Abstract
The advanced use of a pH-responsive biomaterial-based injectable liquid
implant for effective chemotherapeutic delivery in glioblastoma
multiforme brain (GBM) tumour treatment is presented. As an implant, we
proposed a water-in-oil-in-water multiple emulsion with encapsulated
doxorubicin. The effectiveness of the proposed therapy was evaluated by
comparing the cancer cell viability achieved in classical therapy
(chemotherapeutic solution). The experimental study included doxorubicin
release rates and consumption for two emulsions differing in drop sizes
and structures in the presence of GBM-cells (LN229, U87 MG), and a cell
viability. The results showed that the multiple emulsion implant was
significantly more effective than classical therapy when considering the
reduction in cancer cell viability: 85% for the emulsion-implant, and
only 43% for the classical therapy. A diffusion-reaction model was
adapted to predict doxorubicin release kinetics and elimination by
glioblastoma cells. CFD simulations confirmed that the drug release
kinetics depends on multiple emulsion structures and drop sizes.
1. INTRODUCTION
Drug delivery in the treatment of the central nervous system diseases -
CNS (brain tumours, trauma, infections, neurodegenerative problems,
amongst others) requires passing through, or bypassing, the blood-brain
barrier (BBB). The methods of drug administration to the CNS can be
divided into three main groups: invasive techniques, non-invasive
techniques and alternative routes.1,2,3 The
non-invasive techniques explore approaches in which pharmaceuticals are
re-engineered to cross the BBB via: (i) chemical methods (lipophilic
analogues, prodrugs, enzymatic reactions or chemical bonding of drug
molecules with transport facilitated molecules), or (ii) biological
methods (drug attachment to proteins specific for receptors responsible
for transport across the BBB, transport vectors or barrier-crossing
peptides). In addition, nanoparticles, dendrimers, liposomes, micelles,
micro/nanoemulsions, including targeted drug delivery systems, and
stimuli-responsive functional biomaterials in the drug delivery area,
are used to cross the BBB.4,5,6 The invasive
techniques include: (i) local surgical treatment combined with adjuvant
therapy (intracerebral polymer implants or microchips,
intraventricular/intrathecal or interstitial drug delivery, biological
tissue delivery) or (ii) controlled BBB damage with drug delivery (e.g.
convection-enhanced drug delivery, osmotic or ultrasound disruption of
the BBB). Surgical treatment in combination with radio and chemotherapy,
in the case of brain tumours, plays a fundamental role in neurooncology.
If possible, tumours should be removed completely. In most cases, only
part of the tumour is surgically removed, and the remainder is
irradiated or subjected to chemotherapy for destruction. The alternative
methods bypass the cardiovascular system and include transnasal
administration of drugs or iontophoretic delivery. Modern medicine
responds to the needs of the patient based on various strategies
including mathematical modelling for predicting the effect of
chemotherapy on cancer cells. The approaches to simulating cancer
interaction with therapy can generally range from (i) cancer growth
models, then (ii) mass transfer models, which combine drug release,
transport, and elimination in tissue, to (iii) cell-based models at the
molecular level. The first group includes models of tumour growth/volume
change based on the analysis of cell proliferation and cell death, along
with the structure of an avascular (solid) tumour (homogeneous or
heterogeneous), and a vascular heterogeneous tumour, including
tumour-induced angiogenesis.7,8 The second group –
reaction-diffusion/convection models are related to transport mechanisms
for delivering drugs to the tumour and drug elimination in tissue by
chemical reaction. These models provide macroscopic descriptions of the
system based on space- and time-dependent variables such as drug
concentration distribution, pressure in the tumour environment, and drug
release rates.9,10,11 The third group represents
discrete cell-based models describing the dynamics of cancer cells via
individual cell behaviours within the tumour tissue based on the
transformation and transport of substances to the cell at the molecular
level. Models that treat cells individually can combine descriptions at
the subcellular and cellular levels, with a macroscopic description of
the tumour environment. This extended approach via, e.g. the
lattice-based method or cellular automata, creates new hybrid
computational models for simulating the cancer intercellular adhesion
and invasion process.12,13,14,15,16 In the present
study, a diffusion-reaction model was adapted to predict the accurate
chemotherapeutic concentration after its in vitro release from a liquid
implant in the presence of glioblastoma multiforme cells. Glioblastoma
multiforme (GBM) is a primary malignant tumour of the central nervous
system with one of the worst prognoses. Despite huge progress in the
field of oncology, the median survival rate for patients after diagnosis
is less than 2 years.2 As the liquid implant, we
proposed multiple emulsions W/O/W (water-in-oil-in-water) type with
encapsulated doxorubicin (chemotherapeutics) in the internal droplets,
Fig. 1. Multiple emulsions are complex dispersed systems with a
hierarchical structure of droplets of the first liquid dispersed in
larger drops of a second immiscible liquid, which is a dispersion medium
for smaller droplets. The larger drops are then dispersed either in the
continuous phase of the first liquid (double emulsion) or in other still
larger drops of the first or other immiscible liquid and so on (a
multiple emulsion). A multiple emulsion can be then a double, triple,
quadruple, quintuple, or even more structured system. These dispersed
systems offer a wide range of possible applications in separation
processes and environmental protection (alternative fuel),
pharmaceuticals, and medicine, especially for the encapsulation and
controlled release of active ingredients (drugs, living cells, antigen
delivery, cosmetics, food).17,18,19,20,21,22,23,24,25Controlling by multiple emulsions is achieved through the size and
physicochemical parameters of drops forming liquid-permeable membranes
separating the internal droplets from the continuous external phase. The
proposed method involves inserting the liquid implant intraoperatively
into the cavity after surgical resection of the tumour, Fig. 1. This
method is a bypassing of the BBB and belongs to the group of adjuvant
treatments. Administering the drug in the form of multiple emulsions is
intended to support the treatment, i.e. the cytotoxic effect on the
tumour cells remaining after the surgery to prevent the recurrence of
the tumour. The chemotherapeutic agent is released from the liquid
implant into the GBM environment in a manner controlled by the pH of the
tumour environment and also the drop size, structure, or physicochemical
parameters of the emulsion (viscosity, density). The external phase of
the emulsion contains a biopolymer (sodium carboxymethylcellulose)
which, depending on the pH, changes the conformation of the chains and
the viscosity, thus affecting the release rates. Such properties of the
polymer are exploited in this concept for controlled release under the
acidic tumour microenvironment. The use of the implant in a liquid form
also reduces the risk of mechanical damage to healthy tissue during the
intraoperative insertion in comparison to a solid implant. The paper
aims to find optimal brain cancer treatment based on locally controlled
chemotherapeutic release from an emulsion-based implant, including
numerical simulations using a diffusion-reaction model for drug
transport and consumption. To evaluate this model, the paper includes a
comparison of these simulations with experimental data on anti-cancer
drug release and consumption for two emulsions differing in drop sizes
and structures. Also, cancer cell viability was investigated in
comparison with classical chemotherapy involving cells treated with a
drug in a solution.
2. MATERIALS AND METHODS
2.1 Preparation and characterization of multiple emulsions with
anti-cancer drug (drop size, viscosity)
Multiple/double emulsions W1/O/W2 with doxorubicin hydrochloride - DOX
(anti-cancer drug) were prepared in a Couette-Taylor flow (CTF)
apparatus where liquid phases were intensively mixed due to rotational
and axial flows. The emulsions preparation conditions are shown in Table
1. The detailed procedure for the preparation of the emulsion can be
found in the previous authors’ works26,27,28. In
short, the internal water phase (W1) with DOX and soybean oil as an
organic membrane phase (O) were introduced in the inlet cross-section of
the CTF apparatus and intensively mixed to create simple emulsions W1/O.
Then, after introducing the water phase (W2) to the simple emulsions in
the middle section, double emulsions W1/O/W2 were formed. The CTF
apparatus provides high mass transfer parameters and uniform shear flow,
contributing to the high encapsulation efficiency and formation of
stable emulsions.28,29,30 The structures of the
obtained emulsions were analyzed using an Olympus BX60 optical
microscope, with Olympus SC50 digital camera (Olympus, Japan), and image
analysis software, Image Pro Plus 4.5 (Media Cybernetics, USA). For each
of the double emulsions, at least 800 drops of the membrane phase and
1000 drops of the internal phase were measured. Then drop sizes
distributions were determined and the average drop sizes: the Sauter
mean diameter of the internal (d32) and membrane phase
(D32) drops. Also, the volume fractions of the internal
phase drops in the membrane phase drops were determined (packing volume
fraction). The fluorescence spectrofluorometer FLUOstar Optima (BMG
LABTECH, Germany) was used to measure the concentration of the
chemotherapeutic agent in the emulsion continuous phase to determine the
encapsulation efficiency of the DOX in the internal phase droplets. The
emulsions rheological tests were performed with a RheolabQC rotational
rheometer (the measuring system of concentric cylinder geometry - gap
size: 1.64mm, length 60mm, cone angel: 120°, ratio of radii: 1.08, range
of shear rate: 1-1500 s−1, 37°C, Anton Paar, Austria).
Two stable soybean oil-based emulsions, differing in the internal
structure of the drops, with pH-responsive biopolymer (sodium
carboxymethylcellulose - CMC-Na) in the external phase were selected for
further experiments (Table 1). The emulsions were characterized by a
high encapsulation efficiency of (DOX) (>95%), calculated
based on the difference in DOX concentration introduced to the CTF
apparatus and in the external phase of created multiple emulsions
according to the procedure26. All compounds used to
prepare emulsions were supplied by Sigma Aldrich. Detailed data on the
composition and preparation conditions of the emulsions can be found in
Table 1.
2.2 Glioblastoma model cell-lines culturing
The in vitro studies on cell viability and release of the
anti-cancer/cytostatic agent from multiple emulsions were conducted for
selected tumour cell lines of glioblastoma multiforme: U87 MG, LN229.
The cell lines were procured from the Institute of Biochemistry and
Biophysics PAS (Poland). The cells were cultured on 10 cm dishes
(BD-Falcon, USA) to 80-90% confluence in DMEM medium with high glucose
and L-glutamine (HyClone, Poland), with 10% fetal bovine serum (FBS;
Gibco, Poland) and 1% penicillin/streptomycin antibiotics (Life
Technologies, Poland) in an incubator (37°C; 5% CO2).
The cells were passaged 24 hours prior to release and cytotoxicity
experiments (cells were first rinsed with phosphate-buffered saline-PBS
buffer (Lab Empire, Poland), then trypsinized by 0.25% trypsin and
0.1% EDTA (HyClone, Poland).
2.3 Release of anti-cancer drug from multiple emulsions
The in vitro release experiments of the anti-cancer/cytostatic drug
(DOX) from multiple emulsions were carried out in the systems with and
without glioblastoma cells (U87 MG, LN229) to determine the drug
consumption by cells. Standard 12-well plates were used, 4000
cells/wells were seeded 24h before the release experiment and placed in
the incubator (temperature: 37°C, 5% CO2, release
volume: 1 cm3). The release process was analyzed for a
specific concentration of DOX encapsulated in the emulsion (0.1 µM, 0.2
µM). The target concentration of DOX was obtained by diluting the
emulsions with PBS buffer of pH = 7.4 (1:100, 1:50 - volume of emulsion
to PBS buffer). At certain time intervals during the 24h experiment with
cells and 96 h without cells, the entire volume of the diluted emulsion
was taken from each well (1 cm3) and filtered through
a hydrophilic syringe filter (nylon filter membrane, 0.2 µm). The DOX
concentration in the samples was determined using a fluorescence
spectrofluorometer - FLUOstar Optima (BMG LABTECH, Germany), measured at
a wavelength of Ex 488 nm/ Em 593 nm (excitation/emission). Prior to the
release studies, the effect of PBS buffer at pH 7.4 on cell viability
was monitored. It was assumed that the relative viability of cells was
not lower than 80%. The measurement of cell viability was performed in
the presence of cells in PBS buffer for 3h, 6h, 9h, 24h. The
measurements were carried out 24h after re-positioning the cells in the
full culture medium, according to the procedure described in the
cytotoxicity studies. These results determined the limitation of the
experiment time, when the cells were surely in good condition, to the
maximum time of 7h. Measurements of concentration after 24 hours in a
system with cells were excluded due to cell viability below the minimum
required (U87 MG 58±12%.; LN229 65±11%).
2.4 Elimination rate constant of anti-cancer drug in the presence of
cancer cells
The doxorubicin (DOX) elimination/consumption rate constants by GBM
cells (U87 MG, LN229) were determined based on the DOX depletion in the
system. The mass fraction of DOX absorbed by cells was calculated as the
difference between the concentration of DOX released from the multiple
emulsion in a cell-free environment and the presence of cancer cells.
The mass fraction of the drug available was determined by subtracting
from the value 1 (the mass fraction corresponding to the total drug
availability at time t=0) the mass fraction of DOX absorbed (consumed)
by cells for a given time. The obtained values were presented as a
function: ln(1-mass fraction of consumed DOX) vs time with a linear
approximation following the kinetics of the first-order reaction. The
drug elimination rate constant was then found as a slope of this
function. The drug consumption rate constants and the linear function
fit coefficients were determined for each glioblastoma cell line and for
the tested DOX concentrations in emulsions DOX-E1 and DOX-E2, which are
summarised in Table 2.
2.5 Cytotoxicity study of multiple emulsions with DOX
In vitro cytotoxicity tests were performed using the REDOX test based on
Alamar Blue reagent (Invitrogen, USA) according to the manufacturers’
instructions. Cells (U87 MG,
LN229) were seeded on standard 96-well plates with 400 cells/wells to
maintain a proportional number of cells in relation to vessel geometry
for release and cytotoxicity studies. After 24 hours from seeding, cells
were exposed to a potentially cytotoxic agent (DOX). Cytotoxic effects
were determined for the DOX concentrations in the emulsions at 0.1 µM
and 0.2 µM. These dosages of DOX were obtained by the dilution of
emulsion in a culture medium with the proportions 1:100 and 1:50,
respectively. In cytotoxicity studies, the emulsion was diluted with a
full culture medium, with a composition identical to that used for the
cell culture. In earlier studies, the effect of changes in DOX
concentration on U87MG cell viability was checked26.
The cell viability was determined for cells treated with emulsion,
without DOX (E1 and E2 -negative control), emulsion with encapsulated
DOX (DOX-E1 and DOX-E2), and DOX solution in full culture medium. The
measurements were carried out for the three different times of cancer
cell contact with a potentially cytotoxic agent: 24h, 48h, 72h. After
the established contact time, the culture medium was exchanged for a
fresh culture medium with Alamar Blue reagent (1:10 v/v). After 24h and
168h the RFUs (relative fluorescence units) were measured at a
wavelength of Ex 560 nm/Em 590 nm (excitation/emission) using a
multimode detector (DTX880, Beckman Coulter, Canada). The relative cell
viability was calculated as a ratio to that of the untreated control
cells. All obtained values are the result of three independent
experiments. Each of the
parameters was determined in triplicate.
2.6 Emulsion viscosity measurements
A RheolabQC rotational viscometer with a double gap measuring cylinder
(DG42) (Anton Paar, Austria) was used to study the rheological
properties of the emulsions DOX-E1 and DOX-E2, and the external phases
of both emulsions. To study multiple emulsions as systems responding to
changes in a pH environment, emulsions and their external phases were
diluted before measurement with a
phosphate buffer (PBS buffer) at pH 7.4 (alkaline) and pH 6.3 (acidic).
Samples were diluted in a volume dilution ratio of emulsion or external
phase in PBS buffer at 1:10 (volume of tested sample to PBS buffer).
Measurements were performed for 3 independently prepared samples in the
shear rate range 50-2500 s-1 during testing. Samples
of emulsions for rheological tests were placed in the measuring cylinder
immediately after the emulsification process. The tests were performed
after establishing the temperature of the sample at 37°C. During the
measurement, the emulsion structures were microscopically observed to
ensure the stability of the emulsions.
2.7 Statistical analysis
All data in this study were expressed as a mean±standard error (±SD) of
at least three independent experiments. Each of the parameters/values
was determined in triplicate.
2.8 Mathematical model development of drug release from the
emulsion-based implant and drug elimination by cancer cells