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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)

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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)

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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)

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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)

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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

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Cel

l via

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y (%

)

Figure S10. Cell viability (MTT assay) as a function of the bromelain concentration.