Docetaxel-Loaded Fluorescent Liquid-Crystalline Nanoparticles for Cancer Theranostics

Article abstract of DOI:10.1021/acs.langmuir.5b02101

Here, we describe a novel monoolein-based cubosome formulation engineered for possible theranostic applications in oncology. The Docetaxel-loaded nanoparticles were stabilized in water by a mixture of commercial Pluronic (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer) F108 (PF108) and rhodamine- and folate-conjugated PF108 so that the nanoparticles possess targeting, therapeutic, and imaging properties. Nanoparticles were investigated by DLS, cryo-TEM, and SAXS to confirm their structural features. The fluorescent emission characterization of the proposed formulation indicated that the rhodamine conjugated to the PF108 experiences an environment less polar than water (similar to chloroform), suggesting that the fluorescent fragment is buried within the poly(ethylene oxide) corona surrounding the nanoparticle. Furthermore, these nanoparticles were successfully used to image living HeLa cells and demonstrated a significant short-term (4 h incubation) cytotoxicity effect against these cancer cells. Furthermore, given their analogy as nanocarriers for molecules of pharmaceutical interest and to better stress the singularities of these bicontinuous cubic nanoparticles, we also quantitatively evaluated the differences between cubosomes and multilamellar liposomes in terms of surface area and hydrophobic volume.

Full text of DOI:10.1021/acs.langmuir.5b02101

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Article
Docetaxel Loaded Fluorescent Liquid Crystalline
Nanoparticles for Cancer Theranostics
Valeria Meli, Claudia Caltagirone, Angela Maria Falchi, Stephen T Hyde, Vito Lippolis, Maura Monduzzi,
Marc Obiols-Rabasa, Antonella Rosa, Judith Schmidt, Yeshayahu Talmon, and Sergio Murgia
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02101 • Publication Date (Web): 20 Aug 2015
Downloaded from http://pubs.acs.org on August 25, 2015
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Docetaxel Loaded Fluorescent Liquid Crystalline Nanoparticles for
Cancer Theranostics
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Valeria Meli,a Claudia Caltagirone,a* Angela M. Falchi,b Stephen T. Hyde,c Vito Lippolis,a Maura
Monduzzi,a Marc ObiolsꢀRabasa,d Antonella Rosa,b Judith Schmidt,e Yeshayahu Talmon,e and
Sergio Murgia,a*
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a Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Cagliari, S.S. 554
Bivio per Sestu, 09042, Monserrato CA, Italy. Eꢀmail: murgias@unica.it and
ccaltagirone@unica.it, Tel. +39(0)70 675 4453.
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b Dipartimento di Scienze Biomediche, Università di Cagliari, s.s. 554 bivio Sestu, Iꢀ09042
Monserrato (CA), Italy.
c Department of Applied Mathematics, Research School of Physics and Engineering, The Australian
National University, Canberra, A.C.T. 0200, Australia.
d Division of Physical Chemistry, Department of Chemistry, Getingevägen 60, SEꢀ22240
Lund, Sweden.
e Department of Chemical Engineering, Technion – Israel Institute of Technology, Haifa
3200003, Israel.
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Abstract
Here, we describe a novel monooleinꢀbased cubosome formulation engineered for possible
theranostic applications in oncology. The Docetaxel loaded nanoparticles were stabilized in water
by a mixture of commercial Pluronic (polyethylene oxideꢀpolypropylene oxideꢀpolyethylene oxide
triblock copolymer) F108 (PF108), and rhodamineꢀ and folateꢀconjugated PF108, so that the
nanoparticles possess targeting, therapeutic, and imaging properties. Nanoparticles were
investigated by DLS, cryoꢀTEM, and SAXS to confirm their structural features. The fluorescent
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emission characterization of the proposed formulation indicated that the rhodamine conjugated to the
PF108 experiences an environment less polar than water (similar to chloroform), suggesting that the
fluorescent fragment is buried within the polyethylenoxide corona surrounding the nanoparticle.
Furthermore, these nanoparticles were successfully used to image living HeLa cells and demonstrated a
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significant shortꢀterm (4 h incubation) cytotoxicity effect against these cancer cells. Furthermore,
given their analogy as nanocarriers for molecules of pharmaceutical interest, and to better stress the
singularities of these bicontinuous cubic nanoparticles, we also quantitatively evaluated the
differences between cubosomes and multiꢀlamellar liposomes in terms of surface area and
hydrophobic volume.
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1. Introduction
The amphiphilic characteristics of polar lipids drive their selfꢀassembly in water, giving a number
of liquid crystalline phases.1,2 Among them, the most interesting are probably the reverse
bicontinuous cubic phases.3 These are a family of threeꢀdimensional cubic structures constituted by
a curved, nonꢀintersecting lipid bilayer folded to form two disconnected, continuous water channels
with a crystal lattice that can be described by three different types of infinite periodic minimal
surfaces: the double diamond (space group ꢀꢁ3ꢂ), the gyroid (space group ꢃꢄ3ꢅ), and the
primitive (space group ꢃꢂ3ꢂ). These phases have been extensively investigated in recent decades
because they represent versatile nanostructures able to incorporate molecules of biological
relevance.4ꢀ8 Interestingly, cubic phases can be formulated as aqueous nanoparticle dispersions,
known as cubosomes.9 Although various emulsifiers have been suggested,10,11 in general, the
stabilization in water of such liquid crystalline nanoparticles is achieved using amphiphilic
polymers having long polyethylene oxide (PEO) chains, e.g. Pluronics (polyethylene oxideꢀ
polypropylene oxideꢀpolyethylene oxide triblock copolymers) or polysorbate 80.9,12
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Similarly to the reverse liquid crystalline cubic bulk phases, pharmaceutical applications are
predicted for cubosomes,13ꢀ17 and their potential use as nanocarriers for MRI probes has been
investigated.18,19 These nanoparticles have also been proposed for theranostic applications.20ꢀ25
Indeed, several peculiar characteristics make cubosomes appealing as theranostic tools in oncology.
Among these, one of the most distinguishing is the higher hydrophobic volume with respect to
liposomes, often considered as the golden standard for lipidꢀbased nanocarriers in medicine.
However, a quantitative comparison of the hydrophobic portion of these two kinds of nanoparticles
has never been reported in the literature. Other cubosomes specific features are the possibility of
simultaneously host therapeutics and imaging agents, as well as a size (typically in the range 100ꢀ
200 nm) that, at least in principle, allows the exploitation of the enhanced permeation retention
mechanism for the passive targeting to cancer tissues.9,26 In view of their application as
nanomedicine, it is also worth mentioning that the clearance from the bloodstream via the
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mononuclear phagocytic system should be retarded since, in analogy with stealth liposomes, these
nanoparticles are enclosed within a sort of a PEO corona that should prevent efficient recognition
by opsonin, thus hindering phagocytosis.27 Moreover, it was demonstrated that cubosomes (and
hexosomes) can be suitably formulated covering their surface with folate residues20,28 or antiꢀEGFR
Fab29 to actively target these nanocarriers toward cancer cells, while the effects at subcellular level
of pristine monooleinꢀbased cubosomes were recently investigated on HeLa cells.30 Remarkably,
nanoparticles with reverse bicontinuous cubic internal structure endowed of long time stability and
thermoresponsive capabilities can be formulated using polymerꢀsurfactant mixtures.31
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To explore further the potential of lipidꢀbased liquid crystalline nanoparticles as theranostic
nanomedicine in cancer treatments, we report here the loading of monooleinꢀbased cubosomes with
a potent anticancer drug (Docetaxel). Such formulation was stabilized with a mixture of commercial
Pluronic F108 (PF108), folateꢀconjugated PF108 (PF108ꢀFA), and rhodamineꢀconjugated PF108
(PF108ꢀR) to simultaneously confer targeting, therapeutic, and imaging abilities on these
nanoparticles. It deserves noticing that engineering stable, multifunctional cubosome formulations
can be a hard task. While the hydrophobic effect represents the main force that drives the selfꢀ
assembly of the lipids constituting the nanoparticles, their inner phase is basically determined by the
geometrical features of the lipid hydrophobic tail and polar head. This is rationalized by the
molecular effective packing parameter (Peff), given by the ratio v/al, where v and l are, respectively,
the hydrophobic chain volume and length (taken as 80% of the fully extended chain), and a is the
head group area.1 Peff is strongly dependent by the surrounding environment, therefore the type of
phase that characterizes the nanoparticles (and their very existence) is barely predictable in the
presence of additives, since encapsulation within the lipid bilayer of molecules useful to give these
nanoparticles theranostic skills may significantly alter Peff, thus provoking the transition of the inner
phase or the collapse of the formulation.9 In addition, decoration of the cubosome surface with
targeting and/or fluorescent moieties may disturb both the colloidal stability of the formulation and
the cellular uptake of the nanoparticles. Throughout this paper we will demonstrate that the
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proposed cubosome formulation, although prepared with a high portion (40%) of conjugated PF108
is stable, and successfully delivers its cargo to HeLa cells, taken as standard for cancer cells.
Moreover, considering the analogies between liposomes and cubosomes, we also presented here a
theoretical evaluation of the hydrophobic volume of these liquid crystalline nanoparticles.
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2. Materials and methods
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2.1. Materials
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Monoolein (MO, 1ꢀmonooleoylglycerol, RYLO MG 19 PHARMA, glycerol monooleate, 98.1
wt%) was kindly provided by Danisco A/S, DKꢀ7200, Grinsted, Denmark. Pluronic F108 (PEO132−
PPO50−PEO132), Docetaxel (≥ 97%), Nꢀhydroxysuccinimmide (NHS, 98%), N,N′ꢀ
dicyclohexylcarbodiimide (DCC, ≥ 99.5%), triethylamine (≥ 99.5%) and dimethyl sulfoxide (≥
99.7%) were purchased from SigmaꢀAldrich. Distilled water passed through a MilliꢀQ water
purification system (Millipore) was used to prepare the samples.
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2.2. Sample preparation
Monooleinꢀbased cubosomes were prepared and stabilized by dispersing the appropriate amount of
MO in aqueous solutions of a 60/20/20 mixture of Pluronic F108 (PF108), folateꢀconjugated, and
rhodamineꢀconjugated PF108 (PF108/PF108ꢀFA/PF108ꢀR) using an ultrasonic processor (UP100H
by Dr. Hielscher, cycle 0.9, amplitude 90 %) for 10 min. Docetaxel doped cubosomes were
obtained by dispersing the drug in the melted lipids (at 37 °C) with the help of an ultrasonic bath
before mixing with the Pluronic solution. The sample volume was usually 4 mL with approximately
96.4 wt % of water, 3.3 wt % of MO and 0.3 wt % Pluronics mixture.
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2.3. Synthesis of rhodamineꢀconjugated PF108 (PF108ꢀR)
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PF108ꢀNH2 (0.1976 g, 0.013 mmol), Nꢀhydroxysuccinimmide (NHS, 0.0125 g, 0.108 mmol), N,N′ꢀ
dicyclohexylcarbodiimide
(DCC,
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mmol),
and
Nꢀ(9ꢀ(2ꢀ(4ꢀ(3ꢀ
carboxypropanoyl)piperazineꢀ1ꢀcarbonyl)benzyl)ꢀ6ꢀ(diethylamino)ꢀ3Hꢀxanthenꢀ3ꢀylidene)ꢀNꢀ
ethylethanaminium (4) (0.024 g, 0.039 mmol) were dissolved in 8 mL of anhydrous DMSO in the
presence of triethylamine (20 ꢁL). To precipitate DCU (dicyclohexylurea), 20 mL of deionized
water were added, and the mixture was then centrifuged and filtered to obtain a red limpid solution.
This was dialyzed (14 kDa MW cutꢀoff membrane) for 3 days against ultrapure water, which was
changed every 2ꢀ6 hours. The resulting product was lyophilized for 1 day to remove all the residual
water. Yield: 63% (130.3 mg, 0.008 mmol); mp: 48ꢀ50 °C. 1HꢀNMR (400 MHz, DMSOꢀd6, 298 K):
δH 1.045 (3H × 50, ꢀCH3 of PPO), 1.17ꢀ1.33 (m, 3H × 2, ꢀN(CH2ꢀCH3)2), 2.66ꢀ2.69 (m, 4H × 2,
aliphatic protons from rhodamine moiety), 3.29ꢀ3.55 (m, 3H × 50, 4H × 264, ꢀCH2ꢀCH(CH3)ꢀOꢀ of
PPO and ꢀCH2ꢀCH2ꢀOꢀ of PEO), 3.60ꢀ3.73 (m, 2H × 2, ꢀN(CH2ꢀCH3)2), 4.53ꢀ4.56 (m, 1H × 2,
amidic ꢀNH), 6.95ꢀ8.07 (m, 10H × 2, aromatic protons). IR (solid state, cmꢀ1) ν = 2876 (s), 1584,
1635, 1714 (w).
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2.4. Photophysical characterization
Cubosome dispersions were diluted with MilliꢀQ water (1:100) before performing the photophysical
measurements. The emission and excitation spectra were recorded with a Perkin Elmer LS 55
spectrofluorimeter. The fluorescence quantum yield on PF108ꢀR was determined by using
Rhodamine 6G dissolved in EtOH as the reference standard (Φref = 0.94). The absorption spectra
were recorded on a Thermo Nicolet Evolution 300 spectrophotometer.
2.5. Cryogenic transmission electron microscopy (cryoꢀTEM)
Vitrified specimens were prepared in a controlled environment vitrification system (CEVS) at 25 °C
and 100 % relative humidity. A drop (~ 3 ꢀL) of the sample was placed on a perforated carbon
filmꢀcoated copper grid, blotted with filter paper, and plunged into liquid ethane at its freezing
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point. The vitrified specimens were transferred to a 626 Gatan cryoꢀholder and observed at 120 kV
acceleration voltage in an FEI Tecnai T12 G2 transmission electron microscope at about −175 °C in
the lowꢀdose imaging mode to minimize electronꢀbeam radiationꢀdamage. Images were digitally
recorded with a Gatan US1000 highꢀresolution CCD camera.
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2.6. Smallꢀangle xꢀray scattering (SAXS)
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The characterization of the nanoparticles structure as a function of temperature was carried out by
SAXS using a Ganesha 300XL (SAXSLAB ApS, Skovlunde, Denmark). This instrument is
equipped with a 2D 300 K Pilatus detector (Dectris Ltd., Baden, Switzerland) and a Genix 3D Xꢀ
ray source (Xenocs SA, Sassenage, France), generating xꢀrays at a wavelength, λ, of 1.54 Å. The
scattering data was collected at a qꢀrange of 0.014 < q (Åꢀ1) < 0.753, where the magnitude of the
scattering vector, q, is defined as q = (4π/λ) sin (θ/2), where θ is the scattering angle. The twoꢀ
dimensional scattering pattern was radially averaged using SAXSGui software to obtain I(q).
Measurements were performed in 1.5 mm quartz capillaries (Hilgenberg GmbH, Malsfeld,
Germany). The temperature was controlled by an external, recirculating water bath within ± 0.3 °C.
Samples were equilibrated during 1 hour at the measurement temperature prior performing the
experiment.
Water channels radii of the reverse bicontinuous cubic phases were calculated using the relation rw
= [(A0/–2πχ)1/2a] – L, where L is the length of the lipid hydrophobic chain (17 Å, in case of MO), a
is the lattice parameter obtained from the SAXS analysis, and A0 and χ are the surface area and the
Euler characteristic (integral Gaussian curvature scaled by 2π) of the Infinite Periodic Minimal
Surface geometries within the unit cell, explained further in the Appendix (ꢀꢁ3ꢂ, A0 = 1.919, χ =
− 2; ꢃꢂ3ꢂ, A0 = 2.345, χ = − 4). At least two Bragg peaks were used to estimate the errors
associated with a and rw, unless otherwise indicated.
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2.7. Light scattering
Dynamic light scattering (DLS) experiments were performed to obtain the size evolution of the
nanoparticles as a function of temperature. For that we used a light scattering goniometer
instrument (3D LS Spectrometer, LS Instruments, Fribourg, Switzerland), equipped with a 35 mW
HeꢀNe laser light source (wavelength of 632.8 nm). The instrument implements the soꢀcalled crossꢀ
correlation scheme to suppress contributions from multiple scattering32ꢀ34 together with a
modulation unit.35 The samples were placed in a 10 mm diameter cylindrical borosilicate disposable
culture tubes (Fisherbrand, Thermo Fisher Scientific Inc., Waltham, USA) and kept at the
measurement temperature in a temperatureꢀcontrolled indexꢀmatching bath. Samples were
equilibrated at the measurement temperature for 10 minutes before the experiment. Measurements
were performed at a range of scattering angles between 40° and 100° (in triplicate at each scattering
angle). The initial decay rate, Γ, was derived from the secondꢀorder cumulant analysis of the
normalized field autocorrelation function, which was used to calculate the apparent collective
diffusion coefficient, D0, of the nanoparticles:
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Γ = D0q2
(1)
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where q is the module of the scattering vector, q = (4πn/λ)sin(θ/2) , with n the refractive index of
the solvent, λ the wavelength of the incoming laser light and θ the scattering angle. The
hydrodynamic radius, Rh, was obtained by the StokesꢀEinstein relation (D0 = kBT/6πηRh) , where kB
is the Boltzmann constant, T is the absolute temperature and η is the viscosity of the solvent at the
temperature T.
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2.8. Cell Culture
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HeLa cells (ATCC collection) were grown in phenol redꢀfree Dulbecco’s modified Eagle’s medium
(DMEM, Molecular Probes, USA) with high glucose concentration, supplemented with 10 % (v/v)
fetal bovine serum, penicillin (100 U mLꢀ1) and streptomycin (100 µg mLꢀ1) (Invitrogen) in 5% CO2
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incubator at 37 °C. Cells were seeded in 35 mm dishes; experiments were carried out two days after
seeding, when cells reached 90 % confluency. Liquid crystalline nanoparticles were added to the
cells at a concentration of 1: 500 (2 µL in 1 mL of fresh medium) and incubated at 37 °C for 4 h.
For live cell imaging, fresh serumꢀfree medium was used to remove the extracellular particle
suspension before imaging session.
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2.9. Fluorescence microscopy
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Light microscopy observations were made using a Zeiss (Axioskop) upright fluorescence
microscope (Zeiss, Oberkochen, Germany) equipped with 10x, 20x and 40x/0.75 NA water
immersion objectives and a HBO 50 W Lꢀ2 mercury lamp (Osram, Berlin, Germany). Twelveꢀbitꢀ
deep images were acquired with a monochrome cooled CCD camera (QICAM, Qimaging, Canada).
For observation of dye fluorescence, filters were: ex 546 ± 6 nm, em 620 ± 60 nm. Digital images
were obtained with Image Pro Plus software (Media Cybernetics, Silver Springs, MD).
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2.10. Cytotoxic activity (MTT assay)
The cytotoxic effect of cubosome formulations was evaluated in HeLa cells by the MTT assay.
HeLa cells were seeded in 24ꢀwell plates at density of 3×104 cells/well in 500 µL of serumꢀ
containing media. Experiments were carried out two days after seeding when cells had reached 90
% confluence. The different formulations were added to the cells at concentration of 1:500 (2 ꢁL in
1 mL of serumꢀfree medium) and incubated at 37 °C for 4 h. The effect of Docetaxel 10 ꢀM
(specifically, 1 ꢁL of a 10 mM solution of Docetaxel in DMSO was added to 1 mL of serumꢀfree
medium) on HeLa cell viability was also tested for comparison at the same incubation time. A 50
µL portion of MTT solution (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide) (5
mg/mL in H2O), was then added and left for 2 h at 37 °C. The medium was aspirated, 500 µL of
DMSO was added to the wells, and color development was measured at 570 nm with an Infinite 200
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auto microplate reader (Infinite 200, Tecan, Austria). The absorbance is proportional to the number
of viable cells. All measurements were performed in quadruplicate and repeated at least three times.
Results are shown as percent of cell viability in comparison with nonꢀtreated control cells. Data
were expressed as a mean ± standard deviation (SD) of three independent experiments involving
quadruplicate analyses for each sample.
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2.11. Determination of MO and Docetaxel content in cubosomes
After loading with Docetaxel, the cubosome dispersion was purified from the nonꢀencapsulated
drug by dialysis: 2 mL were loaded into a dialysis tubing cellulose membrane (14 kDa MW cutoff)
and dialyzed against water (1000 mL), for 2 h (by replacing the water after 1 hour) at 25 °C. It
should be noticed that this purification step did not influence the concentration of the dye in the
formulation since the fluorophoreꢀconjugated PF108 has a MW higher than the membrane cutoff
(about 16 kDa). Therefore, in comparison with traditional methods that require enclosing the dye
within the lipid bilayer, this new procedure allows a huge saving of the (typically very expensive)
dye. Aliquots (10 ꢀL) of the nanoparticle preparations were dissolved in 300 ꢀL of CH3CN with
0.14 % CH3COOH (v/v). The samples were vortexed to obtain a clear solution, and aliquots (20 ꢁL)
were injected into the Agilent Technologies 1100 liquid chromatograph (Agilent Technologies, Palo
Alto, CA) equipped with a diode array detector (DAD). Analyses of MO (detected at 200 nm) and
Docetaxel (at 230 nm) in nanoparticle dispersions were carried out with a XDB–C18 Eclipse (150 ´
4.6 mm, 3.5 mm particle size) (Agilent Technologies) equipped with a Zorbax XDB–C18 Eclipse
(12.5 ´ 4.6 mm, 5 mm particle size) guard column (Agilent Technologies), with a mobile phase of
CH3CN/H2O/CH3COOH (75/25/0.12, v/v/v), at a flow rate of 2.3 mL/min. The temperature of the
column was maintained at 37 °C. Recording and integration of the chromatogram data was carried
out through an Agilent OpenLAB Chromatography data system. The identification of lipid
components and drug was made using standard compounds and conventional UV spectra.
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Calibration curves of all of the compounds were constructed using standards and were found to be
linear, with correlation coefficients > 0.995.
Drug encapsulation efficacy (EE%) was calculated by the following equation:36 EE% = (weight of
the drug in nanoparticles)/(weight of the feeding drug) × 100 %. Data were expressed as a mean ±
standard deviation (SD) of three independent experiments involving duplicate analyses for each
sample.
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2.12. Statistical analysis
Evaluation of statistical significance of differences was performed using Graph Pad INSTAT
software (GraphPad software, San Diego, CA, USA). Comparison between groups was assessed by
one–way analysis of variance (One–way ANOVA) followed by the Bonferroni Multiple
Comparisons Test.
3. Results
3.1. Synthesis of rhodamineꢀconjugated PF108 and liquid crystalline nanoparticles
characterization
When designing a nanoparticle for diagnostic or theranostic applications, two different approaches
could be adopted: one is doping the nanoparticle with the imaging agent (a fluorescent probe, for
instance); the other is functionalizing the nanoparticle with the imaging agent. This approach is
quite straightforward in the case of silicaꢀbased nanoparticles, which can be easily functionalized by
wellꢀestablished siloxane chemistry.37 Functionalization of nanoparticles as the cubosomes reported
here, can efficiently be achieved conjugating the amphiphilic polymers used to stabilize the
formulation (Pluronic F108, PF108, in our case) with a suitably chemically modified imaging
agent.38 Here, the fluorogenic molecule rhodamine B was conjugated to PF108 via a multiꢀsteps
approach (see Scheme 1). First the fluorophore was synthetically modified as reported in literature39
to obtain its piperazine amide derivative (4) that was subsequently conjugated to the aminated
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derivative of the PF108.40 The reaction was carried out in DMSO in the presence of DCC, NHS and
Et3N. Then, the product was dialyzed against deionized water, and lyophilized.
Since the folate receptor is overexpressed on cancer cell membrane, the PF108 was also conjugated
with folic acid as previously reported20 to actively target the nanoparticles towards cancer tissues.
A mixture of the commercial PF108, folateꢀconjugated PF108 (PF108ꢀFA), and rhodamineꢀ
conjugated PF108 (PF108ꢀR) was used to formulate cubosome dispersions in water also loaded
with the anticancer drug Docetaxel (DTX). According to preliminary tests, it was found that a
mixture having the ratio PF108/PF108ꢀFA/PF108ꢀR = 60/20/20 (wt %) suitably stabilizes the
dispersion for more than two months. This formulation was then investigated for its morphological
and structural aspects by cryoꢀTEM, SAXS, and DLS measurements.
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Figure 1 shows a typical cryoꢀTEM image of the cubosome formulation we proposed. Close
inspection of the image in Figure 1 reveals a number of spherical cubosomes of different sizes and
an inner structure characterized by a dark matrix and alternate bright spots, respectively
representing the lipid bilayer and the water channels. As often occurs, some vesicular materials can
be seen attached to the liquid crystalline nanoparticles while, differently from other cubosome
formulations, only a few vesicles are noticed. Indeed, unilamellar or oligolamellar vesicles typically
form during the process of cubosome production, very likely because of the presence of the
Pluronic used as dispersant (MO in water does not produce small uniꢀlamellar or oligoꢀlamellar
vesicles).41 Actually, depending on the method of preparation, vesicles may exceed cubosomes in
terms of number density.42 Therefore, the absence of vesicular material observed here could be
ascribed to the use of the conjugated Pluronics as stabilizer for the cubosome formulation.
Taking into account the possible use of the system under study as theranostic nanocarrier, variation
of size and inner structure upon temperature changes were monitored through DLS and SAXS
measurements in the range 10ꢀ50 °C. Particularly, the temperatures were chosen to include two
extreme storage temperatures (10 and 50 °C) as well as room and physiological temperatures.
Moreover, to find out if the system nanostructure after temperature cycling is reestablished, SAXS
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measurements were performed in the following order: 25, 37, 50, 10, 25, and 37 °C. Results are
reported in Figure 2 and Table 1.
Table 1. Cubosome lattice parameters (a) and radius of the water channels (rw). Experiments were
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performed at different temperatures in the order reported in the table.
ꢆꢇꢈꢉ
ꢊꢉꢈꢉa
T (°C)
a (Å) rw (Å) a (Å) rw (Å)
25 97.0 ± 0.5 20.9 ± 0.2 129.6 22.6
37 89.2 ± 0.2 17.9 ± 0.1 118.7 19.3
50 80.7 ± 0.2 14.5 ± 0.1 109.6 16.5
25 97.1 ± 0.6 21.2 ± 0.3 123.1 20.6
37 90.5 ± 0.7 18.4 ± 0.3 118.7 19.3
a
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Standard deviations on a and rw pertaining to ꢃꢂ3ꢂ phase were not reported since these
parameters were obtained from the single Bragg peak discernible in the diffraction pattern.
SAXS results confirmed the bicontinuous cubic inner structure of the formulation. Actually, the
diffraction pattern of the cubosome formulation at 25 °C (Figure 2) is dominated by four Bragg
peaks placed in a q ratio of √2:√3:2:√6 that clearly identify the double diamond (ꢀꢁ3ꢂ)
bicontinuous cubic phase. However, a weak reflection at low q reveals the simultaneous presence of
the primitive (ꢃꢂ3ꢂ) bicontinuous cubic phase. The coexistence of the ꢀꢁ3ꢂ and the ꢃꢂ3ꢂ
phases in cubosome formulations is widely reported in literature; it is attributed to a ꢀꢁ3ꢂ to
ꢃꢂ3ꢂ intercubic phase transition caused by the interaction of the hydrophobic moiety of the
Pluronic with the surface of the cubosome nanoparticles.43
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The lattice parameter, a, obtained from the SAXS experiments performed at different temperatures
along with the calculated radius of the water channels rw (see paragraph 2.3) are reported in Table 1.
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As usually detected in liquid crystalline systems, although the nanostructure is retained, the lattice
parameter (and consequently the radius of the water channels) of the cubosomes decreases while the
temperature increases in the sequence 25, 37, 50 °C. This phenomenon is related to the enhanced
disorder of the lipid chain at higher temperatures that, in turn, induces higher negative curvature at
the lipid/water interface.43 The disappearance of the weaker Bragg peaks detected in the diffraction
patterns obtained at 10 °C (see Figure 2) indicates that the inner structure is in some way altered,
although not completely lost. When the temperature is raised again to 25 or 37 °C the nanostructure
is recovered. However some significant differences can be noticed in the intensity of the Bragg
peaks and the general appearance of the diffractograms after the temperature cycle. Therefore, it can
be concluded that while the nanostructure is reestablished, the system evolution upon temperature
changes is characterized by a certain degree of hysteresis.
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The overall trend described by the DLS measurements of the nanoparticles hydrodynamic radius
can be extrapolated from results reported in Table 2. Essentially, the nanoparticle average size does
not vary significantly upon temperature changes.
Table 2. Average hydrodynamic radius (Rh) and polydispersity index (PdI) of the cubosome
formulation at different temperatures.
T (°C) Rh (nm) PdI
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3.2. Rhodamineꢀconjugated liquid crystalline nanoparticles photophysical characterization and
HeLa cells imaging
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The photophysical properties of PF108ꢀR, as well as those of cubosomes, were investigated.
Rhodamine dyes belong to the class of xanthene dyes, characterized by an intense red fluorescence,
high brightness, excellent photostability, and the ability to modulate the properties of the dye
through substitution.44 A key property of these dyes is the equilibrium between an open, colored,
fluorescent quinoid form or a closed, colorless, nonfluorescent lactone, which can be controlled by
appropriate substitution.45 In particular, rhodamine B (bearing two diethyl amino substituents) is
one of the most common derivative.
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The emission/absorption properties of PF108ꢀR were investigated in water and chloroform to test its
photophysical features in media of different polarities. In both solvents the PF108ꢀR has a strong
absorption band at 560 nm (ε = 30660 Mꢀ1cmꢀ1, 31770 Mꢀ1cmꢀ1 in water and chloroform,
respectively) and a shoulder at 522 nm (ε = 13100 Mꢀ1cmꢀ1, 9950 Mꢀ1cmꢀ1 in water and chloroform,
respectively). Upon excitation at 522 nm in water, an emission band at 594 nm is generated.
Conversely, the excitation at the same wavelength in chloroform generates a strong emission band
at 584 nm. The quantum yields measured were 0.16 and 0.58 respectively in water and chloroform.
As expected, water has a quenching effect on the rhodamine fluorescence. The shift of the emission
to higher energy could be explained considering the solvatochromic properties of the fluorophore.
The emission spectrum of the cubosome formulation stabilized with the PF108/PF108ꢀFA/PF108ꢀR =
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60/20/20 (wt %) mixture was also recorded after 1:100 dilution in water. Excitation at 522 nm resulted in
emission bands centered at 581 nm (see Figure 3). The position of the maximum emission wavelength is
similar to that observed for PF108ꢀR in chloroform. These results suggest that the rhodamine fragment
conjugated to the Pluronic F108, rather than protruding towards the bulk water, is hidden within the
polyethylenoxide corona surrounding the nanoparticle, thus experiencing a less polar environment. The
excitation spectrum of cubosomes is in accordance with PF108ꢀR absorption both in water and chloroform.
Due to the turbidity of the solution the determination of the quantum yield of the dye in the nanoparticles
was not possible.
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The uptake of cubosome nanoparticles was investigated by fluorescence microscopy. After 4 h incubation,
faint diffuse fluorescence was distributed within cytoplasm of HeLa cells, whereas in untreatedꢀcontrol cells
no fluorescence was detected (Figure 4). However, it was not possible to determine whether the dye
molecules were localized in specific cell compartments.
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3.3. Monoolein and Docetaxel level in cubosome formulations and cytotoxic activity (MTT assay)
The amount of MO (expressed as mg/mL of dispersion) and Docetaxel (DTX, ꢀg/mL) in different
cubosome formulations was monitored by HPLC. The formulations analyzed were the traditional
cubosomes formulation stabilized with PF108 (CUB), the cubosomes formulation stabilized with
the PF108/PF108ꢀFA/PF108ꢀR = 60/20/20 (wt %) mixture (CUBꢀR), and the latter formulation also
loaded with DTX (CUBꢀR/DTX).
Monoolein content in CUB, CUBꢀR, and CUBꢀR/DTX formulations was 33 ± 3, 35 ± 2, and 29 ± 2
mg/mL, respectively. The CUBꢀR/DTX formulation was loaded with 328 ± 25 ꢀg/mL of drug, with
a DTX/MO molecular ratio of 1/200. The encapsulation efficacy (EE%) was 103 ± 13 %.
These formulations were tested for cytotoxicity (MTT assay) in HeLa cells. Figure 5 shows the
viability, expressed as % of the control, induced in HeLa cancer cells in the presence of the
nanoparticle formulations at concentration 1:500 (2 ꢁL/mL of medium) after 4 h of incubation. The
cytotoxic effect of DTX 10 ꢀM (see paragraph 2.10) was also determined at 4 hꢀincubation for
comparison. Treatment with CUB and CUBꢀR formulations did not induce a significant reduction
in cell viability, in comparison with control cells (Ctrl). Differently, the incubation with CUBꢀ
R/DTX particles induced a statistically significant decrease in HeLa cell viability (36 % of
reduction) with respect to the control as a result of the presence of the anticancer drug. A significant
cytotoxic effect (27 % reduction in cell viability) was also observed in cells treated with the
anticancer drug (DTX) directly added to the cell culture. Remarkably, the HeLa cell treatment with
DTXꢀloaded cubosomes (DTX dose of approximately 0.8 ꢀM) induced a superior cytotoxic effect
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compared to the molecularly dispersed anticancer drug tested at much higher (12.5ꢀfold)
concentration.
DTX is one of the most effective anticancer agents and has activity against a wide range of cancers.
The growth inhibitory effect of DTX was examined in several cancer cell lines and the IC50 (50%
inhibitory concentration) values of this taxoid were in the nanomolar range at longꢀtime incubation
(72 h)46,47 The 24 and 48 h IC50 values of the free DTX in HeLa cells were previously found to be
5.01 and 2.62 ꢀg/mL, respectively.48 Here, the higher cytotoxicity at 4 hꢀincubation of DTX
administered via the nanoparticles (0.66 ꢀg/mL) with respect to free drug molecules (8.08 ꢀg/mL)
can be explained calling into play different uptake mechanisms. Indeed, cubosomes can deliver
DTX into cells via endocytosis, while free DTX can enter cells via passive diffusion with inherent
lipophilicity, and various ABC efflux transporters, expressed at the cellular membrane, can
minimize its effective penetration by efficiently eliminating the drug from the cell.49
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3.4 Cubosomes versus multiꢀlamellar liposome hydrophobic volume
One important feature that distinguishes cubosomes (reverse bicontinuous cubic liquid crystalline
nanoparticles) from liposomes (their lamellar counterpart) is the higher hydrophobic volume of the
former. Since cubosomes are mainly proposed as nanocarriers for hydrophobic molecules of
pharmaceutical interest as an alternative to liposomes, this fact may be extremely relevant in view
of their theranostic applications. Given the very convoluted volumes occupied by lipid chains in
bicontinuous morphologies compared with the shells of liposomes, it is tempting to assume that the
former contains a far larger hydrophobic volume (suitably) normalized than the latter. Surprisingly,
quantification of the hydrophobic volumes of bicontinuous cubosomes with respect to multiꢀ
lamellar liposomes has never been reported.
Assume for simplicity that we have two nanoparticles composed of monoolein in water whose outer
diameter is 100 nm. Assume the liposome is composed of N concentric spherical monoolein
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bilayers, composed of pairs of monolayers, each with hydrophobic thickness of 17 Å, corresponding
to the likely chain length in molten monoolein assemblies. Further, assume that adjacent bilayers
are separated by polar films 16 Å thick, so that each monolayer has an associated polar layer of
thickness of 8 Å (corresponding to dimensions in the fully hydrated lamellar mesophase).50 Each
repeat unit of chains, monoolein headꢀgroups, and water is therefore 50 Å thick. The hydrophobic
volume is then calculated as the chain contribution to the total volume within N concentric nested
shells, whose outermost radius is scaled such that the outermost monolayer and associated polar
layer has diameter of 100 nm. These dimensions imply that the hydrophobic volume fraction within
the liposome (whose total volume is 5.24 × 105 nm3) is approximately 0.18, 0.33, 0.445, 0.53, 0.59
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nm3 for N = 1, 2, 3, 4, 5 bilayers, respectively. The exposed outermost monolayer has area 2.83 ×
104 nm2.
The structural dimensions of a typical cubosome can be estimated as follows. We assume the
cubosome is a single crystalline domain of the ꢃꢂ3ꢂ phase, also contained within a spherical shell
of diameter 100 nm (cubosome nanoparticles, although characterized by a cubic inner
nanostructure, can be spherical in shape, as those shown in Fig. 1). We choose a typical lattice
dimension of the ꢃꢂ3ꢂ unit cell, namely 130 Å, and fix the chain length to be the same as that in
the liposome, 17 Å (the polar dimensions are then fixed by those parameters). The resulting total
exposed area is then 1.68 × 105 nm2 (equal to the summed area of each monolayer displaced 17 Å
from the bilayer midꢀsurface, whose geometry is assumed to lie on the Pꢀsurface, the cubic periodic
ꢃꢂ3ꢂ minimal surface). Surprisingly, the membrane area exposed to water in the liposome is
substantially larger than the total area in the cubosome. The corresponding hydrophobic volume
fraction within the 100 nm cubosomal sphere is 0.59 nm3, higher than that of the liposome with up
to 5 bilayers (see Table 3).
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Table 3. Cubosomeꢀtoꢀliposome hydrophobic volume ratios (Vh) as a function of the number of
concentric bilayers in the liposome (see also the text).
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Vh ratios
3.22 1.79 1.33 1.12 1.00
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4. Conclusion
In this paper we demonstrated that cubosomes loaded with an anticancer drug, namely Docetaxel,
can be formulated decorating their nanoparticle surface with both a cancer cell targeting ligand and
an imaging probe. Worth noticing, this is the first time that Docetaxel is successfully loaded within
cubosomes, and that cubosomes surface is simultaneously decorated with targeting and imaging
moieties. To this aim, the commercial block copolymer Pluronic F108 (PF108), and its folateꢀ and
rhodamineꢀconjugated counterparts were used in a 60/20/20 (wt %) mixture to stabilize the lipid
liquid crystalline dispersion. SAXS, DLS and cryoꢀTEM investigation, at the temperatures range
explored, confirmed the cubic bicontinuous inner structure of the nanoparticles and showed the
physicochemical aspects (morphology and size) akin to those of traditionally prepared cubosome
dispersions. Photophysical measurements suggested that the fluorogenic fragment, although
conjugated to the terminal ethylenoxide moiety of PF108, was very likely exposed to the less polar
environment constituted by the polyethylenoxide corona surrounding the nanoparticles, rather than
protruded toward the bulk water. This formulation, not loaded with the drug, was successfully used
to image living HeLa cells. Besides, viability tests revealed a significant cytotoxic effect (more than
one order of magnitude larger than the molecularly dispersed drug) of the Docetaxel loaded
nanoparticles against HeLa cells. These results evidenced that loading of cubosomes with a
Docetaxel dose able to induce cytotoxic effects on HeLa cells and decoration of their surface with
40% of conjugated PF108 did not compromise the intrinsic nature of these peculiar nanoparticles
and their cellular uptake. We have also evaluated the differences between cubosomes and multiꢀ
lamellar liposomes in terms of surface area and hydrophobic volume. Under the imposed constraints
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(same molecular building block and nanoparticles volume, cubosome nanostructure characterized
by the ꢃꢂ3ꢂ space group with a lattice parameter of 130 Å, and multiꢀlamellar liposome
constituted of adjacent bilayers), these calculations demonstrated that the hydrophobic volume of
cubosomes is more than three times larger than that of singleꢀbilayer liposomes, but the cubosome
to liposome hydrophobic volume ratio rapidly converge to unity as the number of the bilayers in the
liposome increases. It is worth noting that this ratio will be even larger if the liposome contains nonꢀ
adjacent bilayers. Remarkably, the cubosome surface exposed to water is about 60 % with respect
to that of the liposome.
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Taken together, these findings demonstrate the good performance of cubosomes as peculiar
nanocarriers for hydrophobic molecules with therapeutic/diagnostic relevance, and encourage the
investigation of cubic bicontinuous liquid crystalline dispersion for possible applications as
theranostic tools.
5. Appendix: Geometric calculations
Denote the chain length by d, t the thickness of the polar layer (containing water plus head groups)
and the lattice parameter in the cubic mesophase by a. Assume that outer shape of the liposome and
the cubosome is a sphere of outer diameter L.
We assume that all interfaces, lining the monolayers at the chainꢀwater interface and the free chain
ends, are parallel. We then use the formalism described elsewhere1 to deduce dimensions. All
dimensions are calculated from the geometry of the imaginary surface S that run between the chain
ends in opposed monoꢀlayers, at the midꢀsection of the hydrophobic shell for each bilayer. The
“outer” surfaces, exposed to water, are parallel to S, and displaced by d from S. We have two
formulae derived from the geometry of parallel surfaces, both related to the mean and Gaussian
curvatures of S, denoted HS and KS respectively. One is for the area of the surface Sx, parallel to S,
displaced by a distance x:
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ꢓ
ꢍ
ꢐ
ꢄꢋꢌꢄ ꢎꢏ = ꢄꢋꢌꢄꢍꢎꢐꢍ1 + 2ꢑꢏ + ꢒꢏ ꢐ
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6
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8
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(where the sign of x depends whether it is on the outside or the inside of the surface). The second
describes the volume of the film lyingꢀin between S and Sx:
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ꢒꢏꢓ
ꢍ ꢐ
ꢍ ꢐ
ꢔꢕꢖꢗꢂꢌ ꢎꢘ = ꢄꢋꢌꢄ ꢎ ꢏꢍ1 + ꢑꢏ +
ꢐ
3
480
481
482
483
In the liposome, those surfaces are parallel spheres, and if it is made of N bilayers, there are N such
spheres, each one with radius:
ꢚ
ꢍ ꢐ
ꢍ
ꢐꢍ
ꢐ
ꢋ ꢙ = − 2ꢙ − 1 ꢅ + ꢛ
2
484
485
486
These dimensions impose mean and Gaussian curvatures on bilayer i equal to:
ꢍ ꢐ
ꢑ ꢙ = ꢋ ꢙ
ꢍ ꢐꢜꢝ
ꢜꢓ
ꢍ ꢐ
ꢒ ꢙ = ꢋꢍꢙꢐ
487
488
The exposed area of the liposome is that of the outermost spherical monoꢀlayer (displaced distance t
3
4
489
inwards from the 100 nm external shell), namely 4πr(1)2. The total volume is ꢞꢛꢕꢛꢄꢖ 3 ꢟꢍ2ꢚꢐ . The
=
490
491
492
hydrophobic (chain) volume in the liposome is determined by summing over all 2N monolayers, N
displaced x and N displaced ꢀx:
ꢤ
ꢤ
ꢣ
ꢣ
ꢪ
ꢍ ꢐ
∑
ꢍ ꢐꢓ
ꢍ ꢐ
ꢍ ꢐ
493
494
ꢔꢕꢖꢗꢂꢌ ꢠ = 4ꢟꢅ ꢧꢫꢝ ꢋ ꢙ ꢡꢢ1 + ꢑ ꢙ ꢅ + ꢍ ꢐꢨ + ꢢ1 − ꢑ ꢙ ꢅ + ꢍ ꢐꢨꢩ
(1)
ꢥꢦ ꢧ
ꢥꢦ ꢧ
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The corresponding calculation for the cubosome follows directly from the normalised surface to
volume ratio, or “homogeneity index” characteristic of a particular folded surface geometry:
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ꢄꢋꢌꢄꢬꢓ
ℋ ∶=
,
ꢝ
ꢓ
ꢍ
ꢐ
−2ꢟꢭ ꢔꢕꢖꢗꢂꢌ
498
499
500
501
where ꢭ is the Euler characteristic of the hyperbolic bilayer within the particle. If the cubosome is
contained within a spherical shell of diameter L and the membrane within folds onto a periodic
minimal surface forming a cubic lattice of lattice edge a, whose topology per unit cell is ꢭ0, then
ꢯ
502
503
ꢭ = ꢭꢮ ꢰꢱꢰꢲꢳ. The radius of curvature of the minimal surface is given by the expression:
ꢵ
ꢴ
1
ꢸ3
,
ℋ
ꢋꢶ =
ꢷ
−2ꢟꢭ
504
505
506
507
which corresponds to minimal surface curvatures ꢑꢶ = 0 and ꢒꢶ = −ꢋ−ꢶ2.50 It follows that the
minimal surface area in the cubosome,
2
1
2
3
ꢍ
ꢐ
ꢄꢋꢌꢄꢶ =
ꢹ
ℋꢞꢛꢕꢛꢄꢖ −2ꢟꢭ
ꢺ
,
508
509
510
so that the exposed area (at the hydrophobic/hydrophilic interface) is:
ꢄꢋꢌꢄꢌꢏꢻꢕꢼꢌꢅ = 2ꢄꢋꢌꢄꢶ ꢢ1 − ꢒꢅ2
ꢨ
,
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512
and the hydrophobic volume is
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ꢅ
ꢔꢕꢖꢗꢂꢌꢶ = 2ꢄꢋꢌꢄꢌꢏꢻꢕꢼꢌꢅ ꢽ1 +
ꢾ
(2)
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3ꢒ
ꢶ
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517
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If the internal bilayer geometry within the cubosome corresponds to the ꢃꢂ3ꢂ bicontinuous
mesophase, the relevant data are ℋ = 0.7163 and ꢭ0 = −4.51 The ratios of hydrophobic volumes
are calculated from the results of equations (1) and (2) above.
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Acknowledgments
Karin Schillén and Tommy Nylander are kindly thanked for fruitful discussions. The authors
acknowledge financial support from the European Commission under the Seventh Framework
Program by means of the grant agreement for the Integrated Infrastructure Initiative N. 262348
European Soft Matter Infrastructure (ESMI). Financial support by MIUR (Project PRIN
2010BJ23MN_002), Fondazione Banco di Sardegna and Regione Autonoma della Sardegna (CRPꢀ
59699) is gratefully acknowledged. Sardegna Ricerche Scientific Park (Pula, CA, Italy) is
acknowledged for free access to facilities of the Nanobiotechnology Laboratory. The cryoꢀTEM
work was performed at the Technion Laboratory for Electron Microscopy of Soft Matter, supported
by the Technion Russell Berrie Nanotechnology Institute (RBNI).
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