Photodynamic activity of 2,6-diiodo-3,5-dithienylvinyleneBODIPYs and their folate-functionalized chitosan-coated Pluronic F-127 micelles on MCF-7 breast cancer cells

Article abstract of DOI:10.1142/S1088424619501773

A 2,6-diiodo-3,5-dithienylvinyleneBODIPY dye was prepared and encapsulated with folate-chitosan capped Pluronic F-127 to provide drug delivery systems for photodynamic therapy (PDT). Moderately enhanced singlet oxygen quantum yields were observed for the

Full text of DOI:10.1142/S1088424619501773

This is a JAM - Just Accepted Manuscript
Journal of Porphyrins and Phthalocyanines
Article Title:
Photodynamic activity of 2,6-diiodo-3,5-dithienylvinyleneBODIPYs and
their folate-functionalized chitosan-coated Pluronic® F127 micelles on
MCF-7 breast cancer cells
Author(s):
DOI:
Nthabeleng Molupe, Balaji Babu, David O. Oluwole, Earl Prinsloo, Lizhi Gai,
Zhen Shen, John Mack, Tebello Nyokong
10.1142/S1088424619501773
To be cited as:
Nthabeleng Molupe, Balaji Babu, David O. Oluwole, Earl Prinsloo, Lizhi Gai, Zhen Shen, John
Mack, Tebello Nyokong, Photodynamic activity of 2,6-diiodo-3,5-dithienylvinyleneBODIPYs and
their folate-functionalized chitosan-coated Pluronic® F127 micelles on MCF-7 breast cancer
cells, Journal of Porphyrins and Phthalocyanines, doi: 10.1142/S1088424619501773
Received: 9 October, 2019
Accepted: 13 November, 2019
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Accepted manuscript to appear in JPP
2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
Photodynamic
activity
of
2,6-diiodo-3,5-
dithienylvinyleneBODIPYs and their folate-functionalized
chitosan-coated Pluronic® F127 micelles on MCF-7 breast
cancer cells
Nthabeleng Molupe,a Balaji Babua David O. Oluwole,a Earl Prinsloo,b Lizhi Gai,c
Zhen Shen,c John Macka,* and Tebello Nyokonga
a
Centre for Nanotechnology Innovation, Department of Chemistry, Rhodes University, Makhanda 6140, South
Africa.
b Biotechnology Innovation Centre, Rhodes University, Makhanda 6140, South Africa.
c
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing
University, Nanjing 210046, P. R. China
This paper was submitted to a special issue to celebrate the 60th birthday of Professor Roberto Paolesse.
Received date (to be automatically inserted after your manuscript is submitted)
Accepted date (to be automatically inserted after your manuscript is accepted)
ABSTRACT: A 2,6-Diiodo-3,5-dithienylvinyleneBODIPY dye was prepared and encapsulated with folate-chitosan
capped Pluronic® F-127 to provide drug delivery systems for photodynamic therapy (PDT). Moderately enhanced
singlet oxygen quantum yields were observed for the dye encapsulation complexes in water. The in vitro dark
cytotoxicity and photodynamic activity were investigated on the human breast adenocarcinoma (MCF-7) cell line.
Minimal dark cytotoxicity was observed for the BODIPY dyes in 5% DMSO and when encapsulated in folate-
functionalized chitosan-coated Pluronic® F-127 micelles since the cell viability values are consistently greater than
80% over the 0−40 µg.mL−1 concentration range. Upon irradiation of the samples, significant cytocidal activity was
observed for the encapsulation complex of a 2,6-diiodo-8-dimethylaminophenyl-3,5,-dithienylvinyleneBODIPY dye
with less than 50% viable cells observed at concentrations ≥ 20 µg.mL−1.
KEYWORDS: BODIPY dyes, Pluronic® F-127 micelles, folic acid, chitosan; photodynamic therapy, singlet
oxygen
*Correspondence to Dr John Mack, email: j.mack@ru.ac.za, Tel: +27 46-603-7234.
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
INTRODUCTION
Boron dipyrromethene (BODIPY) dyes have received considerable interest in recent years due to their tunable
properties and photostability. The versatility of BODIPY core structure enables the photosensitizers to be designed
to suit a variety of applications such as nonlinear optics, optical labelling, fluorescence imaging, photodynamic
antimicrobial chemotherapy (PACT) and photodynamic therapy (PDT) [1-4]. Where PDT is concerned, halogens are
introduced at 2,6-positions of the core structure of the BODIPY dye to promote faster intersystem crossing into the
lowest triplet excited state (T1) [5, 6]. The T1 state typically transfers its energy to ground state molecular oxygen to
generate reactive oxygen species known as singlet oxygen which is the principal cytotoxic agent responsible for the
selective decimation of the tumor cells [1-4]. Styrylation and vinylation at the 3,5-positions have been widely used
to extend the π-conjugation system of the BODIPY core structure to afford intense absorption within the biological
window (600−800 nm) [7-10]. When the dyes absorb in this spectral region, they become suitable for physiological
applications like PDT for tumor cells [2, 7-10].
The major limitation faced by this group of dyes is their insolubility in aqueous media. This limits their
biocompatibility in the physiological environment, thus resulting in low efficacy. To overcome this drawback,
various solubilization routes have been explored [9-11], including sulfonation and conjugation to solubilising agents,
such as Pluronic® F-127, which has been approved by the Food and Drug Administration for use as a drug delivery
agents due to its aqueous solubility and biocompatibility [12-17]. Pluronic® F-127 is a triblock copolymer with a
hydrophobic polypropylene oxide (PPO) center unit and hydrophilic polyethylene oxide (PEO) end units. Due to the
temperature dependence of this polymer in solution, micelles are formed as temperature increases due to the increase
in solubility of the PPO unit of the copolymer. When Pluronic® F-127 encapsulate the hydrophobic drug molecules,
the solubility and biocompatibility of the dyes are considerably enhanced [12-18].
It has been reported that Pluronic® F-127 micelles aggregate and are unstable when exposed to varying
temperatures. For the use of micelles in prolonged drug delivery, coating with other polymers like chitosan has been
reported to stabilize the micelles [18]. Chitosan is a 2-amino-2-deoxy-β-D-glucan polymer with unique properties
that make it suitable for pharmaceutical applications amongst other polymers. The chitosan structure comprises free
amino groups, which encourage cross-linkages. It also has mucoadhesive properties, which enhance the retention of
the encapsulated drugs in their target substrates. In addition, chitosan is more cationic than other polymers. This
property enables an easy approach of malignant cell membranes and ionic linkages to anions. Due to the ease of
cross-linking, chitosan can be functionalized with folic acid. Since most cancerous cells possess overexpressed
folate receptors, folic acid is used as targeting ligand to enhance the uptake of the drug molecules and the
endocytosis of the malignant tissues [18-24].
We report the use of Pluronic® F-127 micelles encapsulated with folic acid-functionalized chitosan as a delivery
system for 8-dimethylaminophenyl-2,6-diiodo-3,5-di-3-thienylvinyleneBODIPY (1) in PDT studies with the human
breast adenocarcinoma (MCF-7) cell line, Scheme 1. The PDT activities of 1 and 8-Phenyl-2,6-diiodo-3,5-di-2-
thienylvinyleneBODIPY (2) (Scheme 2), which has been studied previously in the context of optical limiting
applications [25, 26], were also studied in 5% DMSO solution.
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
EXPERIMENTAL
Materials
Ultra-pure water was obtained from a Milli-Q Water System (Millipore). Pluronic® F-127, chitosan, folic acid, 1,3-
diphenylisobenzofuran (DPBF), spectroscopic grade dimethylsulfoxide (DMSO), anthracene-9,10-bis-
methylmalonate
(ADMA),
zinc
phthalocyanine
(ZnPc),
trypan blue,
2,4-dimethylpyrrole,
4-
dimethylaminobenzaldehyde, 3-thiophenebenzaldehyde, trifluoroacetic acid (TFA), triethylamine (TEA), p-
chloranil, boron trifluoride diethyl etherate (BF3·OEt2), N-iodosuccinimide, sodium sulfate, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), sodium dodecyl sulfate (SDS), glacial acetic acid and piperidine were
obtained from Sigma Aldrich. Dichloromethane (DCM) was obtained from Saarchem. Hydrochloric acid (32%) was
purchased from Merck. All reagents were used without further purification unless otherwise stated. All atmospheric
air and moisture sensitive reactions were carried out under an argon gas flow, and dry solvents were used
throughout. Cultures of MCF-7 cell were obtained from Cellonex®. Heat-inactivated fetal calf serum (FCS) and 100
unit.mL−1 penicillin-100 µg.mL−1 streptomycin-amphotericin B were obtained from Biowest®. Dulbecco’s
phosphate-buffered saline (DPBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Lonza®.
Cell proliferation neutral red reagent (WST-1 assay) was obtained from Sigma-Aldrich. Zinc
tetrasulfophthalocyanine (ZnPcS4) was prepared using the Weber and Busch method [27].
Instrumentation
UV-visible absorption spectra were measured on a Shimadzu UV−2550 spectrophotometer. Fluorescence emission
spectra were recorded with a Varian Eclipse spectrofluorimeter, while fluorescence lifetimes were measured using a
time-correlated single-photon counting (TCSPC) spectrometer (FluoTime 300, Picoquant GmbH). 1H NMR
measurements obtained from Bruker AMX 600 MHz. Mass spectra data were acquired on a Bruker Auto-FLEX III
Smartbeam TOF/TOF mass spectrometer operated in positive ion mode using α-cyano-4-hydroxycinnamic acid as a
matrix. FT-IR spectra were obtained from a Bruker Alpha FT-IR spectrometer with a universal attenuated total
reflectance (ATR) sampling accessory. Dynamic light scattering (DLS) analysis was performed on a Malvern
Zetasizer nanoseries, Nano-ZS90 instrument.
The MCF-7 cells were cultured in 25 cm2 vented flasks (Porvair) then subcultured in 75 cm2 vented flasks (Porvair)
in a humidified atmosphere incubator with 5% CO2 and physiological temperature at 37°C (Heal Force). In vitro
PDT studies were conducted using the illumination kit of a Modulight® Medical 7710-680 laser system fitted with a
Thorlabs M660L4 660 nm light-emitting diode (LED) that was found to provide an irradiance of 280 mW/cm2
(Coherent FieldmaxII TOP energy/power meter fitted with a Coherent Powermax PM10 sensor) in this context, to
illuminate a 127.76 × 85.48 mm 96 well tissue culture plate. WST1 cell proliferation neutral red reagent (Roche)
was used to measure the cell viability through a Synergy 2 multi-mode microplate reader (BioTek®). A Fluorescence
LED (FL−LED) inverted microscope was used to view the cells under phase contrast (Zeiss-AxioVert).
Synthesis
The BODIPY core dye with a meso-dimethylaminophenyl group (a) that was initially prepared (Scheme 1) has been
reported previously [28]. Iodination and subsequent vinylation to form precursor b and BODIPY 1 followed
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
procedures that have been used to prepared similar dyes [2, 4, 25]. The synthesis of BODIPY 2 (Scheme 2) has been
reported previously [25].
Synthesis of 8-dimethylaminophenylBODIPY (a): 4-Dimethylaminobenzaldehyde (1.052 g, 7.053 mmol) and 2,4-
dimethylpyrrole (1.342 g, 14.11 mmol) were dissolved in dry DCM (30 ml) under argon flow. TFA (0.4 ml) was
added dropwise to the reaction, and the reaction vessel was left to stir at room temperature. The progress of the
reaction was monitored by thin-layer chromatography (TLC). When the aldehyde was fully consumed, the reaction
temperature was lowered to 0°C, and p-chloranil (1.734 g, 7.053 mmol) was added. After the reaction was stirred for
20 min at room temperature, TEA (4.282 g, 42.32 mmol) was added, and the reaction left to stir for a further 10 min.
At 0°C, BF3·OEt2 (10.17 g, 70.53 mmol) was added dropwise over 30 min. The reaction mixture was stirred for 16 h
and was then washed with deionized water, extracted with DCM and then dried over anhydrous sodium sulfate. The
solvent was removed in vacuo. The residue was purified by silica gel column chromatography by eluting with
1
petroleum ether/ethyl acetate (9:1), and a pure orange crystalline product was obtained. Yield: 53%. H NMR (600
MHz, DMSO-d6): δH, ppm 7.11 (d, J = 6.3 Hz, 2H), 6.85 (d, J = 7.12 Hz, 2H), 6.16 (s, 2H), 2.98 (s, 6H), 2.44 (s,
6H), 1.45 (s, 6H). UV-vis (DMSO): λmax, nm (log ε) 502 (4.84).
Synthesis of 2,6-diiodo-8-dimethylaminophenylBODIPY (b): Core dye a (0.29 g, 0.79 mmol) was dissolved in 30
ml of dry DCM. The solution was degassed, and NIS (0.42 g, 2.4 mmol) in DCM (5 ml) was added over 30 min
with stirring. The reaction then proceeded for 8 h and progress was monitored by TLC. The solution was washed
with a 1 M sodium sulfate solution, and the product was extracted with CH2Cl2. The solution was dried with
anhydrous magnesium sulfate, filtered, and the solvent was removed using a rotary evaporator. The product was
isolated by silica gel column chromatography with ethyl acetate/hexane (v/v; 1:9) as the eluent. A dark pink
1
crystalline product was obtained. Yield: 42%. H NMR (600 MHz, DMSO-d6): δH, ppm 7.03 (d, 6.21 Hz, 2H), 6.77
(d, 7.23 Hz, 2H), 3.04 (s, 6H), 2.63 (s, 6H), 1.50 (s, 6H). UV-vis (DMSO): λmax, nm (log ε) 533 (4.64). MALDI
TOF-MS: Calculated: 619.04; Found: 618.34 [M+].
Synthesis of 8-dimethylaminophenyl-2,6-diiodo-3,5-di-3-thienylvinyleneBODIPY (1): Iodinated core dye b
(0.0396 g, 0.0635 mmol) and 3-thiophenecarboxaldehyde (0.02136 g, 0.1905 mmol) were mixed and dissolved in
dry benzene (30 ml). Glacial acetic acid (0.5 ml) and piperidine (0.5 ml) were added under reflux with a Dean-Stark
trap fitted. The reaction was monitored by UV-visible absorption spectroscopy until core dye b was entirely
consumed. The mixture was washed with 1 M HCl and dried in vacuo. The residue was purified by silica gel
1
chromatography with petroleum ether/ethyl acetate (4:1) as the eluent. A blue solid was obtained. Yield: 40%. H
NMR (600 MHz, CDCl3): δH, ppm 8.23−8.16 (m, 2H), 7.55−7.49 (m, 8H), 7.43−7.38 (m, 4H), 3.18 (s, 6H), 1.51 (s,
6H). UV-vis (DMSO): λmax, nm (log ε) 641 (4.69). MALDI TOF-MS m/z: Calculated: 807.30; Found: 807.33 [M+].
Preparation of the 1@PF127 encapsulation complex: A previously reported thin-film hydration method was used
with some modifications for the encapsulation of dyes into Pluronic® F-127 micelles [8]. 1 was co-solubilized with
Pluronic® F-127 (1/40: w/w) in DCM (5 ml), and the solution was sonicated for 15 min. Then, the solution was dried
in vacuo for 1 h at 50°C and dried for 12 h in a desiccator. The solid obtained was hydrated with Millipore water (5
ml) and sonicated for 15 min then further stirred for 2 h. The solution was filtered through a 0.2 µm filter, and the
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
filtrate was freeze-dried to obtain the solid form, which is referred to throughout as 1@PF127. Micelles with no
encapsulated BODIPY dyes were prepared with a similar protocol.
Functionalization of chitosan with folic acid: The functionalization procedure was adapted from Yang et al. [20].
Folic acid (1.3942 g, 3.159 mmol) and EDC (0.6055 g, 3.159 mmol) were dissolved in anhydrous DMSO (20 ml)
and stirred at room temperature for 24 h to activate the carboxylic acid moiety of the folic acid. The activated folic
acid was gradually added to 0.5% (w/v) chitosan in acetic acid solution (0.1 M, pH 4.7) with constant stirring for 16
h in the dark to allow for amide bond formation between the folic acid and chitosan. The solution was adjusted to
pH 9.0 (using 1.0 M aq. NaOH) then centrifuged at 2500 rpm to precipitate out the folate-functionalized chitosan.
The precipitate was dialyzed in phosphate-buffered saline (PBS, pH 7.4) for 3 days then deionised water for 4 days.
The folate-functionalized chitosan solid was obtained through freeze-drying. Yield: 76%. IR cm−1: 1510 (amide I),
1602 (amide II).
Preparation of the 1@PF127@FC encapsulation complex: For further modification with folate-functionalized
chitosan, the 1@PF127 solution was not freeze-dried. Instead, 0.04% (w/v) solution of SDS was added to the dye-
micelle solution and left for 1 h to form closely packed micelles [29]. The solution was filtered through a 0.2 µm
filter to remove unloaded dye molecules. The 1@PF127 solution was mixed slowly for 20 min with 0.005% (w/v)
solution of folate-functionalized chitosan in 0.4 M acetate buffer at pH 4.7 to form a 1:1 1@PF127:folate-
functionalized chitosan solution. The resultant solution was dialyzed for 3 days with deionized water to remove
excess SDS and free BODIPY dye and the acetate buffer. The solution was then freeze-dried for 2 days with a
VirTis bench-top K freeze dryer to obtain solid inclusion complexes coated with folate-functionalized chitosan
(1@PF127@FC). Micelles with no encapsulated BODIPY dyes were prepared with a similar protocol.
Photophysical and photochemical properties
Fluorescence quantum yields (ΦF) and singlet oxygen quantum yields (ΦΔ) were determined using the comparative
method [30, 31] following literature procedures in a manner similar to what has been reported previously for other
BODIPY dyes [2, 28, 32], using Rhodamine 6G (ΦF = 0.95 in ethanol [33, 34]), Rose Bengal (ΦΔ = 0.76 in DMSO
[35, 36]) and zinc phthalocyanine (ΦF = 0.20 [37], ΦΔ = 0.67 in DMSO [38]) as the standards. DPBF was used as a
1O2 scavenger in DMSO and ethanol. The ΦΔ value of the BODIPY dyes in micelles was determined by using
ZnPcS4 (ΦΔ = 0.56 in water) [39] as the standard in aqueous solution with ADMA as the singlet oxygen scavenger
[40].
In vitro dark cytotoxicity and PDT activity
The cell culturing and in vitro studies were carried out as reported in the literature [12, 41]. In vitro cytotoxicity
studies we performed in the dark using human breast adenocarcinoma cell (MCF-7 cell) prior to in vitro
photodynamic therapy studies. The MCF-7 cells were cultured using DMEM containing 4.5 g.L−1 glucose with L-
glutamine and Phenol red, supplemented with 10% (v/v) heat-inactivated FCS and 100 unit.mL−1 penicillin-100
μg.mL−1 streptomycin-amphotericin B. The cells were grown in 75 cm2 vented flasks (Porvair) and incubated at
37°C and 5% CO2 with a humidified atmosphere. Routine subculturing using standard trypsinisation was followed.
Viable trypsinised cells were counted using trypan blue dye exclusion assay (0.4%) using hemocytometer. 10,000
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
cells/well were seeded in supplemented DMEM containing Phenol red in 96-well tissue culture plates (Porvair®).
The cells were incubated at 37°C under 5% CO2 for 24 h to facilitate cell attachment to the wells. The cells adhered
cells were washed with 100 μl DPBS twice, followed by administration of 100 μl supplemented DMEM containing
3, 5, 10, 20 and 40 μg.mL−1 of BODIPYs 1, 2 and 1 in the 1@PF127@FC micelles. Three separate wells were filled
at each concentration for all three photosensitizers to enable a statistical analysis. Stock solutions of BODIPYs 1 and
2 were prepared in DMSO at 1000 µg.mL−1, while the stock solution for the 1@PF127@FC micelles was prepared
in Millipore water. Placebo cells were incubated with supplemented DMEM only and with 5% DMSO (v/v), the
maximum percentage of DMSO that is present. No significant cytotoxic effect was observed on the MCF-7 cells in the
absence of photosensitizer dyes. After 24 h treatment, the wells were rinsed twice with 100 μL DPBS, supplemented
DMEM with Phenol red was added, and the plates were re-incubated for 24 h. A fixed light dose of 336 J.cm−2 was
achieved by illuminating a set of plates for 20 min with a Thorlabs 660 nm LED.
After illumination, the cells were rinsed with PBS then Phenol red-free supplemented DMEM was replaced with
supplemented DMEM with Phenol red. The WST-1 assay was used to quantify the viable cells in the monolayer of
the cells treated with the drugs and the placebo cells, respectively. The quantification was done in accordance with
the manufacturer’s instruction using a Synergy 2 multi-mode microplate reader (BioTek®) at 450 nm. The
percentage cell viability was determined using Eq. (1):
Absorbance of samples at 450 nm
% Cell Viability =
× 100
(1)
Absorbance of control at 450 nm
where the “Absorbance of samples at 450 nm” is the absorbance value for the cells containing drugs, while
“Absorbance of control at 450 nm” is the value for the placebo cells containing only supplemented DMEM with
Phenol red. IC50 values were calculated by nonlinear regression analysis (GraphPad Prism 5). No significant effect
was observed in light control experiments with placebo cells in supplemented DMEM only and with 5% DMSO (v/v).
Triplicate measurements were made for each well, so that nine measurements were made in total at each
concentration.
RESULTS AND DISCUSSION
Synthesis and characterization of 1
Scheme 1 provides the synthetic route for preparing BODIPY 1 through a Knoevenagel condensation reaction with
3-thiophenecarboxaldehyde [10, 42]. Thin-layer chromatography and UV-visible absorption spectroscopy were used
to ensure that 1 was fully separated from the other possible products by silica gel chromatography. The parent peak
in the MS data was in close agreement with the calculated value for the target structure. The 1H NMR spectrum can
be readily assigned since it is broadly similar to that reported previously for 3,5-dithienylBODIPY dyes [25, 26] and
other 3,5-vinylene and 3,5-distyrylBODIPY dyes [43-46].
Ground state UV-visible absorption spectra
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
The UV-visible absorption spectra of core dyes a and b and BODIPYs 1 and 2 in DMSO are shown in Figure 1. A
narrow absorption band is observed at 502 nm in the spectrum of dye a, which is characteristic of BODIPY core
dyes, and at 533 nm for its iodinated analogue b due to a significant bathochromic shift. It has been reported
previously for other halogenated BODIPY dyes, that there is a significant narrowing of the HOMO−LUMO gap due
to the mesomeric interaction between the lone pairs on the halogen atoms at the 2,6-positions and the π-system of
the BODIPY core [2, 8]. After the extension of the conjugation system through vinylation to form BODIPY 1, the
main absorption band of iodinated core dye b was further red-shifted to 641 nm. Theoretical calculations have
demonstrated that there is a relative destabilization of the HOMO when electron-donating styryl or vinylene groups
are introduced at the 3,5-positions, since the HOMO has larger MO coefficients on these carbons than the LUMO [2,
8]. For this reason, the main spectral bands of BODIPYs 1 and 2 (Figure 1) lie within the therapeutic window
(620−1000 nm) and are hence potentially suitable for use as photosensitizer dyes for PDT.
Phase solubility studies
Phase solubility studies were conducted for BODIPY 1 in water over a range of different Pluronic® F-127
concentrations, and the results for BODIPY 1 are provided in Table 1. The phase solubility studies were performed
following a procedure reported by Hadžiabdić et al. [47]. BODIPY 1 solutions were prepared by adding excess
amounts (3 mg) in each Pluronic® F-127 solution; 0.5, 0.8, 1.1, 1.3 and 1.6 × 10−4 mol.L−1. The samples were stirred
at ambient conditions for 24 h to allow an equilibrium to be reached. The samples were filtered using a 0.2 µm pore
membrane filter, and the concentration of the BODIPY dye was determined by UV-visible absorption spectroscopy.
The changes in solubility were plotted, and phase solubility diagrams were obtained. The information from the plots
was used to evaluate the stoichiometry, apparent stability constants (K1:1) and complexation efficiencies (CE) of the
resultant complexes. The apparent stability constants were estimated from the linear portion of the phase solubility
curve using the following equation of Higuchi and Connors [48]:
Slope
K1:1
=
(2)
S0 (1−Slope)
Where K1:1 is the apparent stability constant (L.mol−1), S0 is the saturation concentration of the drug molecule
(BODIPY) in pure water [47]. A 1:1 ratio was assumed for the BODIPY/Pluronic® F-127 encapsulation complexes.
The complexation efficiency (CE) was calculated by using the slope of the phase-solubility diagram by following
Eq. (3):
Slope
CE =
= S0 · K1:1
(3)
1−Slope
where CE is the complexation efficiency.
The overall trend observed for 1 (Figure 2) was an increase in solubility with increasing concentration of Pluronic®
F-127 in water until a critical micellar concentration (CMC) is reached. At the CMC, the solubility of the BODIPY
dye is no longer enhanced by the addition of more surfactant. The CMC for BODIPY 1 was determined to be ca. 1.3
× 10−4 mol.L−1 at both 25 and 37°C, which is similar to the reported CMC values for other Pluronic® F-127 micelles
[49-51]. BODIPY 1 showed a linear increase in solubility as a function of the Pluronic® F-127 concentration, which
is characteristic of an AN-type system. AN-type systems are consistent with the formation of water-soluble
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
encapsulation complexes. The apparent stability constants of the binary complex were determined by using the
linear regression method with Equation 1 at 0.0, 0.5, 0.8, 1.1 and 1.3 × 10−4 mol.L−1 Pluronic® F-127 concentrations,
R2 = 0.9484 @ 25°C and R2 = 0.9979 @ 37°C. A 1:1 complex formation was assumed. High stability constants
were obtained for BODIPY 1 and Pluronic® F-127 complexes, with log(K1:1) values of 4.11 and 4.32 at 25 and
37°C, respectively. The optimal log(K1:1) values have previously been reported to lie in the 1−3 range [49-51]. The
CE values reported in Table 1 demonstrate that there is approximately 2% encapsulation efficiency for BODIPY 1 at
25°C and 3% at 37°C in this context.
It proved impossible to carry out a similar study for BODIPY 2 (Scheme 2), which suggests that the presence of the
functionalized meso-aryl group of 1 (Scheme 1) significantly enhances the interaction with Pluronic® F-127 through
hydrogen bonding interactions.
Determination of the location of dyes in Pluronic® F-127 micelles
The location of the dyes in Pluronic® F-127 micelles was determined using UV-visible absorption spectroscopy. The
UV-visible absorption spectra of the dyes in micellar solutions were compared with the spectra of the dyes in
ethanol and petroleum ether [52, 53] to provide information about the location of the dyes within the micelles. The
UV-visible absorption spectra of BODIPY 1 in micellar solution (1@PF127) and BODIPY 1 in petroleum ether
(1_Pet-Ether) are very similar. In each case, the main absorption band lies at 636 nm, Figure 3. According to the
approach described by Tehrani-Bagha and Holmberg [52], the similarity in the 1@PF127 and petroleum ether
spectra suggests that BODIPY 1 is most likely located in the core region of the micelles [49, 52, 53].
Fourier Transform-Infrared Spectroscopy
FT-IR spectroscopy is a qualitative technique that can be used to determine the presence of functional groups and
identify fingerprint signals for different compounds. In Figure 4, the chitosan FT-IR spectrum shows a broad peak
between 3000 and 3500 cm−1 that arises from the overlap of bands related to the –NH and –OH functional groups. A
C−H stretch is observed at 2873 cm−1, while the amide I, II and III (−N−H) peak lie at 1640−1588, 1419 and 1375
cm−1, respectively. The band at 1061 cm−1 arises from the C−O−C vibration of the chitosan structure. Folic acid has
a complex structure consisting of glutamic acid, p-aminobenzoic acid and heterocyclic pteridine. The broad peak
between 3400−3600 cm−1 was attributed to the –OH and –NH bands of the glutamic and pterinic parts of folic acid,
respectively. Strong peaks at 1689 cm−1 arise from the –C=O stretch, while –NH bends lie at 1602 cm−1 and the
vibrations of the C-C bonds in the pterinic rings are observed at 1481 cm−1 [18-22]. Figure 4(b) shows the
superimposed spectra of chitosan, folic acid and folate-functionalized chitosan. Upon functionalization, there was an
apparent increase in the intensity of the broad peak in the 3000−3500 cm−1 region due to the overlap of the –OH and
–NH bands. The peaks associated with the carbonyl functional group of the folic acid decrease in intensity upon
functionalization. New peaks observed at 1510 and 1602 cm−1 are associated with amide I and II bands, respectively.
This confirms the formation of folate-functionalized chitosan [18-22].
X-ray diffraction
XRD technique is a non-destructive and routinely used for various characterizations. It was used to determine the
crystalline behavior of the respective samples. The diffractograms are shown in Figure 5. The diffraction pattern of
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
the chitosan showed an intense peak at 2θ = 19.9°, and a broad peak was observed for folic acid at 2θ = 10 and 30°.
The folate-functionalized chitosan showed a highly amorphous structure and hence a lack of crystallinity
characterized by a peak at ca. 20°. The diffractograms of BODIPY 1 and its encapsulation complexes showed highly
crystalline patterns. Upon coating of the 1@PF127 micelles with folate-functionalized chitosan to form
1@PF127@FC micelles, there was a change to an amorphous structure (Figure 5) thus indicating a successful
modification of the micelles in this regard [22].
Dynamic light scattering
Dynamic light scattering (DLS) was used to determine the diameters of the micelles (Fig. 6). The values obtained
were 11.7 nm for plain Pluronic® F-127 (A), 37.8 nm for 1@PF127 (B) and 50.7 nm for 1@PF127@FC (C). The
overall trend observed was consistent with an increase in the size of micelles upon encapsulation of the hydrophobic
BODIPY dye [12, 22].
Photophysical properties and singlet oxygen quantum yields
Since PDT is the envisaged application, the photophysical properties (Table 2) and singlet oxygen quantum yields
(Table 3) of the BODIPY dyes were determined in DMSO, since this solvent was used to solubilize the dyes in the
context of the PDT studies. BODIPY core dyes usually have high ΦF values due to the rigid core structure and
minimal intersystem crossing (ISC), which results in very low triplet state quantum yields [7]. The ΦF value for a of
0.03 is unusually low (Table 2), probably due to intramolecular charge transfer that occurs upon formation of the S1
state. It has been reported previously that the lone pairs on the nitrogen atoms of the dimethylaminophenyl group
can act as a donor of electronic charge while the electron-deficient BODIPY core acts as an acceptor [54, 55]. The
introduction of iodine atoms at the 2,6-positions results in a further decrease in the ΦF values of b and 1, due to the
enhanced ISC to the T1 state. The fluorescence lifetime (τF) for 1 of 3.02 ns is similar to those reported previously
for other π-extended BODIPY dyes [56, 57]. Փ∆ values were determined for b, 1 and 2 by using the comparative
method (Table 3). The value of BODIPY 1 was found to be 0.13 in DMSO (Fig. 7) and 0.40 in ethanol, while that of
2 was determined to be 0.40 in DMSO. 1 was found to be highly photostable in this context (Fig. 7), since no
significant degradation was observed in the intensity of the main BODIPY spectral band during the irradiations. The
relatively low value for 1 in DMSO is probably related to an enhanced rate of non-radiative decay due to the
intramolecular charge transfer character of the S1 state caused by incorporating the strongly electron-donating styryl
group [58, 59]. It has been reported previously that the encapsulation of dyes in Pluronic® F-127 micelles can
increase the Փ∆ value due to the effect of polymer encapsulation [12]. There may also be scope for changes in the
photophysical and photochemical properties of the dye if there is an interaction between the lone pair of the
dimethylamino nitrogen and Pluronic® F-127 [60, 61].
Cell studies
Figure 8 shows the in vitro dark cytotoxicity and PDT activity (PDT) data for BODIPY 1 (A), 1@PF127@FC (B)
and BODIPY 2 (C). All three samples showed relatively innocuous behaviour with cell viability greater than 80% in
the absence of irradiation over the 0–40 µg.mL−1 concentration range (Fig. 8). The photodynamic activities of the
samples were tested against MCF-7 cells with a Thorlabs 660 nm LED and 20 min irradiation time at a fixed dose of
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
336 J.cm−2 over the 3–40 µg.mL−1 concentration range. BODIPY 1 in 5% DMSO exhibited relatively high PDT
activity and showed 62% cell viability at a concentration of 40 µg.mL−1, Figure 8A, while the percentage cell
viability decreased to 40% (Fig. 8B) when BODIPY 1 was encapsulated in the context of the 1@PF127@FC
micelles. The IC50 value for the 1@PF127@FC micelles was determined to be 20 ± 1.3 µg.mL−1. The values for 1
and 2 are > 40 µg.mL−1. As anticipated, the PDT activity of the samples was enhanced at higher concentration. The
bar graphs show clearly that the encapsulated dyes have higher PDT activity than the solutions of 1 and 2 in 5%
DMSO. The PDT activity observed is comparable with values obtained previously for phthalocyanines [62]. Figure
9A-C shows the in vitro photodynamic effect on MCF-7 cells when irradiated for 20 min, through a comparison of
the cells in the control solution (A), and cells treated at 40 μg.mL−1 with BODIPY 1 (B) and 1@PF127@FC (C).
These observations are consistent with the PDT effect observed in Figure 8.
Conclusion
Pluronic® F-127 micelles encapsulating BODIPY 1 were characterized by DLS, IR spectroscopy and XRD, and
were found to be stable and water-soluble. BODIPY 1 exhibits low dark toxicity when in solution and when
encapsulated in Pluronic® F-127 micelles. Although there is evidence of a photodynamic effect when the dyes are
introduced in 5% DMSO solution, this becomes more significant when the dyes are encapsulated in Pluronic® F-127
micelles coated with folate-functionalized chitosan with an IC50 value of 20 ± 1.3 µg.mL−1. The enhancement of the
PDT effect can be attributed to the targeting properties of folic acid and the biocompatibility introduced by the
Pluronic® F-127 micelles. The results of this preliminary study demonstrate that the use of folate-functionalized
chitosan micelles of this type as delivery agents for BODIPY dyes merits further in-depth investigation. Future
studies will focus on BODIPY related dyes with higher singlet oxygen quantum yields and spectral bands that are
shifted deeper into the therapeutic window that are suitable for use in this context.
Acknowledgement
Financial support was provided by the National Research Foundation (NRF) and the Department of Science and
Technology (DST) of South Africa through the DST/NRF Research Chairs Initiative for Professor of Medicinal
Chemistry and Nanotechnology (uid: 62620), and a CSUR grant from the NRF of South Africa to JM (uid: 93627).
The photophysical measurements were facilitated by the Laser Rental Pool Programme of the Council for Scientific
and Industrial Research (CSIR) of South Africa.
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
Schemes
Scheme 1. The synthetic procedure for 8-dimethylaminophenyl-2,6-diiodo-3,5,-dithienylvinylene BODIPY (1).
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
Scheme 2. The molecular structure of 8-phenyl-2,6-diiodo-3,5,-dithienylvinyleneBODIPY (2).
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
Figures
Fig. 1. The ground state UV-visible absorption spectra of BODIPY dyes a, b, 1 and 2. Solvent: DMSO.
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
Fig. 2. Phase-solubility diagrams for BODIPY 1 with Pluronic® F-127 at different temperatures. Solvent = water.
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Fig. 3. Normalized UV-visible absorption spectra of BODIPY 1 in Pluronic® F-127 micellar solution (1_PF127)
ethanol (1_EtOH) and petroleum ether (1_Pet-Ether).
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Fig. 4. Infrared spectra of chitosan (black), folic acid (blue) and folate-functionalized chitosan (red).
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
Fig. 5. X-ray diffractograms of chitosan, folic acid, folate-chitosan, BODIPY 1, and the 1@PF127 and
1@PF127@FC micelles.
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
Fig. 6. Dynamic light scattering data for plain Pluronic® F-127 micelles (A), 1@PF127 (B) and 1@PF127@FC
(C). Solvent = water.
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
Fig. 7. Absorption spectra recorded for the determination of the ΦΔ value of BODIPY 1 in the presence of DPBF in
DMSO.
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2,6-Diiodo-3,5-dithienylvinyleneBODIPY cyclodextrin inclusion complexes
Fig. 8. Bar charts showing the in vitro dark cytoxicity (dark) and the effect of 20 min irradiation of BODIPY 1
(A), the 1@PF127@FC micelles (B) and BODIPY 2 (C) with a Thorlabs 660 nm LED. Error bars set at
± 2σ are provided based on nine measurements that were made at each concentration.
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Fig. 9. Images of the MCF-7 cells showing the effect of 20 min of irradiation with a Thorlabs 660 nm LED where
A is the control, and B and C show cells treated with BODIPY 1 and the 1@PF127@FC micelles at 40
μg.mL−1, respectively. Scale = 200 μm.
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Tables
Table 1. A summary of the phase solubility studies for BODIPY 1 with Pluronic® F-127.
Temp. (°C)
slopea
0.0173
0.0248
R2
K1:1 (M−1)b
Log(K1:1)
4.10
CEb
0.02
0.03
%CE
BODIPY 1 and
25
37
0.948 13000
0.998 20800
2
3
Pluronic® F-127
4.30
aValues obtained directly from phase solubility diagram, bvalues calculated from phase solubility diagram data using
Equations 2 and 3.
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Table 2. A summary of the photophysical properties of BODIPYs a, b and 1 in DMSO.
Log ε
4.84
4.64
4.69
λabs / nm
502
λex / nm
502
λem / nm Stokes shift / cm−1
ΦF
0.03
an.d.
<0.01
τF / ns
an.d.
512
---
389
---
a
b
1
533
---
an.d.
641
642
665
515
3.02
a - n.d. implies that the value is too low to be detectable.
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Table 3. A summary of the ΦΔ values for the iodinated BODIPY dyes.
Solvent
DMSO
DMSO
Ethanol
Water
Scavenger
ΦΔ ± 0.01
0.82
DPBF
b
1
0.13
0.40
0.49
0.40
DPBF
1@PF127@FC
ADMA
DPBF
DMSO
2
26