Journal of Drug Delivery Science and Technology 47 (2018) 181–192
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology
journal homepage: www.elsevier.com/locate/jddst
Cancer chemotherapy: Effect of poloxamer modified nanoparticles on T
cellular function
Grace Lovia Allotey-Babingtona,b,∗, Henry Netteyb, Sucheta D’Saa, Kimberly Braz Gomesa,
Martin J. D'Souzaa
a School of Pharmacy Mercer University Atlanta, GA, United States
b School of Pharmacy University of Ghana Legon, P.O. Box LG 43, Legon, Ghana
A B S T R A C T
The use of excipients with the ability to synergize the action of active pharmaceutical ingredients is highly recommended. Although a large spectrum of surfactants
can be used in the preparation of polymeric nanoparticles, the poloxamer surfactants are currently being explored because they have been shown to preferentially
target cancer cells as well as inhibit Multi-Drug Resistant proteins and other drug efflux transporters on the surface of cancer cells. The proper type and concentration
of surfactant used plays a major role in the stability of the nanosuspensions. The aim of the study was to assess the effect of three different poloxamer surfactant
(Pluronic F-108, pluronic F-127 and kolliphor P-188) and chitosan on the stability, immunogenicity, cytotoxicity of dasatinib nanoparticles, as well as cellular uptake
of nanoparticles by various cell types.
A combination of chitosan and the poloxamers enhanced stability of nanoparticles: average size was 190 ± 20 nm just after preparation, changing to
200 ± 40 nm after 28 days of storage. Uptake of dye-loaded nanoparticles into the cells was 1.5 times more than a solution with an equivalent amount of dye. The
uptake of nanoparticles into all the cell lines used was highest for P-188 nanoparticles, whilst F-127 nanoparticles were the least taken up. Poloxamers were observed
to be non-toxic to the cells, however, stimulation of cell growth was observed in some cases. The properties exhibited by the various poloxamers in this study could
guide in the selection of an appropriate poloxamer for the formulation of nanoparticles.
1. Introduction tremendous attention in the field of oncology in the last two decades.
Research has demonstrated that poloxamer surfactants interact with
Nanotechnology for drug delivery has attracted a lot of attention in multidrug-resistant tumors resulting in increased sensitization of these
the past two decades. It has transformed the approach to the design and tumors to various anticancer agents [2,3]. This property is due to their
delivery of medications for the treatment of diseases such as cancer and ability to block P-glycoprotein (P-gp) pump activity as well as overcome
brain disorders. Among the various types of nanoparticles, polymeric glutathione/glutathione-S-transferase (GSH/GST) detoxification system
nanoparticles have found excessive use in the field of oncology not just [4,5]. These properties in combination with their amphiphilic nature
because of its ability to deliver chemotherapeutic agents to cancer cells, promote their use as stabilizers in the fabrication of polymeric nano-
but, also because they provide the flexibility of modifying the release of particles for tumor targeting. Poloxamers are triblock polymers com-
the entrapped drug and additionally, allow the attachment of various posed of a hydrophobic block sandwiched by two hydrophilic polymer
ligands to the nanoparticle's surface thereby producing a targeted effect blocks. Their peculiar structure gives them the ability to coat the sur-
for precision killing of cancer cells and sparing normal cells. The en- face of hydrophobic nanoparticles, leaving the hydrophilic tails to in-
hanced penetration and retention effect (EPR) observed in the micro- duce stealth properties [6]. Manipulation of the length of the individual
environment of solid tumors promote the accumulation of nano-mate- polymer blocks has led to the existence of different types of poloxamers
rials into solid tumors, producing a targeted effect [1]. Maintaining the with varying properties [22]. In this paper, the effect of three polox-
particle size within the desired range is critical for achieving the ne- amer types (F-108, F-127 and P-188) and their concentration on the
cessary effect, making the selection and the amount of the appropriate stability of dasatinib nanoparticles were evaluated. Additionally, com-
surfactant one of the important steps in the formulation process. bination of these poloxamers with chitosan and their effect on some
The nonionic synthetic surfactants - Poloxamers, also known as cellular functions were investigated in order to selection an appropriate
pluronics were invented in the early 1970's [19] and have attracted formulation for further studies. Dasatinib is an inhibitor of Src, tyrosine
∗ Corresponding author. University of Ghana School of Pharmacy, P.O.Box LG 43, Legon, Ghana.
E-mail addresses: Grace.Lovia.Allotey-babington@live.mercer.edu, glallotey-babington@ug.edu.gh (G.L. Allotey-Babington),
dsouza_mj@mercer.edu (M.J. D'Souza).
https://doi.org/10.1016/j.jddst.2018.06.012
Received 9 January 2018; Received in revised form 29 April 2018; Accepted 15 June 2018
Available online 26 June 2018
1773-2247/ © 2018 Elsevier B.V. All rights reserved.
G.L. Allotey-Babington et al. Journal of Drug Delivery Science and Technology 47 (2018) 181–192
kinases, which have been shown to be over-expressed in a number of 2.4. Fourier transform infrared spectra (FT-IR)
cancers including triple negative breast cancer. It was chosen as a
model drug because has been shown to be effective alone and in FT-IR spectra was recorded for each of the twelve formulations
combination with other chemotherapy agents [7,8,20]. prepared using Shimadzu IRAffinity – IS fourier transform infrared
spectrophotometer. Each spectrum was obtained with 50 scans in the
2. Materials and methods range of 600 to 4000cm-1. This was done to determine the effect of
surfactant amount on the spectra.
2.1. Images in this document are presented in color
2.5. Drug content analysis and encapsulation efficiency
Pluronic F-108 and Kolliphor P-188 were gifts from BASF
Corporation (NJ). Pluronic F-127 was a gift from Dr. Banga, Mercer To determine drug content in the formulation, 10mg of nano-
University, Atlanta. Dasatinib was purchased from LC laboratories particles was weighed and added to 0.2mL of acetone to dissolve the
(Woburn, MA). Polycaprolactone, Coumarin VI and Tween 80 were polycaprolactone polymer matrix. Following dissolution, DMSO was
purchased from Sigma-Aldrich (St Louis, MO). Dulbecco's Modified added to solubilize the entrapped dasatinib. The mixture was kept
Eagle Medium (DMEM), Dulbecco's Phosphate Buffer Saline (PBS) and stirring to allow evaporation of the acetone. Following which the
Fetal Bovine Serum (FBS) were purchased from Atlanta Biologicals mixture was then centrifuged, filtered and diluted for further analysis.
(Atlanta GA). 1% Penicillin- Streptomycin was purchased from Cellgro The actual drug content of the particles was determined using a reverse
(Manassas, VA). MDA-MB-231 & HCC-70 breast cancer cell lines were phase HPLC method [21]. Experiments were done in triplicates.
kind donations from Dr. Zhang's lab Mercer University, Atlanta. Murine After determination of the actual amount of drug in 10mg of na-
dendritic cell line DC 2.4 was obtained from Dr. Kenneth Rock at the noparticles, the theoretical amount of drug supposed to be in 10mg of
University of Massachusetts Medical School. MTS cell proliferation nanoparticles was also calculated. The encapsulation efficiency was
assay kit was purchased from Promega Corporation (Madison, WI). determined using the formulae below.
Sulfanilamide, 98% was purchased from Acros Organics (NJ). N-(1- Theoretical Dasatinib amount
Naphthyl)-ethylene-diamine dihydrochloride was purchased from Theoretical Loading (%) = ⎛ ⎞⎜ ⎟xDasatinib + solid content of formulation
Fisher Scientific Company, (NJ). All other materials used were of ana- ⎝ ⎠
lytical grade. 100
Experimental Dasatinib amount obtained
2.2. Preparation and characterization of nanoparticles Actual loading (%) = ⎛ ⎞⎜ ⎟x 100
⎝ Dasatinib + solid content of formulation ⎠
Dasatinib nanoparticles were prepared by the nanoprecipitation Actual loading
method using three poloxamer surfactants (pluronic F-108, pluronic F- Encapsulation Efficiency (%) = ⎛ ⎞⎜ ⎟x 100Theoretical loading
127, kolliphor P-188) and polycaprolactone (PCL) as the polymer ma- ⎝ ⎠
trix. Four different polymer/surfactant ratios (1:1, 1:2, 2:1, 1:5) were
prepared to investigate the effect of poloxamer type and concentration 2.6. Drug release
on size distribution of nanoparticles. Dasatinib was dissolved in 5mL of
a 10mg/mL solution of polycaprolactone in acetone. The mixture ob- To determine the drug released from the nanoparticles in each
tained was added dropwise to 30mL of the aqueous surfactant solution formulation, 10mg each of the various nanoparticles were weighed into
on a stirrer in the ratios listed above while stirring. The milky mixture Eppendorf tubes and suspended in 1mL of phosphate buffered saline
obtained was stirred on a magnetic stir plate for about 6 hours to allow (PBS, 20mM, pH 7.4) supplemented with 0.1% Tween 80 to promote
for the evaporation of all the acetone. The same procedure was used to dissolution of the released dasatinib from the nanoparticle into the
make coumarin-loaded and empty (blank) nanoparticles. Chitosan medium. The Eppendorf tubes were placed on a mechanical shaker with
coated nanoparticles were prepared by adding 1mL of 0.1% chitosan continuous shaking (80 rpm) at 37 °C. At select time intervals, the tubes
solution dropwise into the beaker containing the immediately pre- were centrifuged at 20,000 g for 15min 0.5mL of the supernatant
cipitated nanoparticles. The suspension was left stirring to allow the was collected and processed for analysis using a reverse-phase
evaporation of the acetone. Nanoparticles were washed twice by cen- HPLC method as mentioned above in section 2.4. To maintain sink
trifugation at 20, 000 g for 20minutes, after which the pellet was lyo- conditions, 0.5 mL of fresh medium (0.1% Tween in PBS) was added to
philized and stored for subsequent studies. each tube, vortexed and placed back on the shaker each time a sample
Nanoparticles were characterized by size and charge using a was taken.
Malvern Zetasizer Nano-ZS (Malvern Instruments Inc. UK) which uti-
lizes the principle of dynamic light scattering. About 1mg of drug na- 2.7. Cellular uptake of nanoparticle observed by fluorescence microscopy
noparticles were weighed and suspended in 10mL of filtered deionized
water. 1 mL of the obtained suspension was put in a cuvette and ana- 200 μL of a 105/mL suspension of MDA-MB-231 breast cancer cells
lyzed. The results reported are average values from triplicate runs of and Dendritic cells were each seeded on pretreated cover slips placed in
three independent experiments. petri dishes. The cells were placed in a humidified CO2 incubator over-
night to equilibrate, following which the cells were exposed to cou-
2.3. Colloidal stability marin VI loaded nanoparticles in media (0.1 mg/mL). Each cell type
was exposed to PCL/F-108, PCL/F-127, PCL/P-188 nanoparticles with
To determine physical stability of Dasatinib nanoparticles in sus- and without chitosan. After 2 hours of exposure, nanoparticle suspen-
pension, 20mL of each formulation was stored at two different tem- sion was gently washed off the surface of the cover slip using cold
perature conditions (25 °C and 40 °C) for four weeks (28 days). The size phosphate buffered saline (PBS). Care was taken not to wash away the
distribution of the particles was measured weekly during the study cells from the surface of the cover slip. Cells were then introduced to
period using the Malvern Zetasizer. fresh media, coverslip was flipped onto a glass slide and observed under
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an Olympus CX41 Fluorescence microscope using a 40X objective. 2.10. Effect of PCL-Poloxamer nanoparticles on the release of innate
Experiments were done in duplicates. immune marker (nitric oxide) from antigen presenting cells
Nitric oxide is one of the innate immune markers released by an-
2.8. Cellular uptake of nanoparticle determined by flow cytometry
tigen presenting cells in response to an exogenous antigen. 105 cells/
well of dendritic cells, DC 2.4, were plated in a 24 well plate and al-
Extent of uptake of nanoparticles fabricated with the three polox-
lowed to equilibrate overnight. To determine the antigenicity of the
amers by various cell types was evaluated by flow cytometry. Coumarin
poloxamer-stabilized nanoparticles, the plated cells were treated with
VI, a fluorescent dye was loaded into the nanoparticles and used for the
0.2 mg/mL drug-free nanoparticles in culture media and then incubated
uptake study for easy quantification. Five different cell lines were used,
at 37 °C. After 16 hours the supernatant was analyzed for nitric oxide
namely: (A) MDA-MB-231 breast cancer cells (B) HCC-70 breast cancer
content using the Greiss chemical method [9].
cells (C) B16-F10 melanoma cells (D) WB rat epithelial cells and (E)
Dendritic cells (DC.2.4). 5× 104 cells/well of each cell type were
2.11. Statistical tests
plated in 24 well plates and left to equilibrate in a CO2 incubator. Cells
were then, exposed to two batches of nanoparticles (coumarin VI
Statistical analyses were performed using the SPSS software. T-test
loaded nanoparticles with and without chitosan) in culture media. After
was used to assess the difference between the formulations prepared. P-
2 hours, cells were trypsinized and washed using cold phosphate buf-
value was used as p > 0.05 (non-significant differences), p < 0.05
fered saline. Following the third wash, pellet obtained after cen-
(*), p < 0.01 (**), and p < 0.001 (***). Error bars represent standard
trifugation was re-suspended in 0.5mL of PBS and placed on ice. The
deviation of uncertainty.
mean fluorescence intensity (MFI) of a fixed number of cells was de-
termined by flow cytometry. Three independent wells were analyzed
3. Results
per group.
3.1. Physical characteristics
2.9. Cytotoxicity of blank nanoparticles on different cell types
Poloxamers: F-108 and F-127 nanoparticle suspensions produced a
Cytotoxicity of empty nanoparticles was investigated in-vitro on bell shaped curve with a narrow size range for polymer/surfactant ra-
three different cell types; rat liver epithelial cells (WB), murine kidney tios 1:1 and 1:2. At low concentrations of F-127 (ratio 2:1) a small
cells (MDCK) and triple negative human breast cancer cells (MDA-MB- population of particles with size greater than 1 μm was observed, while
231). About 5×103 cells/well were plated in a 96-well plate. Cells at high surfactant concentrations (ratios 1:5) the distribution was bi-
were treated with various concentrations of empty nanoparticles pre- modal. Notably, a large population of the particles were in the 100 and
pared using the three poloxamers. Percent cell viability was determined 1000 nm size range. Unlike the other two surfactants, kolliphor P-188
after incubating cells with various concentrations of nanoparticles for did not have the characteristic bell shape. Particle size ranged from
24 hours at 37 °C. The viability of each cell type was evaluated using 1 nm to 10 μm as indicated by the line with the triangle symbols along
MTS assay. the size axis (Fig. 1).
Fig. 1. Effect of poloxamer F-108 (red), F-127 (green) and kolliphor P-188 (blue) on size of nanoparticles at fixed polymer/surfactant ratios. (A) Ratio 1:1, (B) Ratio
1:2, (C) Ratio 2:1 (D) ratio 1:5. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Table 1
Physico-chemical characteristics of Dasatinib nanoparticles prepared by the nanoprecipitation method using polycaprolactone as polymer matrix and three different
poloxamers as stabilizers. Four different polymer/surfactant ratios were used. Average size of particles was measured using a Malvern Zetasizer.
Polymer/surfactant Ratio Poloxamer F-108 Poloxamer F-127 Kolliphor P-188
Size (d.nm) PDI Zeta potential (mV) Size (d.nm) PDI Zeta potential (mV) Size (d.nm) PDI Zeta potential (mV)
1:1 182.0 0.20 −11.7 184.7 0.28 −1.6 698.4 0.88 −6.03
1:2 212.0 0.27 −12.9 168.4 0.29 −6.3 210.3 0.35 −15.4
2:1 215.3 0.31 −14.2 202.3 0.34 −8.4 431.6 0.48 −14.0
1:5 187.7 0.65 −7.8 237.9 0.36 −6.3 247.4 0.51 −11.7
Table 2 Table 4
Average size of nanoparticle determined 48hours after preparation. Average Average size of chitosan stabilized nanoparticles after 48 hours of preparation.
size of nanoparticles increased by ≥ 50% of the initial size.
Polymer/ Particle Size (d.nm)
Polymer/ Particle Size (d.nm) Surfactant Ratio
Surfactant Ratio Poloxamer F-108 Poloxamer F-127 Kolliphor P-188
Poloxamer F-108 Poloxamer F-127 Kolliphor P-188
0 h 48 h 0 h 48 h 0 h 48 h
0 h 48 h 0 h 48 h 0 h 48 h
1:1 183.3 185.0 187.2 188.6 170.3 175.1
1:1 182.0 275.5 184.7 252.5 698.4 * 1:2 180.5 182.9 192.0 193.0 175.7 179.0
1:2 212.0 273.2 168.4 189.4 210.3 * 2:1 175.2 182.9 180.5 193.0 173.4 179.0
2:1 215.3 280.1 202.3 335.4 431.6 * 1:5 184.2 190.1 175.0 176.5 170.0 177.8
1:5 187.7 322.4 237.9 287.9 247.4 *
Nanoparticles remained stable within the 48 -hour period. Average increase in
‘*’ - size of nanoparticles could not be accurately determined using the Malvern size was< 5%.
Zetasizer. The size was probably above 6 μm, which is the maximum size of
particle size the Malvern can measure. the increase in surface area and surface free energy and therefore have
a tendency to agglomerate. Aggregation was observed in our formula-
tion 48hours after preparation. Size of the nanoparticles increased by
more than 50% as indicated in Table 2 demanding the need for better
Table 3 stability.
Characteristics of dasatinib nanoparticles stabilized with 0.05% chitosan. The size of the nanoparticles did not change much after stabilization
Polymer/ Poloxamer F-108 Poloxamer F-127 Kolliphor P-188 with chitosan for F-108 and F-127, however, a decrease in particle size
surfactant was observed for P-188 nanoparticles. It is desirable to maintain small
Ratio Size Zeta Size Zeta Size Zeta nanoparticle size, since, larger particles are likely to be taken up by the
(d.nm) potential (d.nm) potential (d.nm) potential immune cells. For all three sets of nanoparticles prepared with the
(mV) (mV) (mV) poloxamers and chitosan, the zeta potential measured gained a positive
1:1 183.3 20.0 187.2 17.2 170.3 26.3 charge (Table 3). The magnitude of the charge was least in all the
1:2 180.5 21.4 192.0 21.1 175.7 17.1 formulation prepared with 1:5 polymer/surfactant ratio.
2:1 175.2 19.3 180.5 19.9 173.4 22.0 To evaluate the effect of chitosan on the stability of the nano-
1:5 184.2 13.0 175.0 14.4 170.9 14.5 particles, the size distribution of nanoparticles coated with chitosan was
Average size of nanoparticles was< 200 nm similar to that prepared using determined just after production and also after 48hours of storage at
poloxamers alone. The zeta potential of the nanoparticles was positive. 25 °C. Nanoparticles remained stable during this period as compared
with the formulations without chitosan (Table 4). The nano-suspensions
were further observed for a period of 28 days under two different
As the surfactant concentration increased in the formulations (ratio temperature conditions (25 °C and 40 °C). The size distribution of the
1:5), the size distribution of the nanoparticles was skewed towards the particles was measured weekly during the study period.
left (smaller particle size). This was observed among all the three po-
loxamers preparations with ratio 1:5. The emergence of the two peaks 3.2. Stability of nanoparticles during storage
indicated two size populations, thus, the increase in Polydispersity
Index (PDI) values. For polymer/surfactant ratios 1:1 and 1:2, polox- Chitosan increased the hydrophilicity on the surface of the nano-
amers: F-108 and F-127 had an acceptable polydispersity index (PDI) particles leading to the introduction of steric hindrance. Aggregation of
value of< 0.3. The PDI values for kolliphor P-188 were high (> 0.34) particles was immensely controlled (Table 4). The average size of na-
for all ratios of the formulation which indicates that size distribution of noparticles after the 28-day study period was 200 ± 40 nm. The
the particles was in a wide range (see Table 1). polydispersity index (PDI), however increased from about 0.09 to 0.5
Nanoparticles in suspension are thermodynamically unstable, due to (Fig. 2).
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Fig. 2. Colloidal stability of the nano-suspension stored under two different temperature conditions (25 °C and 40 °C). Nanoparticles prepared with Poloxamers; F-
108 and F-127 were relatively stable during the storage period. Average size of kolliphor P-188 nanoparticles increased by day 7, however, a drop in average size was
observed after the 21st day which continued gradually to the 28th day.
3.3. Fourier transform infrared Spectroscopy(FTIR) of nanoparticle revealed that the dye loaded nanoparticles were taken up into the cells
(Fig. 6). Results indicate that poloxamers have some effects on cellular
The presence of Dasatinib in the formulations was confirmed by the function. Effect of poloxamer on cell membrane of MDA-MB-231 breast
presence of its characteristic functional groups. The FTIR spectra of the cancer cells is revealed in Fig. 7.
various formulations were similar (Fig. 3). Poloxamers contain poly-
ethylene oxide and poly propylene oxide in various ratios, thus, the
same functional groups. Hence, FTIR spectra could not bring out the 3.7. Cellular uptake of coumarin-loaded nanoparticles determined by flow
differences between the various formulations. cytometry
3.4. HPLC data for dasatinib Flow cytometry analysis of the mean fluorescence intensity of the
cells after internalization of coumarin-loaded nanoparticles and cou-
Dasatinib was quantified using a reverse phase HPLC method. The marin solution revealed that nanoparticles are taken up better than
mobile phase was a combination methanol, acetonile and TEA buffer. solution. Additionally, marked differences in the amounts uptaken by
Dasatinib eluted at 9.02min (Fig. 4). Content analysis was conducted the cells were observed. It was also established that, the charge of the
after the polymer matrix was dissolved. The amount of drug entrapped nanoparticles influenced cellular-uptake in all the cell lines studied
in the PCL matrix was in uenced by the type of poloxamer used as well (Fig. 8).fl
as the ratio of polymer to poloxamer, that is, the amount of surfactant
used.
3.8. Cytotoxicity of blank PCL/Poloxamer nanoparticles using different cell
types
3.5. Release studies
Cytotoxicity of blank nanoparticles was performed to unravel the
The release was conducted from formulations containing PCL/po- effect of the poloxamers (excipients) on the various cell types used. Cell
loxamer ratios 1:1 and 1:2, since they were the most stable. The cu- lines of three different species (mice, rat and human) were used. The
mulative release was conducted over a 5-day period into PBS release- poloxamers were found to be relatively non-cytotoxic (Fig. 9).
media containing 0.01% Tween 80. Dasatinib release from poly-
caprolactone/poloxmer matrices was relatively slow. From PCL/F-108
nanoparticles, release of drug from the 1:2 ratio was about 11%, while 3.9. Effect of nanoparticles on the release of innate immune marker: nitric
that from the 1:1 ratio was about 7%. Addition of chitosan to the for- oxide
mulation did not affect the drug release (see Fig. 5).
Nanoparticles stimulated the release of nitric oxide from dendritic
3.6. Cellular uptake of nanoparticles observed by fluorescence microscopy cells (immune cells). The amount of nitric oxide released was highest
for F-108- nanoparticles, followed by F-127-nanoparticles, with P-188
Fluorescence microscopic images of the cells after internalization nanoparticles producing the least nitric oxide.
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Fig. 3. FT-IR spectra of dasatinib loaded nanoparticles stabilized with poloxamer. (A) PCL/F-108 nanoparticles, (B) PCL/F-127 nanoparticles, (C) PCL/P-188 na-
noparticles. Each graph shows an overlap of spectra taken of nanoparticles with polymer/surfactant ratio of: 1:1, 1:2, 2:1 and 1:5. The characteristic peaks “C]O”
and “O-H” of Dasatinib were observed.
4. Discussion Surfactants play a crucial role in the stabilization of nanoparticles. The
dynamic nature of the interfacial film produced by this group of com-
The stability of polymeric nanoparticle-suspensions has been one of pounds in solution enables them to reduce the surface tension of the
the difficult puzzles formulation scientists have had to solve. nanoparticles, thereby, inducing stability [11]. Thus, the selection of
Nanoparticles are thermodynamically unstable due to the enormous the proper type and concentration of these surface modifiers, are vital
surface free energy they possess. This energy is produced by the in- in the fabrication of this delivery system [12,13]. Van Eerdenbrugh
crease in surface area observed as the particle size decreases [10]. et al. demonstrated no correlation between physical drug properties
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Fig. 4. Chromatographic peak illustrating the retention time of dasatinib during
analysis by a RP-HPLC method. The Y-axis is a measure of the intensity of
absorbance (AU) and the X-axis a measure of the time, in minutes. Dasatinib
eluted at 9.02min.
and nanosuspension stability using 13 different surfactants [14]. Lee
et al. found a correlation between the surface energy of particles, the
polymer used and the stability process [15]. These contradictory find-
ings make the selection of the most appropriate stabilizer a difficult
task. Unfortunately, there is no formula for stabilizer selection, hence,
the preparation processes and the stabilizer system differs from drug to
drug [16]. The physical characteristics of nanoparticles are dictated by
the interactions between the polymer, surfactants as well as the en-
trapped drug. The first part of this study investigated the ability of three
poloxamers (F-108, F-127 and P-188) in stabilizing dasatinib nano-
particles. F-108 is the most hydrophobic among the three poloxamers
and P-188 the least, hence much stronger interaction was expected
between F-108 and the hydrophobic polymer matrix, polycaprolactone
(PCL) used for the formulation. The average size of nanoparticles pre-
pared with F-108 and F-127 were 182 nm and 184 nm, respectively,
with a narrow distribution size range indicated by the low poly-
dispersity index (PDI) value of 0.3 obtained. Just after preparation, P-
Fig. 5. Drug release of Dasatinib (A) PCL/F-108, (B) PCL/F-127, (C) PCL/P-188
188 nanoparticles separated out into two distinct phases, demonstrating matrixes. Ratio of polycaprolactone (PCL) to poloxamer 1:1 (blue stars), 1:2
gross instability. The PDI value of the P-188 formulation was very close (orange squares), 1:1 + chitosan (black circles), 1:2 + chitosan (pink trian-
to the value “1” (0.8). Poloxamer, P -188, being more hydrophilic may gles). (For interpretation of the references to color in this figure legend, the
have had less interaction with the polycaprolactone. Haley et al. ob- reader is referred to the Web version of this article.)
served that particles gain some degree of stability as their surface be-
comes more hydrophilic [17]. As mentioned previously, each polox- (formulation with ratio; 2:1), F-127 produced a bimodal distribution
amer molecule is composed of a hydrophobic units of poly-propylene (Fig. 1). Majority of the produced particles had an average size of about
oxide (PPO) and two hydrophilic block made of poly-ethylene oxide 202.0 nm, while a few of the particles which are represented by the
(PEO). It is reported that, the greater the magnitude of the hydro- smaller peak had an average size greater than 1 μm. A similar ob-
philicity exhibited on the nanoparticle surface the greater the steric servation was made by Ecevit et al. who reported that, low surfactant
hindrance and the better the stability [11]. Thus, during the design of concentrations do not prevent aggregation. The increase in size could
the particle, if the poloxamer can be oriented to have the two hydro- have been provoked by the dispersed system, in an attempt to coun-
philic blocks of PEO on the surface of the particle, a more stable product teract the inefficient surfactant at the nanoparticle surface [18]. Na-
will be achieved. This, notwithstanding, the hydrophobic part of the noparticle formulations prepared using all three surfactants with the
surfactant should be able to interact adequately with the matrix to keep ratio 1:5 (high surfactant content) had more than one population of
the entrapped drug within the particle. As mentioned previously, it was particles, however, in this case, the distributions were skewed towards
observed that poloxamer, P-188-nanoparticles were highly unstable smaller particle sizes. The population of particles with average size
even though it is the most hydrophilic among the three surfactants. It is below 100 nm could be micelles, formed unintended, with subsequent
stipulated here that there was inadequate interaction between itself and entrapment of drug within them (Fig. 1). Poloxamers, similar to other
the polycaprolactone used as the matrix, hence, the poor nanoparticle surfactants are able to form micelles at high concentrations. Thus, it can
formation and entrapment. A good quality nano-formulation is ex- be concluded that very high and very low surfactant concentrations of
pected to have a bell shaped size distribution curve with a narrow size the surfactants, lead to low drug entrapment of in the nanoparticles.
range. The amount of surfactant used, had an influence on the size Nanoparticles in suspension, when left to stand for a period of time, are
distribution. We observed a broadening of the distribution peak as the prone to aggregation due to the increase in Gibbs free energy the
surfactant was reduced. At low concentrations of surfactants
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Fig. 6. Fluorescence microscopic images showing the internalization of coumarin VI loaded nanoparticles prepared with and without chitosan by MDA-MB-231 and
Dendritic cells (phagocytic cells), after incubation with equivalent amount of nanoparticles for 2 hours. Coumarin VI loaded nanoparticles (green dots) can be seen
inside the cells after 2 hours of exposure to culture media containing nanoparticles.
particles possess. After 48 hours of preparation, an increase in average graphs in Fig. 2, indicate a reduction in the average size of the nano-
particle size was observed in all the suspensions, thus the amount of particle after day 21. Polycaprolactone has a low melting point (60 °C),
surfactant used did not prevent aggregation during storage [1]. A 50% hence the reduction in size could have been due to softening of the hard
increase in average size was observed in the formulation of nano- polymer matrix in the aqueous medium over time, followed by its
particles prepared with poloxamers F-108 and F-127, 48 hours after erosion. Based on the results obtained thus far, formulations with ratios
production. Suspension of P-188 nanoparticles, had completely sepa- 1:1 and 1:2 showed much promise and hence the stability and release
rated into two distinct phases and no longer fitted the description of a studies were performed on the formulations prepared with these ratios.
nanosuspension. Stability of nanosuspensions has been shown to be The yield of nanoparticles prepared with pluronic F-108 and F-127
enhanced by inducing steric hindrance to the surface the particles. was ≥80%. Kolliphor, P-188 produced nanoparticles with very low
Stabilizers, such as chitosan have been used to modify the surface of yield (Table 5). The particles produced were precipitating out and
nanoparticles by increasing the hydrophilicity on their surface [11]. sticking to the walls of the container. As mentioned before, P-188 was
Due to the instability observed in the prepared nano-suspension on not able to reduce the surface free energy as the nanoparticles were
standing, chitosan was adsorbed unto the surface of the nanoparticles. forming, explaining why aggregation was observed just after the na-
Following this action, the charge of the nanoparticles became positive noparticles were formed, subsequently, phase separation was observed.
with magnitudes of about 13to 21 units (Table 3), which was expected, The amount of dasatinib entrapped in the PCL matrix was adequate for
however, size was not affected. To evaluate the ability of chitosan to formulations prepared with F-108 and F-127: above 80% for ratios 1:1,
enhance the stability of the nanosuspension, the colloidal stability study 1:2 and 2:1. In formulations with high surfactant concentrations (ratio
was conducted over a to 28-days period. Chitosan-stabilized-nano- 1:5), the percentage of dasatinib entrapped reduced by≥ 10%. This
particles maintained a reasonable degree of stability during the study observed phenomenon run across formulations with all three surfac-
period. Although, aggregation could not be totally halted, about 95% of tants. Since the surfactant concentration in this ratio was above their
the nanoparticle population had an average size of± 240 nm. The CMCs, micelles may have been formed, leading to solubilization of the
Fig. 7. Fluorescence microscopic images showing the blebbing of the cell membrane of MDA-MB-231 cells after exposure to coumarin VI loaded nanoparticles
prepared with and without chitosan.
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G.L. Allotey-Babington et al. Journal of Drug Delivery Science and Technology 47 (2018) 181–192
Fig. 8. Cellular uptake of coumarin loaded nanoparticles by (A) MDA-MB-231 cells, (B) HCC-70 cells, (C) B16 F10, (D) WB rat epithelial cells and (E) DC 2.4 cells by
flow cytometry. A histogram comparing the intensities between the various groups. There was a significant difference between the dye solution and all the nano-
particles. A significant difference in uptake was similarly, observed between nanoparticles with and without chitosan.
(*p < 0.05, **p < 0.01, ***p < 0.005).
drug, which could have been lost during the washing step due to their intracellular enzymes. It is worth mentioning that drug entrapment or
small size. In the preparation of nanoparticles, the optimum amounts of encapsulation masks the drug structure, thereby, preventing its re-
surface modifiers need to be accurately determined and optimized, as cognition by the efflux transporters. From the release profile in Fig. 5, a
too less a concentration will lead to instability of the colloid and too maximum of about 10% of entrapped drug was released after 5 days.
much of it will solubilize the drug leading to low entrapment efficiency. The release of dasatinib was relatively similar between PCL/F-108
In cancer therapy, one of the reasons for entrapping chemotherapy matrix and PCL/F127 matrix. PCL/F-108 matrix with ratio 1:2 released
agents in a matrix is to protect normal cells from the effect of these about 3% more than the same matrix with ratio 1:1. However, after
drugs until they reach the site of action. The lower the amount of drug both formulations were stabilized with chitosan, the release profile of F-
released during the period the delivery system travels to the site of 108 1:1 + chitosan (ch). and F-108 1:2 + chitosan (ch). overlapped
action, the lower the adverse effects patients will experience, which will indicating a normalization of the processes. It was expected that a coat
produce better tolerance. Another advantage of delivering chemother- of chitosan on the nanoparticles will slow down the release, however
apeutic agents as nanoparticles is because of the ability of this delivery that was not observed (Fig. 5). Although PCL/P-188 matrix entrapped
system to produce a passive targeting effect as mentioned above, that is, much less drug than the other two matrices, the rate of release was
by harnessing the enhanced permeability and retention (EPR) effect similar among all three PCL/Poloxamer formulations.
observed in the tumor microenvironment. Nanoparticles after administration into the blood stream, have been
Cells have the ability to uptake micro-, nano-particles by phagocy- shown to accumulate in the tumor microenvironment following which
tosis and or pinocytosis. This also been demonstrated in Figs. 6 and 7. It they stand a greater chance of being taken up into the cells to produce
is postulated that the nanoparticles that enter the tumor micro- the desired effect. The nanoparticles are said to be trapped within the
environment are taken up whole into the cells with subsequent release tumor microenvironment because upon entry into this space from the
of the entrapped drug after the matrix is broken down by the action of blood stream, they are unable to flow back into the blood stream and
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G.L. Allotey-Babington et al. Journal of Drug Delivery Science and Technology 47 (2018) 181–192
Fig. 9. Cytotoxicity profile of nanoparticles without drug prepared with Poloxamers F-108, F-127 and P-188 against MDCK murine kidney cells, WB rat liver cells and
MDA-MB-231 human breast cancer cells.
Table 5
Percentage of dasatinib entrapped in nanoparticles stabilized with poloxamer and chitosan. Entrapment efficiency was> 75% for nanoparticles prepared with F-108
and F-127. Kolliphor P-188 entrapped< 70% of Dasatinib in all ratios used. Its yield was similarly low.
Polymer/Surfactant Ratio Surfactant Type
Poloxamer F-108 Poloxamer F-127 Kolliphor P-188
Yield (%) Entrapment (%) Yield (%) Entrapment Yield (%) Entrapment (%)
(%)
1:1 86.58 88.97 88.68 86.37 69.21 70.0
1:2 81.58 85.06 90.79 92.42 66.58 74.02
2:1 78.82 87.76 89.03 77.49 70.79 73.88
1:5 87.50 66.37 86.18 66.14 76.71 67.14
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G.L. Allotey-Babington et al. Journal of Drug Delivery Science and Technology 47 (2018) 181–192
non-treated cells. Similarly, Sengupta Aritra et al. observed in increase
in cell viability after exposing DU145 prostate cells to laser pulses in the
presence of nanoparticles and poloxamer F-127 treatment.
The resemblance of nanoparticles to microorganisms make them
vulnerable and prone to be attacked by the immune cells. Nitric oxide is
released by innate immune cells following recognition and phagocytosis
of antigens. Opsonization of nanoparticles by immune cells tends to
decrease the concentration of drug intended to reach the target site.
Nanoparticles inducing the least immune responses are preferred in
cancer therapy. A significant difference in nitric oxide release was ob-
served from dendritic cells after exposure to nanoparticles prepared
with the three surfactants. P-188 nanoparticles released the least nitric
oxide, thus, it was less antigenic than F-108 and F-127 (Fig. 10). The
observed stimulation of the immune cells is postulated to be the effect
of the hydrophilic part of the poloxamers: poly-ethylene oxide units,
which orients to the outer part of the nanoparticle.
Fig. 10. Nitric Oxide (NO) released from Dendritic cells (DC 2.4) following 5. Conclusion
incubation with 0.1 mg/mL of nanoparticles stabilized with the three polox-
amer surfactants (F-108, F-127, P-188) with and without chitosan. There was Stability of the nanoparticles increased with an increase in PEO
significant difference in the amount of NO released. (*p < 0.05, **p < 0.01).
units of poloxamer surfactant. Addition of chitosan to the formulation,
increased the stability of PCL/poloxamer nanoparticles. Uptake of ca-
additionally, the lymphatic drainage in solid tumors is usually not well tionic nanoparticles were significantly higher than anionic nano-
developed. Due to this fact, it is important to use biocompatible and particles in all the cell lines studied. Polycaprolactone/poloxamer na-
biodegradable materials like PCL in the manufacturing of nanomater- noparticles did not demonstrate cytotoxicity in the cell lines studied. On
ials for tumor targeting. the contrary, viability of cells in the anionic nanoparticles were slightly
Five cell lines were used to study the cellular uptake of the for- higher than that of the control group, while the cationic nanoparticles
mulations by flow cytometry. Three of them were cancer cell lines, (chitosan coated) had viabilities similar to the control. Among the three
MDA-MB-231, HCC-70 (breast cancer cell lines) and B16 F10 (mela- surfactants studied, poloxamer F-127 nanoparticles were more anti-
noma cell line). A phagocytic cell line DC 2.4 (dendritic cells) was the genic, followed by poloxamer F-108 and then P-188. Chitosan increased
fourth and rat epithelial liver cells (WB cells) representing normal cells the antigenicity of all the nanoparticles in a similar pattern. Thus, po-
the fifth. The uptake of nanoparticle was 1.5 times greater than solution loxamer and chitosan do affect cellular functions. Chitosan will be a
in all the cell lines. Among the three poloxamers, uptake of P-188 and F- useful excipient to consider in formulations, where immunogenicity
108 nanoparticles were relatively, higher than F-127 nanoparticles. need to be enhanced: like vaccines. On the contrary, its use should be
Addition of chitosan to the formulation greatly enhanced cellular up- limited in formulations where increase in immunogenicity is not de-
take. The positive charge inferred on the particle by the chitosan con- sirable. The poloxamer surfactants used in this work were relatively
tributed to the increase in uptake. The surface of most cells are nega- non-cytotoxic, instead, they had the tendency to increase the viability
tively charged, hence the particles adhere to the cell surface by of certain cell types.
electrostatic interaction followed by their subsequent engulfment into
the cell, as revealed by the fluorescence microscopic images in Fig. 6. Conflicts of interest
Poloxamers have been reported to have influence on the cell membrane
of cancer cells. This phenomenon is clearly depicted in the microscopic We wish to indicate that, there are no known conflicts of interest
images. The presence of the poloxamers led to blebbing of cell mem- associated with this publication. All named authors have read and ap-
brane of the cancer cells used (Fig. 7). This observation was not as proved of this manuscript and confirm that there has been no sig-
obvious in the phagocytic (DC 2.4) cell line. Additionally, less blebbing nificant financial support for this work that could have influenced the
was observed in the cells treated with chitosan coated nanoparticles. It results of this project.
is postulated that the chitosan may have masked the hydrophilic wings
of the poloxamer on the surface of the nanoparticles. References
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