Lyotropic Liquid-Crystalline Nanosystems as Drug Delivery Agents for

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Lyotropic Liquid Crystalline Nanosystems as Drug Delivery Agents for 5-Fluorouracil: Structure and Cytotoxicity Paola Astolfi, Elisabetta Giorgini, Valentina Gambini, Barbara Rossi, Lisa Vaccari, Francesco Vita, Oriano Francescangeli, Cristina Marchini, and Michela Pisani Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03173 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Lyotropic Liquid Crystalline Nanosystems as Drug Delivery Agents for 5-Fluorouracil: Structure and Cytotoxicity Paola Astolfi,1 Elisabetta Giorgini,2 Valentina Gambini,3 Barbara Rossi,4 Lisa Vaccari,4 Francesco Vita,1 Oriano Francescangeli,1 Cristina Marchini,3 Michela Pisani1* 1

Dipartimento SIMAU, Università Politecnica delle Marche, via Brecce Bianche, 60131

Ancona, Italy 2

Dipartimento DISVA, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona,

Italy 3

Dipartimento di Bioscienze e Biotecnologie, Università di Camerino, Via Gentile III da Varano,

62032 Camerino, Macerata, Italy 4

Elettra - Sincrotrone Trieste S.C.p.A., S.S. 14 - km 163.5, 34149 Basovizza, Trieste, Italy

ABSTRACT. Lyotropic cubic liquid crystalline systems have received increasing attention due to their unique microstructural and physicochemical properties as efficient nanocarriers for drug delivery. In this study we report the preparation and characterization of bulk phases and cubosome dispersions of phytantriol loaded with the anticancer drug 5-fluorouracil, in neutral and anionic forms. In both cases, a Pn3m cubic phase was observed. The phytantriol phase

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behavior can be influenced by the addition of ionic agents and to this purpose a positively charged lipid, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride salt (DOTAP), was included in the studied formulations. It was found to induce a variation of the spontaneous membrane curvature of the phytantriol lipid bilayer generating a transition from the Pn3m to the Im3m cubic phase. When 5-fluorouracil, in its anionic form (5-FUs), was encapsulated in these latter systems, a further transition to the HII hexagonal phase was observed as a consequence of the formation of a complex phytantriol/DOTAP/5-FUs. The physico-chemical characterization was performed with various complementary techniques including Synchrotron Small Angle X-Ray Scattering, Dynamic Light Scattering, Attenuated Total Reflection Fourier Transform Infrared and UV Resonant Raman spectroscopies. Encapsulation of 5-fluorouracil in the corresponding nanodispersions was evaluated and their in vitro cytotoxicity was assessed in MDA-MB-231 cell line. Phytantriol cubosomes containing 5fluorouracil showed a higher toxicity compared to the bare drug solution and hence they represent potential nanocarriers in the delivery of 5-fluorouracil for cancer therapy. INTRODUCTION The design and development of smart, efficient and biocompatible vectors for drug delivery is among the main goals of research in nanomedicine. Over the last few decades, soft delivery systems based on lyotropic liquid crystals (LLC) have received increasing attention both in scientific and applied research communities. In particular, because of their highly ordered internal structures, inverse bicontinuous cubic and inverse hexagonal mesophases have been investigated for their ability to incorporate different therapeutic agents, ranging from small drug molecules to biomacromolecules, and to control and/or sustain their release.(1-3) In fact, drugs


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must diffuse from intricate three-dimensional networks of aqueous nanochannels, in which both tortuosity and pore size contribute to delayed release. The typical lipids forming bicontinuous inverted phases are amphiphile molecules that, due to intermolecular forces, self-assemble in aqueous environments giving periodic liquid crystalline structures with coexisting separate hydrophobic and hydrophilic regions. These structures are characterized by a curved, bicontinuous lipid bilayer extending in three dimensions, wrapped around two interpenetrating, but non-contacting, networks of aqueous nanochannels. The amphiphilic lipids most commonly used in LLC research are glyceryl monooleate (GMO) and glyceryl monolinoleate (MLO), both forming a bicontinuous cubic phase in excess water. However, the ester-based structure of these lipids may limit their practical application because of undesiderable hydrolysis of the ester linkage within the monoglyceride headgroups upon long term storage or as a result of enzymatic breakdown. Phytantriol (3,7,11,15-tetramethylhexadecane-1,2,3-triol, PHYT, Chart) has the same physicochemical characteristics of GMO or MLO but with a relatively higher resistance to hydrolysis due to the lack of ester linkages and the presence of saturated phytanyl backbone.(4,5) Moreover, being a cosmetic ingredient, PHYT is easily available and relatively cheap, , so that it can be considered a better candidate than GMO or MLO in drug delivery research. Phase behavior of PHYT has been extensively studied:(4,5) just like GMO, it forms an inverse bicontinuous cubic Pn3m phase in excess water at room temperature and a reverse hexagonal phase HII at higher temperatures. The cubic to hexagonal phase transition for PHYT/water system occurs at lower temperature (40-60 °C)(4,6) as compared to GMO/water system (80 °C). The practical implementation of cubic or hexagonal phase gels as drug carriers is limited by their high viscosity, but they can be fragmented, in the presence of steric stabilizers, to obtain


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nanoparticles that retain the complex internal structure of the “parent” bulk phase with much larger surface area and much reduced viscosity. Dispersions from the hexagonal and cubic phases are known as hexosomes and cubosomes, respectively and they are currently studied as delivery systems alternative to the more common liposomes. Their main advantages lie in the inner structure, which determines a high interfacial surface able to encapsulate and protect higher amounts of both hydrophilic and hydrophobic drugs.(7,8) Recently, these nanostructures have received considerable attention as anticancer drugs delivery systems and various antineoplastic molecules have been encapsulated in GMO and PHYT-based lipid formulations in order to improve their stability, biodistribution and therapeutic efficacy. For example, GMO or PHYT cubosomes were demonstrated to improve the therapeutic potential of doxorubicin,(9,10) docetaxel,(11) paclitaxel(12) or other natural compounds(13) for the treatment of various kind of cancers. Among anticancer drugs, 5-fluorouracil (5-FU) is one of the major agents clinically used, alone or in combinations with other chemotherapy agents, for the treatment of stomach, colorectal, gastric, head and neck, and breast cancers. In particular, 5-FU is often used for the treatment of Triple Negative Breast Cancer (TNBC), one of the most aggressive subtypes of breast cancer, associated with the worst prognosis.(14) 5-Fluorouracil is an antimetabolite of the pyrimidine analogue type which, due to its structure, interferes with nucleoside metabolism and can be incorporated into DNA and RNA, leading to cytotoxicity and cell death. The current mode of treatment with intravenous administration is often inconvenient. In fact, due to its high rate of metabolism in the body and to its short half-life (10-20 min),(15) costly and repeated doses are required to maintain a therapeutic serum concentration. In addition, 5-FU is rapidly absorbed


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through blood capillaries into systemic circulation resulting in low levels of drug near the tumor sites and consequent loss of efficacy along with higher toxicity.(16) Therefore, 5-FU requires an effective delivery vector for appropriate therapy and some nanosystems (liposomes and niosomes, hydrogels, polymers, solid lipid nanoparticles, etc.) have been researched.(17-20) Moreover, 5-FU was encapsulated in GMO cubosomes(21) and, on the basis of these preliminary results, also PHYT cubosomes could be good candidates as drug delivery systems to improve the efficacy of low doses of 5-FU. Within this frame, bulk cubic phases and cubosomes dispersions of phytantriol were prepared, in the presence of the steric stabilizer Pluronic F 127, and loaded with the antineoplastic agent 5fluorouracil, in neutral (5-FU) and anionic form (5-FUs) (Chart). Moreover, PHYT cubic phases were co-formulated with a positively charged phospholipid, N-[1-(2,3-dioleoyloxy)propyl]N,N,N-trimethylammonium chloride salt (DOTAP), which can modify the lipid packing, the interfacial curvature and eventually promote phase transitions. These modifications, together with the presence of a cationic charge, could increase the drug entrapment capacity of these structures, especially in the case of the anionic drug and, at this regard, 5-FUs (Chart) was encapsulated. All these systems were characterized by Synchrotron Small Angle X-Ray Scattering (SAXS), Dynamic Light Scattering (DLS), Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) and UV Resonant Raman (UV-RR) spectroscopies and the possible influence of the drug incorporation in the lipid matrix was assessed. Finally, entrapment efficiency of 5-FUs in PHYT and PHYT/DOTAP dispersions, obtained upon fragmentation of the bulk gel phases, was determined and the in vitro antitumor activity in


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human breast cancer MDA-MB-231 cells, showing characteristics of TNBC, was evaluated by means of viable cells assay.

OH OH HO PHYT O N+

O O O

DOTAP

H

O

O

x

Cl-

H

O

xOH

y

Pluronic F127 O

O F 5 6

4 3N 1

H

2

N H

O H

F

N N

3N

and/or

1

O

F N H

O

5-FU

O

5-FUs

Chart. Chemical structures of: Phytantriol (PHYT); N-[1-(2,3-Dioleoyloxy)propyl]-N,N,Ntrimethylammonium chloride salt (DOTAP), Pluronic F127 (x = 100, y = 65); 5-fluorouracil in neutral (5-FU) and anionic (5-FUs) form. EXPERIMENTAL SECTION Chemicals and Reagents. Phytantriol (PHYT) was purchased from TCI Europe; Pluronic F127 and 5-fluorouracil (5-FU) from Sigma-Aldrich. 5-FUs was also used as a 50 mg/mL aqueous solution (NaOH/HCl pH 9, TEVA Pharmaceutical Industries Ltd.) and was kindly provided by Azienda Ospedaliera Universitaria

Ospedali Riuniti Ancona. N-[1-(2,3-

Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride salt (DOTAP) was purchased from


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Avanti Polar Lipids. Chloroform and methanol were obtained from Sigma-Aldrich. All chemicals used in this study were of analytical grade and used without further purification. All solutions were prepared with Milli-Q water (Merck Millipore). Preparation of Blank and Drug-loaded non Dispersed Lipid Phases. Samples of blank bulk LLC phases were prepared by co-dissolving PHYT (50 mg) and Pluronic F127 (10% w/w of PHYT) in chloroform. The solvent was evaporated under a stream of nitrogen and the mixture dried under vacuum. Water (200 µL) was added and the mixture was vortexed to achieve a homogeneous state and equilibrated at room temperature for 24 hours to obtain the cubic phase gel. PHYT/DOTAP blank bulk gels were prepared by adding DOTAP (3-15 mol%) to PHYT and F127 (10% w/w of total lipid) in chloroform. 5-Fluorouracil loaded cubic gel was prepared by adding 200 µL of 50 mg/mL 5-FU alkaline solution (5-FUs) or of 10 mg/mL 5-FU neutral one, instead of water, to the dried PHYT/F127 or PHYT/DOTAP/F127 mixtures prepared as described for the blank gel After vortexing, mixtures were equilibrated at room temperature for 24 hours. PHYT/F127/DOTAP (5 mol%) formulation was used for vibrational analysis and for the characterization and entrapment efficiency determination, as well as for in vitro cytotoxicity test, of the corresponding dispersion. Preparation of Nanostructured Lipid Dispersions. To prepare the lipid dispersions, 1800 µL of water were added to the cubic gels and the samples were sonicated by a probe sonicator (Sonic Vibracell) for 10 min in pulse mode (1 s pulses interrupted by 1 s breaks) at 50 % of the maximum power.


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Small Angle X-Ray Scattering (SAXS) Measurements. Synchrotron SAXS measurements were performed at the SAXS beamline, Elettra Sincrotrone Trieste (Trieste, Italy). 2D diffraction patterns were recorded by a Dectris Pilatus 1M detector using a wavelength of 1.54 Å (8keV). The sample to detector distance was 1279 mm; a vacuum chamber was in place between the sample and the detector to get rid of air scattering. The setup covered the q range from about 0.15 to 5 nm-1 ( q = 4π sinθ / λ , where λ is the wavelength and 2θ is the scattering angle). Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Measurements. ATR-FTIR measurements were carried out at the infrared SISSI beamline, Elettra Sincrotrone Trieste (Trieste, Italy), by using the MIRacle Single Reflection ATR box (PIKE technologies) with diamond crystal, equipped with a Vertex 70 interferometer (Bruker Optics GmbH) and a Deuterated TriGlycine Sulfate (DTGS) detector. Samples were deposited onto the diamond crystal and submitted to a continuous stream of nitrogen gas. ATR-FTIR spectra were collected at room temperature every 5 seconds until sample dehydration, followed by vanishing below the detection limit of the combination band of bending and liberation water modes, centred at about 2100 cm-1. Each spectrum was acquired in the spectral range 5000-550 cm-1 and averaged over 128 scans. A spectral resolution of 4 cm-1 was applied. Before each sample acquisition, the background spectrum was collected on the clean diamond crystal, under the same conditions. Raw spectra were corrected for carbon dioxide and water vapour using OPUS 7.5 routine (Bruker Optics GmbH), and then vector normalized in the entire spectral range of acquisition. UV Resonant Raman Scattering Measurements UV Resonant Raman spectra were collected at the IUVS beamline, Elettra Sicrotrone Trieste (Trieste, Italy) by exploiting the experimental set-up already described.(22) All the samples were placed into a 10 mm path quartz cuvette for


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Raman measurements. UV Raman spectra were excited at 266 nm and recorded in a backscattered geometry by using a triple stage spectrometer (Trivista, Princeton Instruments) with a spectral resolution of about 6 cm-1. In order to prevent any possible photodecomposition of the sample due to the prolonged exposure of the sample to UV radiation, the cuvettes were horizontally moved during the measurements in order to vary the illuminated sample volume through the exciting radiation beam. UV Raman spectra of all the samples were acquired at fixed temperature (20 °C). Particle Size and Zeta Potential Measurements. The particle size (Z-average), polydispersity index (PDI) and zeta potential values (ξ) of the dispersions were determined using the Malvern Zetasizer Nano ZS (Malvern Instruments GmbH). Samples were properly diluted and measured in PMMA cuvettes or DTS1070 capillary cells for the determination of size and zeta potential, respectively. All measurements were performed in triplicate at 25 °C on three independent samples. Data were analyzed using Dispersion Technology Software V5.03 provided by Malvern Instruments. Determination of 5-Fluorouracil Content in Cubosomes. The total amount of 5-FUs (free and incorporated) was determined after addition to an aliquot of the drug loaded dispersions of methanol (1:100) to dissolve the lipid. The resultant solutions were spectrophotometrically assayed at λmax = 266 nm for total 5-FUs content using methanol as the blank. Free 5-FUs (nonincorporated in the cubosomes) was separated by ultrafiltration method: another aliquot of the same dispersions was centrifuged in an Amicon Ultra (Amicon Ultra-4, 10 000 MW, Millipore) 10,000 rpm for 15 min. The obtained filtrates were diluted (1:100) with methanol and spectrophotometrically read at λmax = 266 for free 5-FUs.


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The encapsulation efficiency EE (%) was calculated by the following equation: % =

− 100

where W(T) is the total drug in cubosomes, and W(F) is the free drug in the filtrate. All the experiments were repeated at least three times and measurements were run in triplicate. Cell Culture. Human breast cancer MDA-MB-231 cells were obtained from American Type Culture Collection (Rockville, MD) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA). Cells were grown in a humidified atmosphere with 5% CO2 at 37 °C and were sub-cultured every 2–3 days. MDA-MB-231 showed characteristics of TNBC. Cytotoxic Activity (MTT Assay). The cytotoxic potential of 5-FUs encapsulated in two different formulations of PHYT/F127 and PHYT/F127/DOTAP on MDA-MB-231 cells viability was evaluated by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide Sigma Aldrich, St. Louis, MO] assay, which is based on the conversion of MTT to MTT formazan by mitochondrial enzymes (tetrazolium salt reduction assay).(23) Briefly, 7x103 cells/well (MDA-MB-231) were seeded in octuplicate in 96 wells plates in complete medium (DMEM supplemented with FBS, 100 µL) and incubated at 37 °C in a 5% CO2 atmosphere. After 24 hours, fresh medium containing appropriate concentrations of each formulation or 5FUs was added: empty and 5-FUs loaded dispersions were tested in the 12.5-100 µg lipid/mL concentration range, whereas bare 5-FUs at the 1-8 µg/mL concentration range, corresponding to the quantities present in each dispersion. Pluronic F127 was also tested at the maximum concentration of 10 µg/mL corresponding to F127 concentration in the 100 µg lipid/mL


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dispersion. After 24 and 72 hours of incubation, cell viability was determined by adding MTT (0.5 mg/mL) to each well and incubating the cells at 37 °C for additional 4 hours. The culture medium was then removed, and formazan crystals were dissolved in 100 µL of DMSO per well. Finally, cell viability was determined by reading the absorbance of each well at 540 nm using a 96-well microplate reader. The cytotoxicity of the compounds was reported as percentage of viable cells relative to control cells. Quantitative data were presented as means ± SEM from three independent experiments. The significance of differences was evaluated with one way ANOVA followed by Bonferroni Multiple Comparison Test. Statistical analysis was carried out with GraphPad Prism5 Software (San Diego, CA, USA). p ≤ 0.05 was used as the critical level of significance. RESULTS AND DISCUSSION Structural Investigation of PHYT and PHYT/DOTAP Cubic and Hexagonal Phases. Effect of 5-Fluorouracil Encapsulation. SAXS diffraction experiments were carried out to investigate the nanostructure of PHYT liquid crystal phases and the possible geometrical and topological modifications(24) induced by incorporation of 5-fluorouracil, both neutral and in the anionic form. In order to have bulk phases with the same composition of dispersed nanoparticles (see below), the commonly used block co-polymer Pluronic F127, was added to PHYT bulk lipid. Incorporation of 10% w/w F127 had no effect on the observed mesophases and on the lattice parameter of pure PHYT (data not shown). F127 is likely adsorbed at PHYT surface and do not partition into the lipid bilayer since the branching of methyl groups of the phytyl chains may represent an unfavorable environment for F127 insertion.(5) Figure 1 shows the onedimensional scattering intensity I of PHYT bulk cubic phase vs the scattering vector qhkl.


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Diffraction peaks ((110), (111), (200), (211), (220), and (300)) were observed at qhkl values in accordance with the Pn3m space group.(4) The cubic lattice parameter a was calculated through the linear fit of the plot qhkl vs ( h 2 + k 2 + l 2 ) (Figure 1, inset) and it was found to be 6.51 nm, in agreement with the data reported in the literature for the same system.(25) The Pn3m phase can be described as a continuous curved lipid bilayer with a complex 3-D periodic cubic structure separating two continuous non-intersecting water networks, each one made of channels meeting in four way junctions at the tetrahedral angle of 109°.(26) The radius of water channels was estimated to be rw = 1.14 nm by using the relation:(25)

[

]

rw = (− σ / 2πχ ) a − l 1/ 2

where l is the lipid length (ca. 1.4 nm) and σ and χ are topological constants characteristic of a given cubic phase (for Pn3m structure σ = 1.919 and χ = -2). The correspond diameter dw=2·rw is 2.28 nm, which is in accordance with reference data.(25,27) 3.0

Pn3m

(220)

-1

qhkl (nm )

2.5

(110)

Intensity (arb. units)

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(111)

(211)

2.0 (200)

1.5

(110)

1.5

(111)

2.0

2.5

3.0

(h2+k2+l2)1/2

(200)

(211) (220) (221)

1.0

1.5

2.0

2.5

3.0

3.5

-1

q(nm )


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Figure 1. Synchrotron SAXS diffraction pattern of PHYT/F127 bulk cubic phase. In the inset, plot of the wave-vector qhkl vs (h2 + k2 + l2)1/2 for the observed Bragg reflections and its linear fit by a straight line with slope 2π/a. Addition of 5-FU and 5-FUs to PHYT mixture did not produce any effect on the structure of the cubic phase, as shown in Figure 2 (curves A and B, respectively). The lattice parameter for Pn3m was unchanged, ca. 6.5 nm for both samples. The small drug molecules can be easily accommodated in the aqueous nanochannels where they remain encapsulated, as confirmed by both ATR-FT IR measurements and by entrapment efficiency determinations (see below). However, hydrogen bonds or other intermolecular forces, responsible for the entrapment, are not sufficient to promote any phase transition.

Pn3m (110) (111)

Intensity (arb. units)

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(200) (210) (220)(221)

A

B 1.0

1.5

2.0

2.5

3.0

3.5

-1

q (nm )


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Figure 2. Synchrotron SAXS diffraction patterns of PHYT bulk phase after incorporation of 5FU (A) and 5-FUs (B). The effect of the addition of the positively charged DOTAP on the PHYT phase behavior was investigated by studying PHYT/DOTAP mixtures with different DOTAP contents (3-15 mol%). At DOTAP percentage ≤ 5 mol%, Pn3m and Im3m cubic phases coexisted, whereas only the Im3m cubic phase was detected at 8 mol% DOTAP. Thus, addition of DOTAP resulted in a mesophase characterized by a less negative interfacial curvature and this is likely due to the increased effective size of lipid headgroups and to the electrostatic repulsions between the charged DOTAP headgroups. Table 1 shows the variation in the structure and lattice parameter of PHYT with increasing DOTAP amount: the unit cell dimension of Pn3m phase increases from 6.51 nm in pure PHYT to 7.34 nm in 5 mol% DOTAP, and the lattice parameter of Im3m phase also increases as a function of DOTAP concentrations from 9.72 nm at 3 mol% to 11.38 nm at 8 mol% DOTAP. Both effects likely follow from an enlargement of the cubic phase structure, which is required to accommodate the more bulky DOTAP molecules. In the presence of higher DOTAP concentration (10 or 15 mol%), SAXS diffractograms are characterized by the absence of well-defined peaks, indicating the loss of the ordered structure due to the dominant electrostatic repulsion between charged lipid headgroups at higher loadings.


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Table 1. Phase structure and lattice parameters of PHYT/DOTAP at increasing amount of DOTAP Formulations PHYT/ DOTAP (3 mol%) PHYT/DOTAP (5 mol%) PHYT/DOTAP (8 mol%)

Space group

Cell Parameter, a(nm)

Pn3m Im3m Pn3m Im3m

7.31 9.72 7.34 9.87

Im3m

11.38

Incorporation of 5-FUs in PHYT/DOTAP systems induced the transition to a reverse hexagonal phase (HII, peak spacing ratio 1 : 3 : 4 ) regardless of DOTAP concentration, even for those formulations that did not show any ordered phase in the absence of drug (DOTAP concentration > 8 mol%). Clearly, drug loading resulted in a charge shielding of the cationic DOTAP headgroups by the negatively charged 5-FUs, thus leading to a larger negative curvature of the lipid interface and favoring the HII phase. Moreover, a shift to higher q values of the Bragg peaks associated to HII was observed with increasing DOTAP concentration, indicating a decrease of the lattice parameter: the unit cell dimension decreased from 4.8 nm to 4.3 nm with 15 mol% DOTAP, whereas the estimated water channel varied from 2.0 nm to 1.5 nm.


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Im3m + Pn3m

A Intensity (arb. units)

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Im3m B HII

C 0.5

1.0

1.5

2.0

2.5

3.0

3.5

-1

q (nm )

Figure 3. Synchrotron SAXS diffraction patterns of PHYT/DOTAP (5 mol%) bulk phase: unloaded (A), loaded with 5- FU (B) and loaded with 5-FUs (C). The solid arrows refer to Im3m peaks, the dotted arrows to Pn3m peaks. The reverse hexagonal phase was not observed when neutral 5-FU was encapsulated in PHYT/DOTAP matrix, confirming the critical role of 5-FUs charges in determining the phase structure. For example, an Im3m cubic phase, with a larger lattice parameter of ca. 10.79 nm, was detected when neutral 5-FU was added to the PHYT/DOTAP (5 mol%) bulk phase (Figure 3). Spectroscopic Characterization of PHYT and PHYT/DOTAP Cubic and Hexagonal Phases. Effect of 5-Fluorouracil Encapsulation. In order to have additional information at


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molecular level on the interactions between 5-fluorouracil, both neutral and in anionic form, and PHYT, vibrational spectroscopic analyses were carried out by means of ATR-FTIR (Figure 4) and UV Resonant Raman Spectroscopy (Figure 5) on the following systems: PHYT/5-FU, PHYT/5-FUs and PHYT/DOTAP/5-FUs. As indicated above, the steric stabilizer Pluronic F127 was included in all the formulations: even if not specifically indicated, its presence is implied and does not modify the IR and Raman vibrational profile of PHYT.


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Figure 4. Absorbance average spectra of: (a) 5-FU, PHYT and PHYT/5-FU; (b) 5-FUs, PHYT and PHYT/5-FUs, and (c) 5-FUs, PHYT/DOTAP and PHYT/DOTAP/5-FUs (1900-1000 cm-1 spectral range). The ATR-FTIR spectra of 5-FU, PHYT and 5-FU loaded PHYT samples in the 1900-1000 cm1

spectral region, are reported in Figure 4a. Concerning 5-FU spectrum, some considerations can

be drawn. The spectral band centered at 1245 cm-1 can be undoubtedly assigned to C-F stretching (υ(C-F)), in agreement with data reported in the literature for 5-FU molecules in Ar matrix.(28) In addition, the shoulder of this peak at 1225 cm-1 may reveal the coexistence of some dimeric forms.(29,30) The same dualism can also be inferred by the spectral shape of 5-FU in the 18001500 cm-1 spectral region, where characteristic C=O and C=C vibrations are found. Specifically, i the 1774 and 1723 cm-1 bands are mostly associated to carbonyl stretching of C2=O (υ(C2=O)) and C4=O (υ(C4=O)) groups,(28) while the mode centered at 1659 cm-1 is mostly due to C5=C6 bond (υ(C5=C6)).(31,32) The broad nature of this latter band let us suppose the presence of other contributions, such as the spectral component at ∼1670 cm-1 obtained by second derivative analysis (data not shown), likely due to some hydrogen bonding between 5-FU molecules themselves or with the solvent.(29,30) This is in agreement also with UV Raman scattering measurements, performed on hydrated 5-FU (Figure 5a). The Raman profile of 5FU (solid line) can be decomposed into three major contributions, centred at ~1714, ~1685 and ~1659 cm-1, assigned to υ(C2=O), υ(C4=O) and υ(C5=C6) respectively, strengthening the hypothesis that the solvent may play a role.


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Figure 5. UV Resonant Raman spectra of (a) PHYT/5FU, 5FU; (b) PHYT/DOTAP/5-FUs, PHYT/5-FUs and 5-FUs. The Figure reports also the principal spectral components as derived by a best-fit procedure carried out by using Gaussian curves: experimental curves are reported in grey and the cumulative fit curves in black. In PHYT ATR-FTIR spectrum, the bands corresponding to CH2/3 bending (1463 and 1375 cm1

) together with the absorptions due to C-OH vibrations (1150, 1091, 1061 and 1022 cm-1) can be


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detected (Figure 4a).(33) These bands are clearly discernible also in PHYT/5-FU spectrum, but they do not limit the possibility to verify the presence and the possible interactions of the drug in the system. In fact, in PHYT/5-FU ATR-FTIR spectrum some bands typical of 5-FU are clearly evident, such as the peaks at 1723 and 1249 cm-1. Conversely, a rearrangement of the spectral shape can be appreciated in the band at 1659 cm-1, which shifted to 1690 cm-1. Similar spectral variations were observed by FTIR spectroscopy when 5-FU was included in other kind of vectors, such as zeolites or silica nanoparticles, and it was attributed to the interaction of the 5FU ring, mostly localized at C5=C6, with the functional groups of the matrix.(31,32) Noteworthy, the PHYT/5-FU UV-Raman spectrum strictly resembles the 5-FU one, suggesting that the resonant Raman contribution arising from the matrix is almost negligible in the spectra of the complex (Figure 5a). This is not surprising considering that the exciting wavelength (266 nm) well matches with the energies of electronic transitions of the 5-FU ring. In particular, the bands of 5-FU at ∼1714 and ∼1685 cm-1 were detected almost unaltered in PHYT/5-FU Raman spectrum, whereas the band associated to C5=C6 stretching is more intense and shifted to 1649 cm-1. The same vibrational analysis was carried out on 5-FUs and 5-FUs loaded PHYT samples (Figure 4b). 5-FUs has ATR-FTIR peak positions comparable with 5-FU ones, with an additional peak due to the C=N bond arising from the resonance structures reported in Scheme 1. This latter peak is the result of several components, the most intense of which is centered at 1541 cm-1.(34) Additionally, the C-F moiety shows two bands with similar intensity at 1239 and 1221 cm-1, and two carbonyl stretching components can be also identified at 1733 and 1717 cm-1, beside the one centered at 1765 cm-1.


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O

O H

F

N N

O

O

N N

O

O F

N N H

H

F

O F

N N H

N N

O

O F

O H

F

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O

N N H

O

Scheme 1. Possible resonance structures for 5-FU anion. Encapsulation of 5-FUs in PHYT matrix was confirmed by the presence of some bands of the drug in the spectrum of PHYT/5-FUs system (Figure 4b). The existence of possible interactions between 5-FUs and PHYT can be likely deduced by the splitting of the 1221 cm-1 υ(C-F) band into two components at 1225 and 1214 cm-1, while the component at 1239 cm-1 falls below the detection limit. Moreover, the massif of the C=N stretching band is peaked at 1550 cm-1. The spectral region of C=O and C=C stretching is still characterized by a complex vibrational profile that broadens as a consequence of the encapsulation, as also observed in the Raman spectra (Figure 5b). Overall, these data suggest that the C=N and C-F bonds of 5-FUs play a pivotal role in the interaction with the matrix, while the C5=C6 portion is less involved. In order to improve the encapsulation of 5-FUs in PHYT cubic phases, the cationic lipid DOTAP (5 mol%) was added to the system. PHYT/DOTAP ATR-FTIR spectrum showes an additional band at 1743 cm-1 due to the C=O stretching of the carbonyl ester moiety in DOTAP (Figure 4c). In the complex PHYT/DOTAP/5FUs, the typical bands of 5-FUs were observed. The υ(C-F) at 1239 cm-1 is still detectable in the complex, while the 1221 cm-1 one red-shifts to 1214 cm-1. Moreover, the spectral band associated to C=N stretching modes is peaked in the


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complex at 1543 cm-1, with the relative spectral weight of the component centered at 1571 cm-1 significantly increases. When 5-FU is encapsulated in a more polar matrix, like DOTAP, a modification of the equilibrium among the different resonant forms of 5-FUs can be observed, suggesting that the encapsulation may involves the interaction through C=N and C-F molecular moieties of these different resonant forms. Moreover, an increase of the spectral intensity of C4=O stretching vibrations is clearly observed both in ATR-FTIR (Figure 4c) and in Raman (Figure 5b) spectra. Overall, from the spectral analysis it is clear that 5-FU and 5-FUs are encapsulated in both lipid matrices. In particular, a major involvement of the C5=C6 double bond of the ring can be hypothesized for the interaction of 5FU with PHYT, while the C=N and C-F bonds are more involved in the interactions responsible for the encapsulation of 5-FUs in PHYT. When DOTAP is co-formulated with PHYT, an additional interaction of the carbonyl moieties can be supposed. As a consequence of the complexity of the system, any additional interpretation of the spectral variations highlighted by both techniques cannot be considered reliable.

Characterization of PHYT and PHYT/DOTAP Dispersions. Effect of 5-Fluorouracil Encapsulation. PHYT cubic-phase nanoparticles were prepared by a top down approach,(5) using sonication to disrupt the cubic gel phase of lipid, blank or loaded with 5-FUs, and water in the presence of Pluronic F127(35 as a stabilizer. At a visual inspection, dispersions appeared as uniform opaque white mixtures with no visible large aggregates as confirmed by DLS measurements. For all these samples, the 5-FUs 50 mg/mL solution was used to increase the amount of drug entrapped in the nanoparticles.


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The internal structure of these colloidal dispersions was verified by SAXS measurements and it closely resembled that of the corresponding parent bulk phases, with very similar lattice parameters, even though dispersions’ Bragg diffraction peaks were much less intense (see Supporting Information). Size distribution, polydispersity index (PDI), and zeta potential (ζ) for cubosome formulations, together with entrapment efficiency for those loaded with 5-FUs, are presented in Table 2. The mean diameter for unloaded PHYT cubosomes was approximately 215 nm and encapsulation of 5-FUs resulted only in a slight increase of cubosome size. In both cases, polydispersity index was around 0.2, indicating the effective disruption of bulk gels without formation of large particles. Zeta potentials measured for the prepared formulations were approximately –34 mV and, since PHYT is a neutral molecule, this negative charge may be explained by preferential adsorption of hydroxyl ions at the lipid-water interface.(36,37)

Table 2. Size, Polydispersity index (PDI), Zeta potential (ξ) of PHYT and PYT/DOTAP nanoparticles, empty and loaded with 5-FUs, and Entrapment Efficiency (EE) of 5-FUs in PHYT and PHYT/DOTAP. All measurements were made in triplicate and the data presented are given in mean values ± SD.

Formulations

Size (nm)

PDI

ξ (mV)

PHYT

215.1 ± 1.7

0.224 ± 0.009

-33.8 ± 1.8

PHYT/5-FUs

227.1 ± 1.1

0.249 ± 0.014

-31.9 ± 0.8

PHYT/DOTAP

109.6 ± 0.9

0.220 ± 0.010

+36.8 ± 3.5

PHYT/DOTAP/5-FUs

187.2 ± 2.1

0.233 ± 0.008

+26.0 ± 1.2


EE (%)

25.6 ± 3.2

35.8 ± 1.8

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Entrapment efficiency of 5-FUs in PHYT cubosomes was determined after separation of the unloaded 5-FUs by ultrafiltration method and by measuring its UV-absorbance at 266 nm. As indicated in Table 2, it was found that only 25% of 5-FUs was entrapped in the cubosomes. This low EE% is not surprising since 5-fluorouracil is a small hydrophilic molecule, as indicated by its logPoctanol/water partition coefficient value of -0.85.(38) Moreover, due to their mobile character, 5-FUs molecules can easily flow through the pores without anchoring and a rapid leakage of the drug from the cubosome aqueous channels cannot be excluded during preparation and centrifugation procedures.(39) Given that 5-FUs molecules in the 50 mg/mL solution (pH 9) used in this study are negatively charged (pKa = 7.8-8),(40) it should be much easier for 5-FUs to get trapped inside positively charged cubosomes via electrostatic interactions. For this reason, PHYT cubosomes containing 5 mol% of the cationic lipid DOTAP were prepared and characterized. The entrapment efficiency actually improved (EE = 35 %) even though the effect of DOTAP was not dramatic. This is probably due the hydrophobic methyl groups surrounding charged nitrogen in DOTAP, which may limit interactions with 5-FUs.(41,42) In the case of unloaded cubosomes, the obtained charged dispersions were much less milky and almost transparent, as already observed for the addition of other ionic surfactants to PHYT.(43,44) The transparency of these dispersions may be attributed(43,44) to the formation of Im3m cubosomes coexisting with vescicles. even if the correlation peaks due to vescicle scattering were not observed in SAXS diffractogram (Figure S4, Supporting Information) Addition of DOTAP caused a considerable increase of the Zeta Potential to + 37 mV, confirming the presence of the cationic lipid in the structure. Concerning the size, PHYT/DOTAP cubosomes were significantly smaller than PHYT ones (110 vs. 215


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nm), probably because of the higher Zeta Potential, which could prevent the nanoparticle aggregation by means of stronger repulsive forces.(45) Inclusion of 5-FUs in PHYT/DOTAP cubosomes resulted in larger nanoparticles (187 nm) than empty ones: in this case, 5-FUs alkalyne molecules are electrostatically linked to the DOTAP headgroups, thus partially neutralizing its positive charge, reducing the repulsive forces and favoring the formation of larger structures. As expected, also Zeta Potential decreased upon addition of negative 5-FUs (+ 26 mV vs. + 36 mV).

In Vitro Cytotoxicity of 5-Fluorouracil Loaded PHYT and PHYT/DOTAP Dispersions. To determine the anticancer activity of 5-FUs loaded PHYT and PHYT/DOTAP formulations, in vitro cellular cytotoxicity against MDA-MB-231 cells was evaluated by MTT assay and compared with that of different concentrations of bare 5-FUs (Figure 6).


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Figure 6. Impact of bare 5-FUs (black) and 5-FUs encapsulated in (A) PHYT (PHYT/5-FUs, red) and (B) PHYT/DOTAP (PHYT/DOTAP/5-FUs, green) nanoparticles on MDA-MB-231 cell viability. Cells were treated with various concentrations of PHYT/5-FUs or PHYT/DOTAP/5FUs dispersions and bare 5-FUs for 24h (left panels) and 72 h (right panels). The respective concentrations of 5-FUs encapsulated in the two different formulations have been reported above each panel. In addition, the corresponding unloaded PHYT (blue) and PHYT/DOTAP (yellow) dispersions were tested. Cell viability was determined by MTT assay. Values are Mean ± SEM, n = 8. The significance of differences between the impact of bare and encapsulated 5-FUs, is indicated by dots above the compared columns (*p

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