Accepted Manuscript

Title: Drug-loaded nanoemulsion as positive control is an

alternative to DMSO solutions for in vitro evaluation of
curcumin delivery to MCF-7 cells

Authors: Eduarda Schultze, Karine Coradini, Paula dos Santos

Chaves, Liziane Pereira da Silva, Julieti Buss, Silvia S.
Guterres, Tiago Collares, Ruy Carlos Ruver Beck, Adriana R.
Pohlmann, Fabiana Kommling Seixas

PII: S1734-1140(16)30377-2
DOI: http://dx.doi.org/doi:10.1016/j.pharep.2017.05.003
Reference: PHAREP 721

To appear in:

Received date: 17-11-2016

Revised date: 4-5-2017
Accepted date: 9-5-2017

Please cite this article as: Eduarda Schultze, Karine Coradini, Paula dos
Santos Chaves, Liziane Pereira da Silva, Julieti Buss, Silvia S.Guterres, Tiago
Collares, Ruy Carlos Ruver Beck, Adriana R.Pohlmann, Fabiana Kommling
Seixas, Drug-loaded nanoemulsion as positive control is an alternative to DMSO
solutions for in vitro evaluation of curcumin delivery to MCF-7 cells (2010),
http://dx.doi.org/10.1016/j.pharep.2017.05.003

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Drug-loaded nanoemulsion as positive control is an alternative to DMSO

solutions for in vitro evaluation of curcumin delivery to MCF-7 cells

Eduarda Schultzea, Karine Coradinib, Paula dos Santos Chavesc, Liziane

Pereira da Silvaa, Julieti Bussa, Silvia S. Guterresb,c, Tiago Collaresa, Ruy

Carlos Ruver Beckb,c, Adriana R. Pohlmannb,c,d, Fabiana Kmmling Seixasa*

a
Programa de Ps-Graduao em Biotecnologia (PPGB), Grupo de Pesquisa

em Oncologia Celular e Molecular, Laboratrio de Genmica Funcional,

Biotecnologia/Centro de Desenvolvimento Tecnolgico, Universidade Federal

de Pelotas, Pelotas, RS, Brazil

b
Programa de Ps-Graduao em Nanotecnologia Farmacutica, Faculdade de

Farmcia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752,

Porto Alegre, 90610-000, RS, Brazil

c
Programa de Ps-Graduao em Cincias Farmacuticas, Faculdade de

Farmcia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752,

Porto Alegre, 90610-000, RS, Brazil

d
Departamento de Qumica Orgnica, Instituto de Qumica, Universidade

Federal do Rio Grande do Sul, PBox 15003, Porto Alegre, 91501-970, RS,

Brazil

Correspondence author: Dra. Fabiana Seixas, seixas.fk@gmail.com

1
Abstract

Background: In vitro evaluation of toxicity and/or efficacy of nanostructured drug

delivery systems involves the uses of different controls, including positive and

negative controls, as well as a solution or dispersion of the drug in water. One

of the most frequently solvent used to dilute poorly water soluble drugs to in

vitro tests are dimethylsulfoxide (DMSO). However, its different specific surface

area and different diffusion coefficients could make the comparative effects

difficult. We proposed that a solvent-free dispersions having similar specific

surface area could be a better control than drug in solution against cell lines.

Methods: We evaluate the effect of curcumin-loaded lipid-core nanocapsules,

curcumin-loaded nanoemulsion and curcumin DMSO-water solution on viability

and colony forming efficiency of human breast cancer cell line, MCF7.

Results: The cytotoxic effect of nanocapsules at 24-72 h was similar to

nanoemulsion and lower than drug solution. However, the nanocapsules had a

superior anticancer activity when long periods (10 days) were evaluated, which

highlight the sustained drug release by nanocapsules.

Conclusions: Our results showed a superior anticancer activity of curcumin-

loaded lipid-core nanocapsules compared to curcumin-loaded nanoemulsion

and curcumin dissolved in DMSO in long exposition time assay, wihch is not

observed in short exposition time assays like MTT. When a poorly water-soluble

drug is under investigation, the nanoemulsion prepared with the same

compounds of the nanocapsules, except the polymer, could be a better control

than DMSO-solution of drug.

2
Keywords:
Nanocapsules, nanoemulsion, control, drug delivery, lipid-core
Introduction

The very low bioavailability of poorly water-soluble compounds is a

challenge for therapeutic use [1-3]. These compounds generally have low

absorption after oral administration, rapid metabolism and rapid systemic

elimination. Even though high hydrophobic compounds, such as curcumin,

present desirable pharmacodynamics properties, its poor pharmacokinetic

characteristics limit clinical application [1, 4-6].

Much attention has been given to the development and study of the

characteristics of nanostructured drug delivery system as an alternative to

improve the bioavailability of active molecules [7-9]. Nanostructured drug

delivery systems include nanoparticles (nanocapsules and nanospheres),

liposomes, micelles and phospholipid complexes [1].

Curcumin is a compound which has antioxidant, anti-inflammatory,

anticarcinogenic, antimicrobial, antispasmodic, antiviral, hepatoprotective and

many others biologic activities [10]. The chemical name of Curcumin is bis-,-

unsaturated -diketone and is also called diferuloylmethane. This compound

has been used as antitumoral agent against many types of tumor cells [1].

In general, the in vitro evaluation of drug delivery systems is carried out to

access either efficacy or toxicity using different cell cultures as models [11-13].

This evaluation involves the use of diverse controls, such as a negative control,

a positive control, as well as a solution or dispersion of the drug in water. The

latter is assayed to access the influence of nanoencapsulation on the in vitro

response of the drug. Different solvents and dispersants are used to prepare

3
these aqueous solutions or dispersions [14-17]. One of the most commonly

used solvent is dimethylsulfoxide (DMSO) since its physicochemical

characteristics allow the dissolution of a variety of poorly water-soluble drugs

[18, 19].

It is hypothesized that the in vitro efficacy of drug-loaded nanoparticulate

systems might be compared to solvent-free dispersions having similar specific

surface area instead of being compared to drug in solution. The hypothesis is

based on different diffusion coefficients of the drug when entrapped in a

nanocarrier and freely diffusion in solution. Our objective was to evaluate the

cytotoxic effect of curcumin-loaded lipid-core nanocapsules over short and

extended periods of time on human breast cancer cell line, MCF7, compared to

curcumin-loaded nanoemulsion and curcumin DMSO-water solution. The drug-

unloaded formulations were also evaluated as controls.

Material and Methods

Nanocapsules and nanoemulsion preparation

Curcumin-loaded lipid-core nanocapsules and nanoemulsion at a drug

concentration of 1.0 mg/ml were prepared by nanoprecipitation [20] and

spontaneous emulsification [21], respectively. To prepare the polymeric

particles, 0.1 g of polymer [poly(-caprolactone)] (PCL), 0.0385 g of

sorbitanmonostearate,0.165 ml caprylic/capric triglyceride mixture (CCT), and

0.01 g of curcumin were dissolved in 27 ml of acetone. This organic phase was

injected into 54 ml of an aqueous phase containing 0.077 g of polysorbate 80

under moderate magnetic stirring. After this, acetone was removed and the

aqueous phase concentrated under reduced pressure. The formulations

4
containing caprylic/capric triglyceride mixture (CCT) as oil core were named C-

LNCCCT. To prepare the nanoemulsion with CCT as core (C-NECCT), the same

materials and proportion were used, except for adding polymer PCL. Non-

encapsulated curcumin were prepared at a stock concentration of 10 mg/ml in

dimethyl sulfoxide (DMSO) and diluted in medium for treatments of cells. The

concentration of DMSO in the treatments did not exceed 0.5 %.

Mean particle size

Nanoparticles were analyzed regarding the mean particle size (D[4, 3]) values

by laser diffraction (Wet method, Mastersizer 2000, Malvern Instruments, UK).

The size distribution, dispersion index of the particle size (SPAN) and surface

area were also evaluated. All samples were analyzed in triplicate batches (n=3).

Cell culture

MCF7 breast cancer cell line was obtained from the Rio de Janeiro Cell

Bank (PABCAM, Federal University of Rio de Janeiro, RJ, Brazil). Cells were

cultivated in Dulbeccos modified Eagles medium (DMEM), supplemented with

10% fetal bovine serum (FBS), purchased, respectively from Vitrocell Embriolife

(Campinas, Brazil) and Gibco (Grand Island, NY, USA). Cells were grown at 37

C in an atmosphere of 95% humidified air and 5% CO2. The experiments were

performed with cell in the logarithmic phase of growth.

Viability assay

To evaluate the toxicity of curcumin-loaded lipid-core nanocapsules and

nanoemulsion against breast cancer cell, a MTT assay was performed. Cells

were cultivated on a 96 well plate in a density of 2 x 104 cells per well and

allowed to adhere for 24 h in a controlled atmosphere of 5% CO2 at 37 C. Cells

5
were treated with different concentrations of non-encapsulated curcumin

(diluted in DMSO), curcumin-loaded lipid-core nanocapsules and curcumin-

loaded nanoemulsion for 24 h, 48 h and 72 h. After the incubation time, cells

were washed twice with phosphate-buffered saline (PBS; Gibco, Carlsbad,

USA) and 5 mg/ml of MTT solution was added to each well. After an incubation

of 3 h at 37 C in 5% CO2, the formazan crystals produced by viable cells by

reduction of MTT were dissolved in DMSO under agitation.

Clonogenic assay

To determine the efficacy of exposure to different formulations of curcumin

over extended periods of time, the clonogenic assay was performed as

previously described [22]. MCF7 cells were seeded at a density of 400 cells per

well in duplicate six-well plate and allowed to adhere for 24 h without any

treatment. After this period, 10 M of non-encapsulated curcumin, curcumin-

loaded lipid-core nanocapsules and curcumin-loaded nanoemulsion were added

and cells were cultivated for additional 10 days. Cells that grew without any

treatment, only with medium were the negative control. Colonies were stained

with crystal violet and counted.

Statistical analyses

Results were expressed as mean standard error median (SEM). The

experimental data were analyzed for statistical significance by factorial ANOVA

followed by Tukey test for multiple comparisons. Signicance was considered at

p value < 0.05 in all analyses.

6
Results

Mean particle size

The homogeneity of submicrometric nanoparticle population was

determined by laser diffraction technique. C-LNCCCT, and C-NECCT presented

unimodal size distributions at granulometric profiles (Fig. 1). Table 1

summarizes the average diameter, surface areas and size distribution index.

Viability assay

To evaluate the acute effect of different formulations of curcumin on

breast cancer cells, the MTT assay was performed at 24 h, 48 h and 72 h of

incubation. Considering the high hydrophobicity of curcumin, nanocapsules with

different core content were evaluated: caprylic/capric triglycerides mixture or

grape seed oil.

Statistical analyses showed a significant difference (p < 0.001) among

non-encapsulated curcumin treatment group and other treatment groups. The

group treated with nanoemulsion showed the lowest inhibition rates at 24 h.

Differences between groups, except for non-encapsulated curcumin,

disappeared at 48 h. At 72 h, the highest inhibition rates were observed in the

group treated with non-encapsulated curcumin. However, curcumin-loaded lipid-

core (formulation with caprylic/capric triglycerides mixture in core) showed

significant difference from the other groups: F (3,16) = 21.01, p < 0.001 at 24h,

F(3,16) = 6.287, p < 0.01 at 48 h and F(3,14) = 9.098, p < 0.01 at 72h (Fig. 2).

Clonogenic assay

7
 To evaluate the effect of long-term exposure to curcumin-loaded lipid-

core nanocapsules on breast cancer cells, a clonogenic assay with MCF-7 cells

was performed. This assay showed that C-LNCCCT was more efficient in

inhibiting colony formation than C and C-NECCT (F(5,11) = 191.1, p < 0.001).

However, LNC were not inert, and the colony number in this treatment group

was about 50% related to control (Fig. 3).

Discussion

C-LNCCCT and C-NECCT presented nanometric average diameter with

unimodal size distribution. The slightly wider size distribution profile by volume

observed for the C-NECCT (Fig. 1E) can be explained by absence of the

polymeric layer, which could protect the coalescence of some oily droplets [23].

These results coupled with low span values show that the formulations are

homogeneous.

Our group had previously shown that release of curcumin was controlled

and sustained over time [4]. We have also observed that the effect of C-LNCCCT

can be compared to C in some cells only after long periods [4]. Sun J et al also

have shown a better inhibition rate of curcumin dissolved in DMSO compared to

nanoformulations [5]. However, this inhibition does not remain over time. In this

assay, C was quickly uptaken by cells (10-30 min), contrasting with the slow

uptake by the cells of encapsulated curcumin (3 h). In fact, Yu et al reported

that solubilized curcumin permeated across the monolayers rapidly by passive

diffusion [24] and LNC seems to be internalized by endocytosis [4, 25]. Other

authors demonstrated better inhibition of cell growth by C dissolved in DMSO

compared to nanoformulation, arguing that this lower activity was due to

8
incomplete release of curcumin by de nanoformulation during the incubation

time (24-72 h) [26].

Another study that does not shown significant differences between C and
nanoformulations evaluated these results in longer times (5 days),
strengthening the characteristics of controlled release by the nanoformulations
[6]. Besides that, C seems to be rapidly degraded in medium contrary to
nanoformulations [4].

Polymeric nanocapsules differ to nanoemulsion due to the presence of a

polymer coating the oil core, which gives a gradual release of active compound
dissolved in the oil core [1, 27]. On the other hand, both formulations are coated
with surfactant (in this case, Polysorbate 80), which can allow similar
endocytosis-mediated uptake by cells [25, 28, 29].

Conclusions

We proposed in this study that nanoemulsion prepared with the same

compounds of nanocapsules, excepting the polymer is a better vehicle control
than DMSO for evaluation of poorly water soluble drugs in vitro. Our results
showed a superior anticancer activity of curcumin-loaded lipid-core
nanocapsules compared to curcumin-loaded nanoemulsion and curcumin
dissolved in DMSO in long exposition time assay, which is not observed in short
exposition time assays like MTT. This may indicate that the specific surface
area confers a similar drug diffusion in medium and similar uptake of the drug
by cells.
Sources of support: This research was supported in part by CAPES, CNPq
and FAPERGS.
Conflict of interest statement: None of the authors declare a conflict of
interest

9
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Figure captions:

Fig. 1. Size distribution profile by laser diffraction (by volume and number): (A
and B) C-LNCCCT, and (C and D) C-NECCT.

12
Fig. 2. Viability assay. MCF-7 cells were treated with curcumin-loaded lipid-core
nanocapsules (C-LNCCCT), curcumin nanoemulsion (C-NECCT), free curcumin
(C) and blank-lipid-core nanocapsules (LNCCCT) for 24 h, 48 h and 72 h. Bars
represents mean SEM of three independent experiments performed in
triplicate. Different uppercase represents significant difference between groups
(p < 0.001). Different lowercase represents significant difference between
concentrations within the same group (p < 0.001).

13
Fig. 3. Clonogenic assay. Data are expressed as mean SEM from two

independent experiments performed in duplicate. Different letters represent

significant difference between treatments (p < 0.001).

14
15
Tables:

Table 1 - Characterization of formulations by laser diffraction (LD)

Parameter C-LNCCCT C-NECCT

D(4,3) (nm)a 270 6 233 4

D(0,1) (nm)b 130 4 81 8

D(0,5) (nm)c 240 5 172 3

D(0,9) (nm)d 430 8 455 1

SPANe 1.27 0.1 2.12 0.8

Specific surface area m/g 28.3 7 39.6 5

a
particle volume mean; b mean diameter of 10% of the particles; c mean
diameter of 50% of the particles; d mean diameter of 90% of the particles; e
express particle size distribution

16