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Supporting Information
Bromelain-functionalized multiple-wall lipid-core nanocapsules: formulation,
chemical structure and antiproliferative effect against human breast cancer cells
(MCF-7)
Catiúscia P. Oliveira1,‡,*, Willian A. Prado2, ‡, Vladimir Lavayen2, Sabrina L. Büttenbender2,
Aline Beckenkamp1, Bruna S. Martins2, Diogo S. Lüdtke2, Leandra F. Campo2, Fabiano S.
Rodembusch2, Andréia Buffon1, Adalberto Pessoa Jr3, Silvia S. Guterres1, Adriana R.
Pohlmann1,2,*
Supporting information for section
2.3.1.1 Coupling of 2-(5'-amino-2'-hydroxyphenyl)benzoxazole with iodooctane
Benzazole probes have been previously synthesized from aminohydroxyphenyl derivatives28.
To improve the lipophilicity of 2-(5'-amino-2'-hydroxyphenyl)benzoxazole (5AHBO) we
coupled it with iodooctane as previously described29. Briefly, 5AHBO (2.21 mmol), iodooctane
(2.21 mmol) and potassium carbonate (in excess) were solubilized in 2-butanone (20 mL) under
refluxe for 22 h. Then, part of the solvent was evaporated. The mixture was cooled at 4 ºC
(refrigerator) and, after precipitation, hexane (25 mL) was added. The mixture was filtered and
the filtrate evaporated. The residue was added of hexane to purification by chromatography
using a column of silica gel (70-200 mesh) and a gradient of hexane and dichloromethane (1:1;
7:3 and 0:10, v/v). 2-(5’-N-Octylamino-2’-hydroxyphenyl)benzoxazole (5AHBO-C8) was
obtained in 60% (mol/mol) of yield. UV-visible and fluorescence analyses were performed in
Varian (Cary® 50 and Cary Eclypse) instruments after dissolving 5AHBO-C8 in
acetonitrile/water (1:1, v/v), showing absorption maximum located in the violet region of the
visible spectrum (395 nm) and emission maximum located in the cyano-green region (496 nm).
The chemical identity of 5AHBO-C8 was confirmed by 1H NMR.
7.80 7.75 7.70 7.65 7.60 7.55 7.50 7.45 7.40 7.35 7.30 7.25 7.20 7.15 7.10 7.05 7.00 6.95 6.90 6.85 6.80 6.75 6.70Chemical Shift (ppm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Intens
ity
2.151.01 1.000.99 0.980.97
11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Inte
nsity
3.0010.341.990.782.111.000.991.010.80
N
O
OH
NH CH3a
b
c d
e
f
g
h
i
j
k
l
m
n
o
Figure S1. 1H NMR spectrum of 5AHBO-C8, CDCl3, VARIAN INOVA 300 MHz.
Supporting information for section
2.3.1.2 Coupling of rhodamine B with poly(ε-caprolactone)
The coupling of RhoB with PCL was performed as previously reported30. Briefly, RhoB
(0.320 mmol) and EDC.HCl (0.320 mmol) were dissolved in dry dichloromethane (10 mL) at 5
°C (ice bath) under magnetic stirring. After 1 hour of reaction, DMAP (0.032 mmol) was
added, and the reaction medium was stirred at 5 °C for 1 h. PCL (0.200 mmol) solubilized in
dry dichloromethane (15 mL) was added to the reaction medium. After 5 days at 30 °C under
argon, the reaction medium was extracted with 1 mol L-1 HCl followed by NaHCO3 saturated
solution. The organic solvent was removed under reduced pressure and the residue purified by
liquid column chromatography using silica gel (70-200 mesh) as stationary phase and a
gradient of CH2Cl2/CH3OH from 98:2 to 95:5 (v/v) was used as eluent. The product was
characterized by thin layer chromatography, eluted with CH2Cl2:CH3OH (90:10, v/v).
Absorption spectroscopy (Varian Cary® 50) and fluorescence emission (Varian, Cary Eclypse)
were performed using chloroform as solvent, and the average molar weight was determined by
size exclusion chromatography (Viscotec, GPCMax Triple Detector) using refraction index
detector.
Figure S2. Thin layer chromatography carried out in siílica gel using CH2Cl2/CH3OH (9:1,
v/v): spot left = RhoB; co-spots center = RhoB and PCL-RhoB conjugate; spot right = PCL-
RhoB conjugate.
Supporting information for section
2.3.1.3 Synthesis of acridine orange-chitosan conjugate
Step 1: Alkylation of acridine orange
Alkylation of acridine orange (AO) was carried out using an alkyl dihalide previously
described for the synthesis of trifunctional 99mTc based radiopharmaceuticals31. After
complete solubilization of acridine orange (free base) (0.19 mmol) in dry toluene, diiodobutane
(3.80 mmol) was added. The reaction was refluxed for 24 h under argon. Then, toluene was
added (15 mL) and the reaction medium was maintained at 4 ºC, overnight, to precipitate the
product. The precipitate was filtered and extracted with toluene (2 x 5 mL) and toluene added
of traces of NH4OH (2 x 8 mL). The product was solubilized in dichloromethane and the
orange solution was diluted with hexane until precipitation of the product. The medium was
alkalinized using NH4OH and stirred for 10 min at room temperature. The mixture was filtered.
The precipitate was extracted with hexane and the dried product (alkylated-AO) had an aspect
of dark orange powder. 1H NMR (300 MHz, CDCl3) (δ, ppm): 8.49 (s, 1H); 7.77 (d, 2H, J = 9.4
Hz); 6.97 (dd, 2H, J = 9.4, 2.3 Hz); 6.65-6.59 (m, 2H); 4.92-4.85 (m, 2H); 3.45 (t, 2H, J = 5.8
Hz); 3.29 (s, 12H); 2.32-2.21 (m, 2H); 2.14-2.02 (m, 2H).
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5Chemical Shift (ppm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Inte
nsity
12.00 2.392.06 2.05 2.03 1.970.98
3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0Chemical Shift (ppm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Inte
nsity
12.00 2.392.041.45 1.05
N+
I
N
CH3
CH3
NCH3
CH3
Figure S3. 1H NMR spectrum of iodobutyl acridine orange, CDCl3, VARIAN INOVA 300
MHz.
Step 2: Coupling of alkylated-AO with chitosan
Chitosan (100.0 mg) was dispersed in dimethylformamide (DMF) (9.0 mL) and 50% acetic
acid aqueous solution (1.5 mL). After the addition of K2CO3 (0.20 mmol) the medium was kept
under stirring for 30 minutes at 80 °C. The reactant was solubilized, and, then, the alkylated-
AO (0.036 mmol), previously dissolved in DMF (1.0 mL), was added. After 72h, the reaction
medium was alkalinized. The medium was extracted with CH2Cl2 (8 x 25 mL) to remove free
alkylated-AO and a colorless CH2Cl2 phase was obtained. The medium containing AO-labeled
chitosan (CS-AO) was evaporated under reduced pressure. The solid product was extracted
with distilled water and residual DMF was removed by dispersing the product in a saturated
NaCl aqueous solution as previously reported for DMF-water binary mixtures32. The product
was characterized by absorption spectroscopy in the UV-Vis region, fluorescence emission
(triangular cuvette) and FTIR in KBr pellet.
Supporting information for section
2.3.2 Synthesis of multifluorescent-labeled multi-wall lipid-core nanocapsules
The lecithin-polysorbate 80-coated oil- and polymer-labeled lipid-core nanocapsules were
prepared with the conjugates: 5AHBO-C8 and PCL-RhoB. The formulation was named
5AHBO-RhoB-LNC. Soybean lecithin (60 mg) was dissolved in ethanol (4 mL) and poured
into the acetone solution (25 mL) containing PCL (99.5 mg), PCL-RhoB (0.5 mg), 5AHBO-C8
(1.0 mg), sorbitan monostearate (40 mg), and CCT (120 mg). The obtained solution was
injected into the aqueous solution containing polysorbate 80 (80 mg) under moderate magnetic
stirring at 40 °C. The organic solvents and the excess of water were removed under reduced
pressure at 40 °C. The final volume was adjusted to 10 mL in a volumetric flask. The 5AHBO-
RhoB-LNC was coated using a blend of CS and CS-AO (1:1, w/w). The chitosan coating was
performed as described in section 2.2.2 using 1% CS.CS-AO blend in acetic acid aqueous
solution. The formulation was named 5AHBO-RhoB-LNC-CS.CS-AO.
Supporting Information for section
2.4 Pre-formulation study to obtain phenylalanine-functionalized multi-wall lipid-core
nanocapsules
After obtaining the lecithin-chitosan-polysorbate 80-coated lipid-core nanocapsules as
reported above in sections 2.2.1 and 2.2.2, zinc acetate and phenylalanine (Phe) were used at
three concentrations (Table SI) maintaining constant the molar proportion 1:3 of Zn+2 and Phe.
In this way, 1 mL of the zinc acetate aqueous solution was added to 9 mL of LNC+60.100
formulation under moderate magnetic stirring. Immediately after the zinc-II addition, in
aqueous solution (1 mL) was added phenylalanine. The reaction medium remained under
moderate magnetic stirring for 2 h at room temperature (20 ºC). The formulations were named
Phe-MLNC-Zn25, Phe-MLNC-Zn50 and Phe-MLNC-Zn100.
Table SI. Lipid-core nanocapsules: formulations and their compositions.
Formulation MaterialsPCL 100±1 mgSorbitan monoestearate 39±2 mgCCT 0.120 mLAcetone 25 mL
LNC-6 Lecithin 60±2 mg
Ethanol 4 mLPolysorbate 80 81±1 mgWater 50 mL
LNC+60.100 Chitosan 100.3±1.5 mg
Phe-MLNC-Zn25 ZincPhenylalanine
250.0±1.5 µg mL-1
1902.5±2.5 μg mL-1
Phe-MLNC-Zn50 ZincPhenylalanine
500.0±1.0 µg mL-1
3806.7±5.8 μg mL-1
Phe-MLNC-Zn100 ZincPhenylalanine
1000.7±1.2 μg mL-1
7610.0±10.0 μg mL-1
* Each formulation was prepared in triplicate batches (n=3).
Supporting information for section
2.6 Laser diffraction analysis
Laser diffraction analysis (Mie scattering) was performed using a Mastersizer® 2000
(Malvern Instruments Ltd. UK). Measurements give particle size distribution in the range from
0.020 to 2000 µm. Each sample was directly inserted, without previous treatment, into the wet
sample dispersion unit (Hydro 2000SM - AWM2002, Malvern) containing about 100 to 150
mL of distilled water (2,000 rpm). Background, recorded before each analysis, was discounted.
Specific surface area (SA) was determined considering the density of the colloids as the unit.
The results are expressed as volume weighted mean diameter (D[4,3]), and the width of the
particle size distribution (Span) was calculated using Equation 1.
Span = [d(0.9)-d(0.1)]/d(0.5) (1)
where d(0.1), d(0.5) and d(0.9) are the particle diameters at 10%, 50% and 90% of the
undersized particle distribution curves determined by the software furnished by Malvern.
Supporting information for section
2.7 Dynamic light scattering and Zeta Potential
Dynamic light scattering (DLS) and zeta Potential were performed using a Zetasizer® Nano
ZS (Malvern Instruments, UK). Hydrodynamic mean diameter was determined by the method
of Cumulants for unimodal size distributions and expressed as z-average diameter. The relative
variance was determined and expressed as polydispersity index (PDI). In parallel, for
comparison hydrodynamic mean diameter (Dh) and peak width were also calculated by the
CONTIN algorithm. Samples were diluted 250 times in ultrapure water (MilliQ®) and analyzed
at 25 ºC. The samples for Zeta potential were diluted (250 times) in 10 mmol L−1 NaCl aqueous
solution, and analyzed at 25 °C and the results obtained from electrophoretic mobility.
Supporting information for section
2.8 Nanoparticle tracking analysis
Nanoparticle tracking analysis (NTA) was used to determine the mean diameter, the D50
(median diameter), the D90 (particle diameter at percentile 90 under the particle size
distribution curves) and the concentration of nanocapsules per volume, expressed as particle
number density (PND). The scattered light of individual nanoparticles under Brownian motion
is registered for 60 seconds using automatic shutter and gain adjustments, and tracked by the
software (NTA 2.0 Analytical software) using Nanosight® LM10 (Nanosight Amesbury, UK)
equipped with a sample chamber with a 640 nm laser as source. Formulations were diluted
5000 times in ultrapure water (pre-filtered, 0.45 µm) and injected into the sample chamber.
Supporting information for section
2.11 Zinc quantification
To validate the analytical methodology, in 10 mL volumetric flasks containing zinc-II at 200,
300, 450, 600, and 800 ng mL-1 were added of 1 mL of dithizone (13 ng mL-1) in methanol
solution. After complex formation, each solution was poured into an optical cell to analysis (λ =
514 nm). The linear regression coefficient (r) was higher than 0.999. The intermediate precision
and repeatability had coefficient of variations of 0.61% and 0.51%, respectively. The accuracy
at 0.200 μg mL-1, 0.450 μg mL-1 and 0.600 μg mL-1, were respectively 100±3%, 100±1% and
98±1%. The limit of quantification was 28 ng mL-1.
Dialysis of formulations was performed to isolate the amount of zinc-II soluble in the
continuous phase of the formulation. In this way, 5 mL of the formulation (n=3) was placed in
regenerated cellulose dialysis membranes (10 kDa, USA). Each dialysis bag was placed in 50
mL of ultrapure water (MilliQ®). The experiment was conducted under magnetic stirring at 25
°C for 6 hours. Every 2 hours, the water in contact with the dialysis bag was removed (solution
Dn) and each bag was immersed into new 50 mL of ultrapure water. Sample of the solutions
Dn were analyzed at each time interval by UV-Vis spectroscopy (UV-1800 PC EC, Pro-
Analise, Brazil). The percentage of zinc-II in the formulations was calculated considering
equation 2,
CZn=[T−( x+ y+z )]
Tx 100 (2)
where CZn is the percentage (%) of Zn+2 coordinated to the nanocapsules (w/v), T is the total
concentration of Zn+2 added in the formulation and x, y and z are the concentrations of zinc-II
in each solution Dn analyzed.
Supporting Information
0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 0.01 1 100 10000
LNC+3 LNC+
6 LNC+9
A)
B)
C) C) C)
B)B)
A)A)
D)
H) H) H)
G)
F)
E)
D)
G)
F)
E)
D)
F)
E)
G)
Figure S4. Laser diffraction diameter profiles expressed in micrometers by versus percentage
of volume of particles: chitosan-lecithin-polysorbate 80-coated lipid-core nanocapsules (LNC+)
prepared using different concentrations of soybean lecithin (3, 6 and 9 mg mL-1) and chitosan
(CS): A) 0.001% CS; B) 0.003% CS; C) 0.005% CS; D) 0.010% CS; E) 0.030% CS; F) 0.050%
CS; G) 0.080% CS; H) 0.100% CS.
Figure S5. Infrared spectra recorded for CS and CS-AO.
Figure S6. Secondary light colors produced by the simultaneous emissions of the probes in
dried 5AHBO-RhoB-LNC-CS.CS-AO formulation observed at the same sites on the slide by
confocal microscopy: a) cyano (blue and green, 5AHBO-C8 and CS-AO), b) magenta (blue and
red, 5AHBO-C8 and PCL-RhoB), and c) yellow (red and green, PCL-RhoB and CS-AO).
Table SII. Volume-weighted mean diameter (D[4,3]), polydispersity (Span) and zeta potential
of Bro-MLNC-Zn using different concentrations of the bromelain and Zn+2 by laser diffraction
and electrophoretic mobility.
Formulation D[4,3] (nm) Span Zeta potential (mV)
Bro10-MLNC-Zn50 4761±4007 45.17±76.38 +13±6
Bro25-MLNC-Zn50 773±96 0.95±0.04 +18±5
Bro50-MLNC-Zn50 126±1 0.95±0.05 +23±3
Bro100-MLNC-Zn50 704±482 0.96±0.01 +22±9
Bro200-MLNC-Zn50 1949±1728 35.60±60.04 +15±4
Bro100-MLNC-Zn100 965±24 1.37±0.20 +15±5
Note: Data are expressed as mean ± standard deviation.
c)
a)
b)a)
Supporting Information
Transmission electron microscopy analysis
The analysis of transmission electron microscopy (TEM) supplementary figures S7, S8 and
S9.
Figure S7. A) TEM image of a LNC+6 0.001 nanocapsule. B) Cross section dashed line of the
image A. C) Fast Fourier Transformation (FFT) spot image of the nanocapsule. D) A view in 3D on nanocapsule. Bar equals 90 nm.
Figure S8. A) TEM image of a Phe-MLNC-Zn50 nanocapsule. B) Cross section dashed line of the image A. C) Fast Fourier Transformation (FFT) spot image of the nanocapsule. D) A view in 3D on nanocapsule. Bar equals 120 nm.
Figure S9. A) TEM image of a Bro50-MLNC-Zn50 nanocapsule. B) Cross section dashed line of the image A. C) Fast Fourier Transformation (FFT) spot image of the nanocapsule. D) A view in 3D on nanocapsule. Bar equals 70 nm.
Cell viability study
0 50 100 150 200 250 300 350 400 4500
20
40
60
80
100
120
Bromelain concentration (µg mL-1)
Cel
l via
bilit
y (%
)
Figure S10. Cell viability (MTT assay) as a function of the bromelain concentration.