CH2)3CH3 and
+
+
+
+NCC(CH3)2), 1.41 (m, 2H, N(CH2)2CH2(CH2)2CH3), 1.28
NH(CH2)2(CH2)3CH3), 0.91 (t, 6H, N(CH2)5CH3), 0.87 (t,
+
(m, 4H, N(CH2)3(CH2)2CH3), 0.85 (t, J = 7.0 Hz, 3H,
3H, NH(CH2)5CH3). 13C NMR (100 MHz, DMSO‒d6), δ
(ppm): 169.2 (+NCC(CH3)2), 166.9 (+NCCHCHCCNH),
+N(CH2)5CH3). 13C NMR (100 MHz, DMSO‒d6), δ (ppm):
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143.2 (arom. +NCq), 140.1 (arom. +NCqCq), 138.0
determined by dynamic light scattering (DLS).
(+NCCHCHCCNH), 128.0 (+NCCHCHCCNH), 122.5
(arom. CH), 122.0 (arom. CH), 120.1 (arom. CH), 108.22
Measurements were carried out at 25 ºC on a Zetasizer
Nano-ZS ZEN3600 instrument (Malvern Instruments)
equipped with a 4 mW He‒Ne laser with light wavelength
of 632.8 nm, and backscattering angle of 173°, using a
disposable DTS 1070 cell. Nanoparticle samples were
dispersed in dd-H2O (pH 8.0) at a concentration of 1
mg.mL‒1 and each sample was analyzed in triplicate. Data
are presented as % by number.
(arom.
CH),
94.0
(+NCCHCHCCNH),
(+NCC(CH3)2),
50.1
43.2
(+NCH2(CH2)4CH3),
47.7
(NHCH2(CH2)4CH3), 31.5 and 31.4 (+NCH2CH2(CH2)3CH3
and NHCH2CH2(CH2)3CH3), 29.1 (+NCC(CH3)2 and
NHCCCH2CH2), 26.8, 26.5 and 26.4 (NHCCCH2CH2,
+N(CH2)2CH2(CH2)2CH3 and NH(CH2)2CH2(CH2)2CH3),
25.2 (+N(CH2)3CH2CH2CH3), 22.6, 22.5 and 21.6
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2.10. Surface Morphology. The surface morphology of
Ac-Dex NPs was examined using a Quanta 250 field
emission scanning electron microscope (FESEM) (FEI
Ltd.) operating at an accelerating voltage of 5 kV. Particle
samples were prepared by dropping 10 μL of a nanoparticle
suspension (1 mg.mL‒1 in dd-H2O, pH 8.0) onto a polished
silicon wafer. Samples were allowed to dry overnight in the
air, and sputter coated with a layer of iridium in a Bal-Tec
MD 020 instrument (Balzers).
(+N(CH2)4CH2CH3,
NH(CH2)3CH2CH2CH3
and
NH(CH2)4CH2CH3), 14.1 and 14.0 (+N(CH2)5CH3 and
NH(CH2)5CH3). HRMS (ESI+) m/z [M]+ calcd for
+
C48H70N3 : 688.5564, found: 688.5483.
2.5. Preparation of empty Ac-Dex NPs (Blank Ac-Dex
NPs). The preparation of blank Ac-Dex NPs was performed
by adapting the oil-in-water (o/w) nanoemulsion solvent
evaporation procedure described by Ornelas et al.38 Ac-Dex
(25 mg) dissolved in 1 mL of ethyl acetate was added to 5
mL of polyvinyl alcohol (PVA; MW 13,000-23,000 g/mol,
87‒89% hydrolyzed) solution (3% wt/wt in PBS). The
mixture was emulsified through ultrasonication for 30 s in
an ice bath by using a probe sonicator (Branson Digital
Sonifier 250) with 0.5 s pulses intercalated with 0.1 s
intervals, and amplitude of 50%. A white milky solution
was obtained, and the organic solvent was evaporated under
a gentle flow of dry nitrogen for 2 h. Nanoparticles were
isolated by centrifugation (6,500 × g, 30 min), and washed
with dd-H2O (pH 8.0). Three cycles of redispersion in
dd‒H2O (5 mL, pH 8.0), vortexing, bath sonication (15-20
min), centrifugation (6,500 × g, 20 min) and removal of
supernatant were performed. Finally, residual water was
removed under vacuum to yield a white fluffy powder (13
mg).
2.11. Quantification of PtIV complex 3 encapsulated in
Ac-Dex NPs. The concentration of compound
3
encapsulated in the Ac-Dex NPs (PtIV/Ac-Dex NPs and co-
loaded/Ac-Dex NPs) was determined by inductively
coupled plasma-optical emission spectroscopy (ICP-OES)
measurements of free platinum using a Perkin Elmer
Optima 8300 optical emission spectrometer. Samples were
prepared through digesting the nanoparticle suspensions in
HNO3 and diluting in Milli-Q water to a final acid content
of 2%. Intensity of spectral line at 265.945 nm was
measured for all samples and standards. The platinum
concentration in the samples was determined by comparing
the measured intensity with a calibration curve of platinum.
All ICP-OES measurements were carried out in triplicate.
2.12. Quantification of dye 9 encapsulated in Ac-Dex
NPs. The amount of dye 9 encapsulated in the NIR-dye
9/Ac-Dex NPs and co-loaded/Ac-Dex NPs was determined
using an UV-vis spectrometer (Agilent HP 8453). The
particles were weighted out in triplicate and dissolved in
THF to release the cargo. Then, the absorbance of dye 9 at
633 nm was measured, and its concentration in solution was
calculated from a pre-established calibration curve. All UV-
vis measurements were carried out in triplicate.
2.6. Preparation of Ac-Dex NPs encapsulating the PtIV
Prodrug 3 (PtIV/Ac-Dex NPs). PtIV-loaded Ac-Dex NPs
were prepared following a similar procedure to that
described for blank NPs. In this case, 7.5 mg of the PtIV
compound 3 (corresponding to an initial feed of 30% (w/w),
defined as (mg of PtIV complex/mg of polymer) × 100) was
dissolved in the organic phase together with Ac-Dex prior
to sonication.
2.7. Preparation of Ac-Dex NPs encapsulating the
NIR dye 9 (NIR dye 9/Ac-Dex NPs). Dye 9-loaded Ac-
Dex NPs were prepared in the same manner as PtIV/Ac-Dex
NPs, but in this case dichloromethane was used as the
organic solvent instead of ethyl acetate.
2.8. Preparation of Ac-Dex NPs co-encapsulating the
PtIV Prodrug 3 and the NIR-dye 9 (co-loaded Ac-Dex
NPs). Co-loaded Ac-Dex NPs were prepared in a similar
manner as the single loaded Ac-Dex NPs, but in this case
both 7.5 mg of PtIV compound 3 and 0.5 mg of dye 9 were
dissolved in ethyl acetate together Ac-Dex prior to
sonication.
2.13. Loading Content and Encapsulation Efficiency.
The loading content (LC) and encapsulation efficiency (EE)
of the nanoparticles were calculated as follows:
LC % =
푤푒푖푔ℎ푡 표푓 푐표푚푝표푢푛푑 푒푛푐푎푝푠푢푙푎푡푒푑 푖푛 푛푎푛표푝푎푟푡푖푐푙푒푠
× 100
푡표푡푎푙 푤푒푖푔ℎ푡 표푓 푛푎푛표푝푎푟푡푖푐푙푒푠
EE % =
푤푒푖푔ℎ푡 표푓 푐표푚푝표푢푛푑 푒푛푐푎푝푠푢푙푎푡푒푑 푖푛 푛푎푛표푝푎푟푡푖푐푙푒푠
× 100
푡표푡푎푙 푤푒푖푔ℎ푡 표푓 푐표푚푝표푢푛푑
2.14. Stability Study. The physical stability of Ac-Dex
NPs was evaluated by monitoring the changes in their size
over time. Particles were incubated under mildly basic (dd-
H2O, pH 8.0) and acidic (0.1 M acetate buffer pH 5.5), and
physiological conditions (0.01 M PBS buffer pH 7.4 and
DMEM containing 10% FBS pH 7.4), at 37 °C with
2.9. Nanoparticle Size, Polydispersity Index and Zeta
Potential.
Particle’s
hydrodynamic
diameter,
polydispersity index (PDI) and zeta potential were
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constant agitation. At determined time points (0, 0.4, 1, 2,
4, 8, 12, 24, 48 and 72 h), the particles’ size was monitored
by DLS.
Alexa Fluor 555 Phalloidin were excited at 405 nm and 543
nm, whereas 633 nm was used to excite dye 9.
2.18. Cell Viability Assay. Cells were seeded into
96‒well plates (7.5 × 103 cells/well) containing 200 μL of
complete cell culture medium, and incubated for 24 h at 37
°C in a humidified atmosphere with 5% CO2. Then, the
cells were treated with different concentrations of PtIV/Ac-
2.15. In Vitro Release Study. PtIV/Ac-Dex NPs and NIR-
dye 9/Ac-Dex NPs were suspended in either 3% Tween 80
in 0.1 M acetate buffer (pH 5.5) or 3% Tween 80 in 0.01 M
PBS buffer (pH 7.4), and incubated at 37 °C under gentle
shaking. At selected time intervals (0, 2, 4, 6, 8, 12, 24, 48
and 72 h), the suspension was centrifuged at 11,300 × g for
20 min to separate pellet from supernatant. The supernatant
was removed and replaced with the same volume of fresh
buffer solution containing 3% Tween 80, in which the pellet
was resuspended and returned for further incubation. The
amount of PtIV complex 3 and NIR-dye 9 was quantified by
ICP-OES and UV-vis spectroscopy, respectively. The
cumulative amount of released compound was calculated,
and the percentages of compound released from Ac-Dex
NPs were plotted against time. The release experiments
were conducted in triplicate.
Dex NPs (concentration of PtIV compound 3: 2.9 × 10-4
-
10
11
12
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28.8 μM), NIR-dye9/Ac-Dex NPs (concentration of dye 9:
2.0 × 10-5- 2.0 μM), and co-loaded Ac-Dex NPs
(concentration of PtIV compound 3: 2.9 × 10-4 - 28.8 μM
and concentration of dye 9: 2.0 × 10-5- 2 μM) for further 48
h. Cisplatin, free PtIV complex and empty Ac-Dex NPs were
also tested for comparison, using the same procedure. After
incubation, the growth medium was removed, the cells were
washed twice with PBS, and the cell viability was evaluated
using the Cell Counting Kit-8 (CCK-8) according to the
manufacturer’s instructions. The absorbance of the solution
in each well was measured at 450 nm in a microplate reader
(FlashScan 530 Analitic Jena). The negative control was
designed as 100% of dehydrogenase activity, and cell
viability was expressed as a percentage of the untreated
cells.
2.16. Cell Culture. The human MCF-7 (breast cancer),
PC3 (prostate cancer) and PNT2 (normal prostate) cells
were cultured in RPMI-1640 supplemented with 10% FBS
and 1% penicillin‒streptomycin. HeLa (human cervical
cancer) cells were cultured in DMEM (Dulbecco’s
Modified Eagle Medium) with high glucose supplemented
with 10% FBS and 1% penicillin‒streptomycin. Caco-2
(human colorectal cancer) cells were maintained in DMEM
with high glucose containing 1% nonessential amino acids,
3. RESULTS AND DISCUSSION
The use of PtIV prodrugs instead of injecting directly
highly toxic PtII cisplatin has been recently investigated as a
potential strategy to overcome resistance of cisplatin and
minimize its harmful side effects while maintain its potency
against cancer cells. This strategy relies on the fact that PtIV
complexes present low toxicity; however, inside the cells
they are reduced by the intracellular medium to highly toxic
PtII.49 In order to increase the efficiency of anticancer
therapy and increase its selectivity while minimizing its
harmful side effects, in this study we designed a drug
delivery system that combines the advantages of Ac-Dex
NPs with the advantages of using a PtIV prodrug. The
current design is expected to provide a nanomaterial system
that will be more selective and more effective against
cancer cells through EPR effect while being harmless to
healthy tissues (Scheme 2).
2
mM
L‒glutamine,
10%
FBS
and
1%
penicillin‒streptomycin. All cells were incubated at 37 °C
in a humidified atmosphere with 5% CO2. The cell culture
medium was changed every 2-3 days, and cells were
passaged at 80-90% confluency using 0.25% trypsin-0.02%
EDTA solution.
2.17. In Vitro Cellular Uptake Study. Confocal laser
scanning microscopy (CLSM) was used to visualize cellular
uptake of the NPs. MCF-7 cells were seeded into 6-well
plates at a density of 3 × 105 cells/well in 3 mL of
RPMI‒1640 supplemented with 10% FBS and 1%
penicillin‒streptomycin. After culturing for 24 h at 37 °C in
a humidified atmosphere with 5% CO2, the cells were
further incubated for 6 h with free NIR-dye 9 and with
NIR-dye9/Ac-Dex NPs at 0.1 μM (final concentration of
dye). The cells were washed three times with PBS, fixed
with 4% p‒formaldehyde for 15 min at room temperature,
and washed again with PBS. Then, the cells were
permeabilized with 0.2% Triton X-100 for 5 min, washed
with PBS, and incubated with Hoechst 33342 to label the
nuclei and with Alexa Fluor 555 Phalloidin for staining the
cytoskeleton, according to the standard protocol provided
by the supplier. The slides containing the cells were rinsed
once with PBS and mounted with antifading solution.
Finally, the fluorescence images were obtained using an
Upright LSM780‒NLO Zeiss confocal laser scanning
microscope equipped with 405, 458, 488, 514, 543, 561,
and 633 nm laser excitation sources. Hoechst 33342 and
Scheme 2. Schematic representation of the new Ac-Dex drug
delivery system for the sustained release of the PtIV prodrug 3 to
cancer cells, and NIR imaging with dye 9.
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O
3.1. Synthesis
and
characterization
of
the
hydrophobic PtIV prodrug 3
4
Complex cis,cis,trans-[Pt(NH3)2Cl2(CO2(CH2)4CH3)2] 3
(Scheme 3) was chosen as the PtIV prodrug because its
hydrophobicity allows encapsulation in Ac-Dex NPs using
o/w emulsion procedures, and because this complex is
reduced to highly cytotoxic cisplatin by the intracellular
medium.49
6
N
5
6
7
8
(i) POCl3, DMF
reflux, 1 h
(ii) aniline, EtOH
r.t., 1 h
I
MeCN, reflux, 24 h
9
Cl
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N+
H
N
N
H
Cl-
7
5
NaOAc, EtOH
reflux, 12 h
Cl
Scheme 3. Synthesis of the hydrophobic PtIV prodrug 3 using
cisplatin (1) as starting material. PtII was oxidized to PtIV in the
presence of hydrogen peroxide, and the resulting compound 2
reacted with hexanoic anhydride to yield the hydrophobic PtIV
complex 3.
+
N
N
Cl-
8
H2N
DMF, 80˚C,16 h
The hydrophobic PtIV prodrug 3 having two aliphatic
carboxylate axial ligands was synthesized from cisplatin 1
in two steps (Scheme 3), with a global yield of 40%. IR and
NMR spectroscopies were employed to confirm the
structures of 2 and 3, and the corresponding spectra are
shown in Figures S1 to S3 of the Supporting Information
(S.I.). Since the compound 3 dissolves in the organic
solvent ethyl acetate, it was a suitable candidate for
encapsulation in Ac-Dex NPs by single emulsion process.
NH
+
N
N
Cl-
9
Scheme 4. Synthesis of the hydrophobic NIR-fluorescent
aminocyanine dye 9.
3.2. Synthesis
and
characterization
of
the
hydrophobic NIR-fluorescent dye 9
The photophysical properties of dye 9 were evaluated
in six different solvents: tetrahydrofuran (THF), chloroform
(CHCl3), dichloromethane (DCM), acetonitrile (ACN),
dimethylsulfoxide (DMSO) and methanol (MeOH). The
UV-vis absorption and emission spectra of the dye 9 are
shown in Figure 1, and the relevant data are summarized in
Table 1. The absorption spectra of dye 9 in solution showed
an absorption band with λmax at around 635 nm. Dye 9
presents a large Stokes shift (ranging from 97 in
dichloromethane to 133 in DMSO), which can be attributed
to an intramolecular charge transfer (ICT) transition.52, 53
A new hydrophobic dye from the tricarbocyanine family
that is fluorescent in the near infrared region (NIR) was
synthesized to be efficiently encapsulated in the Ac-Dex
NPs by single nanoemulsion process. We focused on the
synthesis of a tricarbocyanine dye since this class of
compounds have been widely explored as imaging agents
due to their strong fluorescence in the NIR region and low
cytotoxicity.51 The synthetic route to synthesize dye 9
involved four steps (Scheme 4).50, 52
The precursor N-[5-anilino-3-chloro-2,4-(propane-1,3-
diyil)-2,4-pentadiene-1-ylidene]anilinium chloride (5) was
synthesized by Vilsmeier‒Haack formylation of
cyclohexanone 4. In parallel, 1-hexyl-2,3,3-trimethyl-3H-
indol-1-ium iodide (7) was prepared by alkylation of 2,3,3-
trimethylindolinine (6) with hexyl iodide in refluxing
acetonitrile. The iminium salt 5 was condensed with the
indolium derivative 7 to afford the chlorocyanine dye 8 in
Table 1. Photophysical data from absorption and emissiona
spectra of the aminocyanine dye 9 in six solvents.
λmax
Solvent (absorption)
(nm)
λmax
(emission)
(nm)
Stokes
shift
(nm)
ε
(M-1 cm-1)
THF
CHCl3
DCM
633
635
665
635
633
633
29503
20329
79693
22505
81741
99394
758
764
762
750
766
757
125
129
97
98% yield as a bright green solid.
Finally, the
aminocyanine dye 9 was obtained as a deep blue solid from
the SRN1 reaction of 8 with hexylamine. All compounds
were fully characterized using NMR spectroscopy and
high-resolution mass spectrometry (HRMS). The detailed
structural characterization of the compounds is provided in
Figures S4 to S14 (SI).
ACN
115
133
124
DMSO
MeOH
aExcitation wavelength: 635 nm.
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One of the major disadvantages of tricarbocyanine
percentage of cyclic acetal groups than the acyclic ones.31
Considering that the higher thermodynamic stability of the
cyclic acetals generates NPs with a slow degradation rate,
indeed, Ac-Dex60 is expected to release the content within
12 h. The nanoemulsion procedure was adapted38 and
optimized, in order to afford NPs with sizes of around 100
nm, in order to avoid clearance by the kidneys and liver,
improve the drug pharmokinetic properties and to enhance
tumor selectivity through the EPR effect.
Four different Ac-Dex NPs were prepared using a single
o/w nanoemulsion procedure: (i) blank Ac-Dex NPs, (ii)
Ac-Dex NPs containing the PtIV prodrug 3 (PtIV/Ac-Dex
NPs), (iii) Ac-Dex NPs containing the NIR-dye 9 (NIR-
dye9/Ac-Dex NPs), and (iv) Ac-Dex NPs containing both
PtIV prodrug 3 and NIR-dye 9 (co-loaded Ac-Dex NPs).
The size, polydispersity index (PDI) and zeta potential of
the resulting nanoparticles were characterized by dynamic
light scattering (DLS) (Figure 2a and Table 2). In addition,
size and shape were confirmed by scanning electron
microscopy (SEM, Figures 2b and 2c). For all formulations
prepared, the Ac-Dex NPs obtained were spherical, with
sizes ranging between 100 and 113 nm, with low dispersity
(PDI < 0.155).
dyes is their tendency to aggregate in solution; however, it
is interesting to note that dye 9 has low tendency to
aggregate in the tested solvents. The absorption spectra of
dye 9 in DCM, ACN, MeOH and DMSO show mainly the
absorption band at 635 nm that corresponds to the
monomeric species. Only in THF and CHCl3 the absorption
spectra show the presence of significant bands at lower
wavelengths (hypsochromic shift) that correspond to
parallel aggregates, which are known as H-type
aggregates.52 The high molar absorption coefficients (ε) of
dye 9 (around 8x104 - 1x105 M-1 cm-1) decrease significantly
in THF and CHCl3 probably due to the presence of the H-
type aggregates. These photophysical properties of dye 9
together with its strong fluorescence in the NIR region are
promising features for the use of this dye as an imaging
agent when encapsulated in Ac-Dex NPs.
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(a)
Due to the different solubility of the PtIV complex 3
and dye 9, the nanoemulsion procedures to encapsulate
each guest was formulated using different solvents. To
encapsulate PtIV ethyl acetate was used as the organic
solvent (similar to formulation of empty Ac-Dex NPs),
whereas dichloromethane was used to encapsulate dye 9.
Nevertheless, minimal variability among the three distinct
formulations was observed, even using different organic
solvents during the preparation of blank NPs, PtIV/Ac-Dex
NPs, and NIR-dye9/Ac-Dex NPs. Therefore, our data show
that our formulations present a very reproducible and
versatile behavior. Furthermore, the zeta potential of Ac-
Dex NPs varies between -16.7 and -26.9 mV (Table 2),
suggesting low tendency of the Ac-Dex NPs to aggregate in
solution.
(b)
The encapsulation efficiencies (EE) of PtIV prodrug 3
and NIR dye 9 in Ac-Dex NPs were determined using ICP-
OES and UV-vis spectroscopy (Table 2), respectively.
When adding an initial feed of 30% (w/w) of 3 and 9 to Ac-
Dex, 100% of 3 and 77% of 9 were encapsulated in the
single-loaded NPs, which correspond to loading levels of
30 and 23% (w/w), respectively. Very high LC and EE
were also observed by co-loaded Ac-Dex NPs. These high
loadings are most likely the result from the high
compatibility between the hydrophobic nature of Ac-Dex
polymer and the high hydrophobicity of both guest
compounds. The high drug loading capability of Ac-Dex
NPs is very attractive for therapeutic applications, since
high drug loading enables the use of lower amounts of
nanoparticle in a given dose of treatment, as well as a lower
number of doses.
Figure 1. Absorption (a) and emission (b) spectra of dye 9 in six
different solvents.
3.3. Preparation and characterization of Ac-Dex NPs
Ac-Dex NPs were tailored to enhance tumor
accumulation and to release the encapsulated prodrug in a
sustained manner within 12 h. The Ac-Dex polymer was
synthesized using an acetalation time of 60 min (Ac-
Dex60), in order to contain in its structure a higher
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Table 2. Size, polydispersity index (PDI), zeta potential, loading content (LC) and encapsulation efficiency (EE) for all Ac-Dex NPs.
Zeta
potential
(mV)
Initial feed
drug/polymer
ratio (w/w) (%)
Sizea
(nm)
Polydispersity
index
Ac-Dex NPs
LC (%)
EE (%)
Blank Ac-Dex NPs
PtIV/Ac-Dex NPs
113 ± 44
100 ± 35
102 ± 38
0.152
0.126
0.137
-25.7 ± 3.8
-16.7 ± 2.2
-26.9 ± 0.4
--
--
--
30
30
30 ± 0
23 ± 1
30 ± 0
2 ± 0
100
77
NIR-dye9/Ac-Dex NPs
30 (PtIV complex)
100
100
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Co-loaded Ac-Dex NPs
108 ± 43
0.155
-21.1 ± 0.8
2 (NIR-dye9)
aData are presented as the mean ± standard deviation (n = 3). The standard deviation was calculated using the formula PDI = (/D)2, in
which PDI is the polydispersity index,  is the standard deviation, and D is the mean diameter.
(b)
(a)
(d)
(c)
200 nm
Figure 2. Size characterization of Ac-Dex NPs: (a) Size distribution of empty and loaded Ac-Dex nanoparticles by DLS. (b) SEM image of
blank Ac-Dex NPs. (c) Magnified SEM image of PtIV/Ac-Dex NPs. (d) SEM image of NIR-dye9/Ac-Dex NPs.
3.4. pH-responsiveness and stability of Ac-Dex NPs
The stability of Ac-Dex NPs was evaluated in
different pH conditions because pH plays an important role
in storage conditions and in guest release responsiveness,
which are crucial parameters in therapeutic applications.
Particles were incubated in vitro at 37 ºC under: (i) PBS at
physiological pH (pH = 7.4), (ii) cell culture medium
DMEM with 10% FBS (pH = 7.4) mimicking the
physiological medium, (iii) slightly alkaline water (pH =
8.0), (iv) mildly acidic aqueous solution mimicking
lysosomal pH (pH = 5.5). For all medium conditions, the
nanoparticles’ size was monitored by DLS at predetermined
time points (Figure 3). As shown in Figure 3a no significant
variations in size were observed for PtIV/Ac-Dex NPs in
PBS at pH 7.4 and in H2O at pH 8.0 over three days. The
particles remained in a size range of 113 - 165 nm, with
changes smaller than their standard deviation, indicating
that the NPs have high stability under such conditions.
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When suspended in a protein-containing cell culture
size populations with high dispersity, confirming the
expected degradation and disassembly of the PtIV/Ac-Dex
NPs in these conditions.
medium (DMEM supplemented with 10% FBS, pH 7.4)
mimicking the physiological medium, the PtIV/Ac-Dex NPs
increased in size within 12 h to 190 - 220 nm, which
remained constant for three days. These data suggest the
formation of a small protein corona in the particle surface,
with low tendency to aggregate in solution, reflecting the
good stability of Ac-Dex NPs in this protein-enriched
medium.
3.5. In Vitro drug release kinetics
The release profile of PtIV prodrug 3 from the pH-
responsive Ac-Dex NPs was quantitatively assessed using
ICP-OES at both pH 7.4 and pH 5.5, at 37 °C (Figure 4). At
pH 5.5, about 80% of PtIV was released from Ac-Dex NPs
within the first 12 h, whereas during this same period, only
ca. of 15% of the prodrug 3 was released at pH 7.4.
Moreover, PtIV/Ac‒Dex NPs reached nearly complete
release of PtIV after 72 hours at pH 5.5. In contrast, the
amount of compound 3 released from the NPs after three
days was lower than 25%. It is worth noting that within the
first 12 hours PtIV/Ac‒Dex NPs exhibit a continuous
sustained release of 3 (Figure 4a, inset). A very similar
release profile was observed for NIR-dye 9 from NIR-
dye9/Ac-Dex NPs (Figure 4b), thus confirming that for this
type of NP, the release rate is controlled by the Ac-Dex
degradation in acidic environment, and it does not depend
significantly on the nature of the encapsulated molecules.
This release profile is suitable for anti-cancer drug delivery
applications.
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600
PBS (pH 7.4)
H2O (pH 8.0)
(a)
500
DMEM (pH 7.4) with 10% FBS
400
300
200
100
0
0
20
40
60
80
Time (hours)
(a)
(b)
Acetate-Tween (pH 5.5)
PBS-Tween (pH 7.4)
(b)
100
Figure 3. Stability study of Ac-Dex NPs in solution. DLS data
obtained for PtIV/Ac-Dex NPs as a function of time in different
media. (a) Size changes of PtIV/Ac-Dex NPs in PBS (pH 7.4),
DMEM with 10% FBS (pH 7.4) and H2O (pH 8.0) at 37 ºC in the
period of 72 h measured by DLS (error bars represent the standard
deviation of the mean size of three measurements). (b) Size
changes of PtIV/Ac-Dex NPs at pH 7.4 and pH 5.5 at 37 ºC, over
24 h measured by DLS.
80
60
40
20
0
When dispersed in acetate buffer at pH 5.5 (Figure 2b),
a significant increase in size of the NPs was observed
within fifteen minutes up to 2 h. This change in size
suggests the initial degradation of the acetal groups
increasing the hydrophilicity of the polymer, and
consequent disentanglement of the polymer chains and
disassembly of the Ac-Dex NPs. DLS measurements after 8
and 24 h, at pH 5.5 show inconsistent results with several
0
10
20
30
40
50
60
70
80
Time (h)
Figure 4. In vitro release profile of PtIV prodrug 3 from PtIV/Ac-
Dex NPs (a), and dye 9 from NIR-dye 9/Ac-Dex NPs (b), in
acetate buffer-Tween (pH 5.5) and phosphate buffered saline-
Tween (pH 7.4) at 37 °C.
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Figure 5. Comparison of cellular uptake of free and encapsulated NIR-dye 9 by observing intracellular dye 9 distribution in MCF-7 cells
°
with confocal laser scanning microscopy. MCF-7 cells were incubated with NIR-dye 9 and with NIR-Dye9/Ac-Dex NPs for 6 h, at 37 C.
Nuclei and cytoskeleton were stained with Hoechst 33342 (blue) and Alexa Fluor 555-Phalloidin (green), respectively. The red color
presented in the third channel results from dye 9 fluorescence. In both assays, the concentration of both free and encapsulated NIR-dye 9
was equivalent to 0.1 μM.
3.6. Cellular uptake by confocal microscopy
dye9/Ac-Dex NPs, present a strong NIR fluorescence
(presented in red, Figure 5). The CLSM images enabled the
visualization of a homogeneous distribution pattern of the
fluorescent dye in the interior of the cells delivered by the
Ac-Dex NPs.
Cellular internalization of anticancer agents and
nanomaterials plays a crucial role in therapeutic efficacy.
To evaluate the internalization of Ac-Dex NPs, we have
used the NIR-dye 9 as a fluorescent probe. The cellular
uptake of the NIR-dye9/Ac-Dex NPs was compared with
the cellular uptake of the free NIR-dye 9. To carry out this
assay, the cytotoxicity of the free and encapsulated dye was
pre-determined towards different cell lines (Figures S15
and S16 of S.I.). The cytotoxicity studies clearly indicate
that dye 9 and NIR-dye9/Ac-Dex NPs present no significant
toxicity in the tested cells up to 2 μM. Therefore, the
cellular uptake of dye 9 and and NIR-dye9/Ac-Dex NPs by
MCF-7 cells was evaluated through confocal laser
fluorescence microscopy (CLSM, Figure 5). The nuclei of
MCF-7 cells were stained using Hoechst 33342 (presented
in blue), while the cytoskeleton was stained with Alexa
Fluor 555 Phalloidin (presented in green). MCF-7 cells
were incubated for 6 h at 37 °C with an equal concentration
(0.1 μM) of free and encapsulated dye 9 (presented in red).
Upon incubation with free dye, the MCF-7 cells present a
minimal intensity of NIR fluorescence, attesting a low
cellular internalization of the free dye 9. The low
internalization of the free dye 9 is probably due to its highly
hydrophobic nature that results in low solubility in the
aqueous medium, and might enable the formation of dye
aggregates that cannot penetrate efficiently the cell
membrane. In contrast, MCF-7 cells incubated with NIR-
Figure 6. Orthogonal projection of confocal laser fluorescence
microscopy image of MCF-7 incubated with NIR-dye9/Ac-Dex
NPs for 6h. Blue and green colors stain the nuclei and
cytoskeleton, respectively. The red fluorescence highlights the
presence of NIR-dye 9 distributed within the cells.
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To further confirm the cellular uptake of NIR-dye 9/Ac-
nanoparticles the PtIV prodrug 3 presented much higher
cytotoxic activity against all cancer cells tested when
compared with the free compound. For example, free PtIV
prodrug 3 and cisplatin display IC50 values of 2.3 and 8.3
μM for MCF-7 cells, whereas the encapsulated PtIV prodrug
3 in the Ac-Dex NPs presented an IC50 of around 0.2 μM in
these cells. This suggest that the encapsulated prodrug 3
was about 12- and 44-fold more potent towards MCF-7
cells than the free PtIV compound and cisplatin,
respectively. In PC-3 cells, the encapsulated anticancer
compound (IC50 = 390 nM in PtIV/Ac-Dex NPs) exhibited a
6- and 41- fold higher cytotoxic activity compared to the
Dex NPs, a z-stacking acquisition and an orthogonal
projection of confocal images were performed (Figure 6).
The images endorse the previous findings, indicating the
successful uptake of NIR-dye 9 encapsulated in Ac-Dex
NPs. Moreover, the stacked images revealed the presence of
red fluorescence inside the MCF-7 nuclei, as well as in the
cytoplasm. This outcome has significant importance since
the mode of action of cisplatin take place in the nuclei,
through the DNA-cisplatin interaction to form DNA
adducts, which rises signal transduction pathways until the
activation of apoptosis.54 Altogether, these results suggest
that Ac-Dex NPs are a suitable delivery platform for
anticancer drugs and imaging agents.
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free PtIV complex 3 (IC50 = 2.3 μM) and cisplatin (IC50
=
16.0 μM), respectively. Similar behavior in PC3 cells was
showed for the PtIV complex 3 in co-loaded NPs. These
results confirm the higher therapeutic efficacy of the new
nanosystem when compared to both the free prodrug and
cisplatin.
3.7. Cytotoxic activity and IC50 for all compounds
The cytotoxicity of the blank and loaded Ac-Dex NPs
was assessed in four different cancer cell lines: MCF-7
(breast), HeLa (cervical), Caco-2 (colon) and PC-3
(prostate), and in one healthy cell line (PNT2, prostate)
using the CCK-8 assay. As showed in Figure 7, the blank
Ac-Dex NPs displayed no significant cytotoxicity against
all tested cell lines, even at the fairly high concentration of
1 mg.mL-1, which exceeds the maximum concentration used
during the cytotoxicity tests with loaded PtIV/Ac-Dex NPs.
These results are comparable to other well-known
biocompatible materials,16 and are very promising, since the
biocompatibility of delivery vehicles intended for
therapeutic use is of critical importance to its eventual
success.
Table 3. IC50 values obtained for cisplatin, free PtIV prodrug and
PtIV in Ac-Dex NPs against five cell lines.a
Compound
Cisplatin
PtIV prodrug 3
MCF-7 HeLa Caco-2 PC3 PNT2
8.3
2.3
36.9
2.9
5.9
1.1
16.0
2.3
14.7
3.5
PtIV in PtIV/Ac-Dex
NPs
0.2
0.2
0.4
0.3
0.4
0.3
0.4
0.3
0.5
0.4
PtIV in co-loaded
Ac-Dex NPs
aCells were treated for 48 h with the indicated compounds, and the effect
on cell growth was analyzed. The half-maximal inhibitory concentration
(IC50) is expressed in µM. Data are presented as the mean of at least one
experiment carried out in triplicate. The IC50 values were determined from
nonlinear regression analysis calculated with the GraphPad Prism 5.0
software.
MCF-7
HeLa
Caco-2
PC-3
PNT2
100
80
60
40
20
0
CONCLUSION
We have demonstrated for the first time that Ac-Dex
NPs are valid candidates as drug delivery platform for anti-
cancer applications. Specifically, a PtIV prodrug (3), named
cis,cis,trans-[Pt(NH3)2Cl2(CO2(CH2)4CH3)2],
and
a
Control
0.001
0.01
0.1
1.0
tricarbocyanine derivative dye (9), with NIR fluorescent
properties and low cytotoxicity, were efficiently
encapsulated in different Ac-Dex NPs, and co-encapsulated
in the Ac-Dex NPs using an oil-in-water nanoemulsion
method. These NPs were obtained as small polymeric
spheres with around 100 nm with low polydispersity and
high encapsulation efficacy. Confocal laser fluorescence
images demonstrated efficient cellular uptake Ac-Dex NPs
in MCF-7 cells. Combination of the advantages of using a
PtIV prodrug with the advantages of using Ac-Dex NPs
resulted in a significant enhancement of the cytotoxic
activity of the encapsulated PtIV prodrug against different
cell lines when compared to free PtIV and cisplatin. The
current results indicate that the Ac-Dex NPs provide
protection for the encapsulated prodrug 3 against fast
chemical or enzymatic degradation in the blood. Once the
PtIV-loaded nanoparticles were internalized by cancer cells,
Concentration of blank Ac-Dex NPs (mg/mL)
Figure 7. Cell viability data obtained for the blank Ac-Dex NPs
against five different cells lines: MCF-7, HeLa, Caco-2, PC-3 and
PNT2. Data were determined by CCK-8 assay and are presented
as the mean from two independent experiments performed in
triplicate (the bars represent the standard deviations from n = 6).
The cytotoxic activity of new nanosystems PtIV/Ac-Dex
NPs and co-loaded Ac-Dex NPs was also assessed against
MCF-7, HeLa, Caco-2, PC-3 and PNT2 cell lines, and the
IC50 values (expressed in μM) were compared to the ones
from free PtIV complex 3 and cisplatin (Table 3, Figures
S17-S36 of S.I.). The free PtIV prodrug showed higher
cytotoxic activity against all cell lines tested when
comparing with the PtII parental compound cisplatin.
Moreover, when delivered via the Ac-Dex polymeric
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PtIV was released from Ac-Dex particles into the cytosol in
INFABIC is co-funded by Fundação de Amparo a Pesquisa
response to the mildly acidic environment. Finally, PtIV was
reduced by the intracellular medium releasing cisplatin,
which inhibited DNA replication and cell proliferation.
Moreover, the co-encapsulation of both the NIR-dye and
the PtIV prodrug enables the possibility to use the Ac-Dex
NPs for theranostics applications.
do Estado de São Paulo (FAPESP) (08/57906-3) and
Conselho Nacional de Desenvolvimento Cientifico e
Tecnológico (CNPq) (573913/2008-0).
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100 nm Ac-Dex NPs will result in in vivo selectivity
through the EPR effect.
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biocompatibility, high drug loading capability, high
encapsulation efficiency, 100 nm size, excellent stability in
physiological conditions, pH-responsiveness, and tunable
release rate of the Ac-Dex NPs make them a promising
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delivery of antineoplastic drugs and imaging agents to
tumor cells. Ongoing and future studies in our lab focuses
on the encapsulation and co-encapsulation of different
antineoplastic agents to test their anti-cancer activity in
vitro and in vivo.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website at DOI 10.1021/acs.xxxxxxx
Spectroscopic characterization of the compounds; cell
viability data of the free and encapsulated NIR dye 9; IC50
plots obtained for all compounds against the five cell lines
(PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: catiaornelas@catiaornelaslab.com
ORCID
Carolyne B. Braga: 0000-0003-1800-386X
Gabriel Perli: 0000-0003-2588-5100
Catia Ornelas: 0000-0001-8020-9776
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Sao Paulo Research
Foundation - FAPESP (grant #2018/02093-0 for C.O.;
fellowship #2017/06146-8 for C.B.B.; scholarship
#2017/24488-3 for G.P.), and Coordination for the
Improvement of Higher Education Personnel - CAPES
(PhD scholarship for T.B.B.) for the financial support. This
study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nivel Superior - Brasil
(CAPES), finance code 001. We also thank the access to
equipment and assistance provided by the National Institute
of Science and Technology on Photonics Applied to Cell
Biology (INFABIC) at the State University of Campinas;
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