- Email: [email protected]
Particle size tailoring of ursolic acid nanosuspensions for improved anticancer activity by controlled antisolvent precipitation
Accepted Manuscript Title: Particle size tailoring of ursolic acid nanosuspensions for improved anticancer activity by controlled antisolvent precipitation Author: Yancai Wang Ju Song Shing Fung Chow Albert H.L. Chow Ying Zheng PII: DOI: Reference:
S0378-5173(15)30149-6 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.08.052 IJP 15135
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
6-5-2015 10-8-2015 18-8-2015
Please cite this article as: Wang, Yancai, Song, Ju, Chow, Shing Fung, Chow, Albert H.L., Zheng, Ying, Particle size tailoring of ursolic acid nanosuspensions for improved anticancer activity by controlled antisolvent precipitation.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.08.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Particle size tailoring of ursolic acid nanosuspensions for improved
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anticancer activity by controlled antisolvent precipitation
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Yancai Wang a,b,#, Ju Song a,c,#, Shing Fung Chowd, Albert H. L. Chow d,*, Ying Zheng a,*
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a
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Sciences, University of Macau, Macao
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b
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250353, China
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c
Beijing Aohe Pharmaceutical Research Institute Co. Ltd., Beijing, 101113, China
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d
School of Pharmacy, The Chinese University of Hong Kong, Hong Kong
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#
These authors contributed equally to this work.
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical
School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology, Jinan
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*To whom correspondence should be addressed:
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1. Ying Zheng, PhD
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Institute of Chinese Medical Sciences, University of Macau
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3/F, Rm 204A, Block 3, Av. Padre Tomás Pereira, S.J. Taipa, Macao
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Tel: (853) 83974687; Fax: (853) 28841358
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E-mail: [email protected]
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2. Prof. Albert H. L. CHOW, BPharm, MSc, PhD, MRPharmS
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School of Pharmacy, Faculty of Medicine
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The Chinese University of Hong Kong
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Tel: (852) 3943 6829; Fax: (852) 2603 5295
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Email: [email protected]
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Graphical abstract
25 26
Abstract
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The present study was aimed at tailoring the particle size of ursolic acid (UA)
28
nanosuspension for improved anticancer activity. UA nanosuspensions were prepared
29
by antisolvent precipitation using a four-stream multi-inlet vortex mixer (MIVM)
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under defined conditions of varying solvent composition, drug feeding concentration
31
or stream flow rate. The resulting products were characterized for particle size and
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polydispersity. Two of the UA nanosuspensions with mean particle sizes of 100 and
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300 nm were further assessed for their in-vitro activity against MCF-7 breast cancer
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cells using fluorescence microscopy with 4',6-diamidino-2-phenylindole (DAPI)
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staining, as well as flow cytometry with propidium (PI) staining and with double
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staining by fluorescein isothiocyanate. It was revealed that the solvent composition,
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drug feeding concentration and stream flow rate were critical parameters for particle
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size control of the UA nanosuspensions generated with the MIVM. Specifically,
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decreasing the UA feeding concentration or increasing the stream flow rate or ethanol
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content resulted in a reduction of particle size. Excellent reproducibility for
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nanosuspension production was demonstrated for the 100 and 300 nm UA
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preparations with a deviation of not more than 5% in particle size from the mean
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value of three independent batches. Fluorescence microscopy and flow cytometry
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revealed that these two different sized UA nanosuspensions, particularly the 300 nm
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45
sample, exhibited a higher anti-proliferation activity against the MCF-7 cells and
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afforded a larger population of these cells in both early and late apoptotic phases. In
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conclusion, MIVM is a robust and pragmatic tool for tailoring the particle size of the
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UA nanosuspension. Particle size appears to be a critical determinant of the anticancer
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activity of the UA nanoparticles.
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Keywords: nanoparticles; antisolvent nanoprecipitation; multi-inlet vortex mixer
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(MIVM); ursolic acid; in-vitro anticancer activity; breast cancer
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1. Introduction
54
Nanotechnology-based formulations, notably nanosuspensions, offer an effective
55
strategy for in vivo delivery of water-insoluble drugs (Ma et al., 2013; Wang et al.,
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2014). In pharmacy, nanosuspensions are defined as colloidal dispersions of nano- or
57
submicron-sized drug particles (i.e., ≤ 1000 nm) in a liquid medium stabilized by a
58
suitable polymer and/or surfactant (Müller et al., 2011; Müller and Keck, 2012). They
59
may be prepared by both “top-down” and “bottom-up” technologies (Ghosh et al.,
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2011). The former technology has been most extensively investigated and applied in
61
nanosuspension production, as exemplified by the preparation of silybin (Wang et al.,
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2010) and ascorbyl palmitate (Teeranachaideekul et al., 2008) nanosuspensions, while
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the latter technology is still at the exploratory stage (Thorat and Dalvi, 2012) with the
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evaporative precipitation of nanosuspension (EPN) and sonoprecipitation methods
65
being the most widely studied (Jiang et al., 2012; Kakran et al., 2010). Despite the
66
growing interest in the bottom-up technology in recent years, the top-down method,
67
notably high pressure homogenization, is still the preferred approach for
68
nanosuspension product development in the pharmaceutical industry since it is well
69
validated and relatively free from lengthy regulatory hurdles.
70
The major obstacle that has precluded the widespread utilization of conventional
71
bottom-up techniques in nanosuspension production is their relatively poor
72
reproducibility (Shegokar and Müller, 2010). As proper control of the operating
73
conditions in such processes is often operator-dependent, frequent interbatch
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variations of the generated nanoparticles particularly with regard to their particle size
75
have been a major concern in the application of this technology in industry
76
(Sievens-Figueroa et al., 2012). Furthermore, the production is often limited by the
77
relatively small batch size and high energy consumption of the associated equipment
78
(Singare et al., 2010).
79
Flash nanoprecipitation (FNP) utilizing a multi-inlet vortex mixer (MIVM) is a
80
novel bottom-up technique based on antisolvent precipitation for nanoparticle
81
preparation (D'Addio and Prud'homme, 2011; Han et al., 2012). The mixer consists of
82
a central circular chamber with four tangentially positioned peripheral inlets for
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admitting separate solvent or solution streams. Premised on the vortex mixing
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principle, FNP is a simple, scalable process that relies on rapid micromixing to create
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a high supersaturation level for inducing rapid precipitation of a water-insoluble
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solute together with a stabilizer, normally an amphiphilic copolymer (Pustulka et al.,
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2013). The technique offers two major advantages for nanosuspension production.
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Firstly, the process allows tight control of nanoparticle properties. For instance, the
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particle size of the preparation can be tailored by adjusting the Reynolds number (Re)
90
of the fluid mixture. Re is a parameter reflecting the physical properties of the fluid,
91
and is determined by the viscosity and velocity of the fluid (see Eq. 1) (Lubbersen et
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al., 2012; Mohseni and Bazargan, 2011), in addition to the internal dimensions of the
93
mixer. The viscosity of the fluid is in turn governed by its composition and solute
94
concentration. Hence, Re can, in principle, be regulated by altering the properties of
95
the fluid (Liu et al., 2008). Secondly, the process offers continuous nanoparticle
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production. With a constant infusion of the solvent and anti-solvent phases into the
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MIVM, nanosuspensions can be produced over a prolonged time period without
98
interruption. This can substantially increase both the manufacturing efficiency and
99
product yield, which is particularly vital for large-scale industrial production (Van
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Eerdenbrugh et al., 2008).
101
The present study aimed to evaluate the viability of tailoring the particle size of
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nanosuspensions of organic materials (including drugs) by FNP using an
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engineering-designed four-stream MIVM. For this purpose, ursolic acid (UA), a
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naturally occurring pentacyclic triterpenoid with poor water solubility (approximately
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5.6 μg ml-1 in water) and high lipophilicity (log P = 6.5), was employed as the model
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compound in the nanosuspension development studies (Zhang et al., 2013a). Our
107
previous work has demonstrated that UA nanocrystals with mean particle size of ~200
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nm displayed enhanced anticancer activity against MCF-7 cells compared with a
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solution formulation at equivalent UA concentration (Song et al., 2013), suggesting
110
that nanoparticles of a particular size may be advantageous in improving the delivery
111
and bioactivity of UA. Thus another aim of the current study was to further
112
investigate the impact of particle size on the anticancer activity of the UA
113
nanoparticles.
114
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2. Materials & methods
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2.1 Materials
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A four-stream multi-inlet vortex mixer was fabricated at the mechanical
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workshop of the Chinese University of Hong Kong based on the design, geometry and
119
dimension of the mixer reported previously (Liu et al., 2008). Ursolic acid (UA) of
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purity>98% was purchased from Nanjing Zelang Pharmaceutical Co. Ltd., China.
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Polyvinylpyrrolidone K 90 (PVP K90) was supplied by Sigma-Aldrich (Saint Louis,
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USA). SDS and ethanol (analytical grade) were obtained from Merck (Darmstadt,
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Germany). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum
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(FBS) were purchased from Life Technologies (New York, USA). Analytical grade
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dimethyl sulfoxide (DMSO) was supplied by Fisher Scientific (Paris, France). FITC
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Annexin V/Dead Cell Apoptosis Kit and DAPI (4,6-Diamidino-2-phenylindole) were
127
purchased from Invitrogen (Eugene, USA). All chemicals and solvents were used as
128
received. Water used was from a Millipore Q purification unit.
129
130
2.2 Preparation of UA nanosuspension
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As depicted in Figure 1, fluids were separately admitted into the mixer via its
132
four inlets from Syringes A, B, C and D. Syringes A, B and C were filled with water
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containing dissolved stabilizers as the anti-solvent phase while Syringe D was filled
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with ethanol containing 4 mg ml-1 UA as the solvent phase. The stabilizers consisted
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of PVP K90 (0.05% w/v) and SDS (0.05% w/v) in MilliQ water (Song et al., 2013).
136
The fluid flow rate was controlled by means of two precision programmable syringe
137
pumps (PHD2000 Infusion, Harvard Apparatus USA). Prior to mixing, all solutions
138
were filtered through 0.45-μm filters.
139
Figure 1.
140 141 142
For ease of comparison between different mixing systems, Reynolds number
143
(Re), a dimensionless parameter reflecting the flow properties of fluids, was employed
144
and calculated by the following expression:
Re
145
vd
Eq. 1
146
where ρ and μ are the density and the dynamic viscosity of the fluid, respectively. The
147
variable ν is the inlet velocity, and d is the hydraulic diameter of the channel. The Re
148
in Equation 1 refers to the overall Re of the final fluid mixture, which is different
149
from that obtained by summation of Re’s of individual streams. The density and
150
dynamic viscosity of the final mixture were assumed to be 978 kg m3 and 1003 Pa s,
151
respectively in the present study (Quijada-Maldonado E, 2013).
152
To investigate the impact of operating parameters on particle size, UA
153
nanosuspensions were prepared by varying the following conditions while keeping the
154
other variables constant:
155
a) the volume ratio of solvent and antisolvent phases was varied at 1:7, 1:11, 1:15 and
156
1:19 while the UA feeding concentration in the solvent phase was kept at 3 mg ml-1 -8-
157
and the mixing rate of the solvent phase at 10 ml min-1;
158
b) the mixing rates for the solvent/antisolvent phases were varied at 3/33, 10/110,
159
17/187 and 24/264 (ml min-1/ml min-1) (i.e., at a fixed mixing rate or volume ratio of
160
1:11) with the UA feeding concentration maintained at 1, 2, 3 or 4 mg ml-1 ; and
161
c) the mixing rates for the solvent/antisolvent phases were further varied at 2/22, 4/44,
162
6/66, and 8/88 (ml min-1/ml min-1) with the UA feeding solution kept at 3 mg ml-1.
163
Finally, to assess the effect of particle s ize on anticancer activity, two batches of
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nanoparticles with mean sizes of 100 and 300 nm were prepared and the conditions
165
for preparing them were tested for reproducibility of particle size and other physical
166
properties. Production of two different-sized nanoparticle samples employed the same
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UA feeding concentration (3 mg ml-1 UA), but different mixing rates for the
168
solvent/antisolvent phases (2:22 ml min-1 and 8:88 ml min-1 for the 300 nm and 100
169
nm samples, respectively). These conditions were selected based on the results from
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the aforementioned studies with regards to the effect of processing variables on the
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particle size of the resulting nanosuspensions.
172
Samples were analyzed for particle size, PDI and zeta potential immediately after
173
preparation, and then stored in a refrigerator at 4°C for five weeks. Stability was
174
monitored during the storage period by measuring the particle size and PDI of the
175
samples at defined time intervals.
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2.3 Determination of particle size, polydispersity and zeta potential
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The particle size and polydispersity (PDI) of sample were determined by photon
179
correlation spectroscopy (PCS) using a Malvern Zetasizer (Nano ZS system, Malvern
180
UK). The same equipment was also employed to determine the zeta potential of
181
nanoparticles through measurement of the net velocity and electrophoretic mobility of
182
the nanoparticles in the liquid that results when an electric field is applied. For
183
electrical characterization, zeta potential (ζ) was calculated from electrophoretic
184
mobility (μ) using Henry’s equation as follows:
185
2 f (r ) 3 0
Eq. 2
186
where κ is the Debye-Huckel parameter and r is the particle radius. The term f(kr)
187
refers to Henry’s function; this function takes the value of 3/2 (according to the
188
Smoluchowski approximation which generally applies to aqueous media) (Tantra et
189
al., 2010). The values of dielectric constant (or permittivity) (ε) and viscosity (η0)
190
were used as supplied by the instrument computer program. All measurements were
191
reproducible and average values of triplicate measurements for each condition were
192
reported.
193
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2.4 Transmission electron microscopy (TEM)
195
The particle morphology of representative UA nanosuspensions was examined
196
by a transmission electron microscope (JEOL, JEM-1400). Fresh nanosuspension was
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diluted 20 fold with water, and a drop of the diluted nanosuspension was then
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transferred to the surface of carbon grid. After drying, TEM was conducted at
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magnification between 50,000x and 8,000x under 120-kV accelerating voltage.
200
201
2.5 MCF-7 cell cultures
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The MCF-7 cell line was obtained from American Type Culture Collection
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(Rockville, USA) and cultured with DMEM containing FBS (10%), streptomycin
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(100 μg ml-1) and penicillin G (10 μg ml-1) at 37°C under a humidified atmosphere of
205
5% CO2.
206
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2.6 MCF-7 cell morphology
208
MCF-7 cells were seeded in 96-well plates for 24 h and then treated with 5 μM
209
of either DMSO or the UA nanosuspensions for 24 h. Following the treatment, the
210
cells were harvested, incubated with DAPI (5 μg ml-1) in DMEM medium, washed six
211
times with PBS at 4°C, and resuspended in PBS prior to testing. The whole
212
experimental procedure was performed in a darkroom. Morphological changes and
213
apoptosis of MCF-7 cells were examined by DAPI staining and IN Cell Analyzer
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2000 software (GE Healthcare Life Sciences). The DAPI stained the cell nuclei, and
215
the apoptotic cells exhibited a bright blue fluorescence. All experiments were
216
performed in triplicate.
- 11 -
217
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2.7 Cell cycle determination
219
The effect of UA nanosuspensions on cell cycle phase arrest was studied by flow
220
cytometry (BD FACS CantoTM, BD Biosciences, San Jose, USA). Briefly, MCF-7
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cells were cultured in six-well plates for 24 h. Then, the cells were treated with either
222
UA nanosuspension or UA solution at a concentration of 5 μM. Following the 24-h
223
treatment, the cells were trypsinized. The harvested cells were washed twice with PBS
224
(4°C) and fixed in 70% ethanol (4°C) overnight. The cells were then resuspended in
225
100 μl of binding buffer, followed by addition of 5 μl of propidium iodide (PI, 100 μg
226
ml-1). Flow cytometric measurements of cellular DNA content were conducted with
227
ethanol-fixed cells using the intercalating DNA fluorochrome PI as described earlier
228
(Bhardwaj et al., 2012). The experiments were performed in triplicate.
229
230
2.8 Apoptosis determination
231
Cell apoptosis was analyzed by flow cytometry using double staining with
232
fluorescein isothiocyanate (FITC Annexin V/Dead Cell Apoptosis Invitrogen, USA)
233
in accordance with the manufacturer’s protocol. Briefly, the MCF-7 cells were seeded
234
in six-well plates and incubated at 37°C. After culturing the cells for 24 h, the cells
235
were exposed to the UA nanosuspension or the UA solution at equivalent UA
236
concentration (i.e., 5 μM) for 24 h. The treated cells were harvested by trypsinization
- 12 -
237
and washed twice with PBS at 4°C. The cells were resuspended in 100 μl of binding
238
buffer, 5 μl FITC Annexin V and 5 μl PI (100 μg ml-1). The cell samples were stained
239
in the dark at room temperature for 15 min. Before analysis, 400 μl of binding buffer
240
was added to each sample, and the samples were analyzed by flow cytometry. The
241
percentages of FITC Annexin V-positive and -negative cells were estimated by
242
applying the appropriate gates and the regional statistical analysis provided in the
243
software. Early apoptotic cells were defined as cells that were positive for FITC
244
Annexin V, but negative for PI staining. The experiments were performed in triplicate.
245
246
247 248
2.9 Statistical analysis
All data were expressed as the means ± SD. A one-way ANOVA was used for the statistical analysis. A p value of <0.05 was considered statistically significant.
249
250
3. Results
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3.1 Influence of processing variables on particle size and particle size distribution
252 253 254
Figure 2.
255 256
As shown in Figure 2, an increase in the relative amount of water to ethanol in - 13 -
257
the final mixture resulted in an increase in mean particle size. Furthermore, the PDI of
258
all nanosuspensions was less than 0.3, indicative of a narrow particle size distribution
259
of the MIVM-generated nanosuspensions. The PDIs of nanosuspensions prepared at
260
the ethanol: water ratio of 1:7, 1:11, 1:15, 1:19 were 0.18, 0.21, 0.17 and 0.21,
261
respectively. While the nanosuspension prepared at the solvent:antisolvent
262
(ethanol:water) volume ratio of 1:7 displayed the smallest mean particle size (90 nm)
263
among all the samples, its physical stability was the poorest, which could be attributed
264
to the relatively high surface volume ratio and surface free energy of the dispersed
265
particles. Since the 1:11 ethanol:water ratio afforded the most desired mean particle
266
size (~100 nm) for the nanosuspension compared with the other two ethanol:water
267
ratios at 1:15 and 1:19 (with mean particle sizes of around 300 nm and 550 nm
268
respectively), this ethanol:water ratio was employed in all subsequent formulation
269
studies unless indicated otherwise.
270 271 272
Figure 3.
273 274 275
In addition to the solvent-to-antisolvent (ethanol:water) volume ratio, the UA
276
feeding concentration in the ethanol phase can exert a significant impact on particle
277
size via its link to the density and viscosity of the fluid mixture. As revealed in Figure
278
3, an increase in UA feeding concentration from 1 mg ml-1 to 2 mg ml-1 resulted in an
- 14 -
279
increase in particle size irrespective of the mixing rates of the solvent and antisolvent
280
phases. However, no apparent particle size change could be observed on raising the
281
UA feeding concentration further from 2 mg ml-1 to 4 mg ml-1.
282
Figure 4.
283 284 285
The effect of mixing rate on particle size was further investigated at a constant
286
UA feeding concentration of 3 mg ml-1. As shown in Figure 4, increasing the flow
287
rates of solvent and antisolvent streams from 3/33 to 8/88 (ml min-1/ml min-1) resulted
288
in a gradual reduction of particle size, reflecting the dependence of particle size on the
289
flow rates of the liquid streams. Such dependence enabled the tailoring of the particle
290
size of nanosuspension through manipulation of the stream flow rate. To further
291
evaluate the influence of stream flow rate (expressed as Re for relative comparison
292
between different mixing systems) on the mixing efficiency and particle size of
293
nanosuspension, the flowrates of pumps A and B were adjusted in the working ranges
294
of 10 to 130 ml min-1 and 2 to 26 ml min-1, respectively. Figure 4 shows that the
295
particle size of nanosuspension drops substantially when Re is increased to the
296
transition boundary from laminar to turbulent flow at around 2,000. Above this
297
transition limit (Re > 2,000), the flow behaviors of both ethanol and water phases
298
become fully turbulent. In this flow region, the particle size of nanosuspension is
299
independent of both flow rate and Re.
300
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301
3.2 Reproducibility of nanoparticle preparation
302
To study the impact of particle size on the anticancer activity of the generated
303
nanosuspensions, two batches of nanosuspensions with mean particle sizes of 100 nm
304
and 300 nm were employed, and the conditions for preparing them were tested for
305
reproducibility of particle size and other physical properties.
306
Presented in Tables 1 and 2 are the particle sizes, PDIs and zeta potentials of the
307
two UA nanosuspensions. Both batches (100 nm and 300 nm) exhibited high particle
308
size reproducibility, with less than 5% deviation from the average value. The PDIs of
309
the two batches were less than 0.25, indicative of a narrow particle size distribution.
310
Interbatch variations of the observed PDI and zeta potential were about 15% and 10 %,
311
respectively.
312
nanoprecipitation using the four-stream MIVM is a simple, pragmatic and consistent
313
technique for fabricating drug nanoparticles in the desired particle size range.
All
these
observations
suggest
that
controlled
antisolvent
314 315
Table 1.
316 317
Table 2.
318
319
3.3 Physical stability and particle morphology of UA nanosuspensions
320
The particle morphology and short-term physical stability of the two UA
321
nanosuspensions are presented in Figure 5. The particles in the 100 nm UA - 16 -
322
nanosuspension displayed a mixture of tabular and somewhat elongated or prismatic
323
morphology, while the particles in the 300 nm UA nanosuspension appeared as
324
irregular, roughly spherical clusters. In addition, the particle sizes observed in the
325
TEM micrographs were slightly smaller than those determined by the Malvern
326
Zetasizer with PCS model. This is perhaps not unexpected, as the intensity diameter
327
obtained by conventional PCS model tends to be an overestimate of the actual particle
328
diameter (Zhu et al., 2010).
329
The particles in both nanosuspensions exhibited a slight increase in size with
330
storage time, which could be brought about by a combination of interdependent events
331
including particle aggregation, solute recrystallization and Ostwald ripening (Zhu,
332
2013). In addition, the residual ethanol present in the final mixture could reduce the
333
adsorption of the amphiphilic stabilizers on the particle surface, thus destabilizing the
334
nanoparticle system (Wang et al., 2013).
335 336
Figure 5.
337
338
3.4 Influence of particle size on cell morphology
339
The morphology changes in cell nuclei were examined by DAPI staining and
340
visualized under a fluorescence microscope. The DAPI reagent would stain the nuclei
341
of apoptotic cells and display a bright blue fluorescence. After the treatment of DAPI
342
agent, the cell chromosomes with nanosuspension samples exhibited a dark-blue or
- 17 -
343
blue-black color (Figure 6). Compared with the control group and the solution group,
344
nucleus fragment and irregular nucleus were observed in the nanosuspensions groups,
345
especially for the 100 nm nanosuspensions. It was worth noting that some apoptosis,
346
featured by cytoplasmic shrinkage, was also observed in some sense (Zhang et al.,
347
2012).
348 349
Figure 6.
350
351
3.5 Influence of particle size on cell cycle and apoptosis
352
The distribution of cell cycle was determined by propidium iodide (PI) staining
353
followed by flow cytometry analysis. The results revealed that the UA
354
nanosuspensions showed only a slight cell cycle arrest at the G2/M checkpoint
355
(Figure 7). Compared with either the UA solution or the nanosuspensions, the
356
excipients (0.05% (w/v) PVP K90 and 0.05% (w/v) SDS) used in the preparation of
357
nanosuspensions did not induce apparent cell cycle arrest (data not shown).
358
The percentages of MCF-7 cells with apoptosis and necrosis (post-apoptosis)
359
induced by treatment (incubation at 37ºC for 24 h) with UA nanosuspensions and
360
solution at equivalent dosing concentration were determined by double staining with
361
FITC-labeled Annexin-V and PI and flow cytometry analysis. Compared with the UA
362
solution-treated cells, the population of MCF-7 cells in the early and late apoptotic
363
phases was increased respectively by 49% and 52% when treated with the 100 nm
- 18 -
364
nanosuspension and 82% and 69% when treated with the 300 nm nanosuspension. No
365
significant changes in cell apoptosis were found between the control and excipient
366
groups (data not shown).
367
Figure 7.
368 369
370
4. Discussion
371
4.1 Particle size control of nanosuspensions prepared by MIVM
372
While current studies on nanoformulations have mainly focused on active tumor
373
targeting based on the ligand-receptor-mediated uptake, passive targeting by enhanced
374
permeation and retention (EPR) effect is also very important in drug delivery. To
375
utilize the EPR effect, the particle size should be greater than 70 nm to prevent rapid
376
clearance through renal filtration but smaller than 300 nm to escape capture by the
377
phagocytic cells located in the reticuloendothelial system (Wang et al., 2011). Thus, a
378
nanoparticle preparation method capable of tailoring particle size in this particular
379
range with good reproducibility would be highly desirable for drug targeting
380
purposes.
381
The MIVM is specifically designed for achieving extremely rapid micromixing
382
of the reacting fluids so as to induce rapid nucleation and growth of nanoparticles
383
(Dong et al., 2010). By controlling the supersaturation level and mixing efficiency, the
384
MIVM is capable of tailoring the particle size of the generated nanosuspensions. In - 19 -
385
the present study, a number of key processing parameters have been investigated to
386
evaluate the quality of mixing, product characteristics and performance of the MIVM.
387
Broadly speaking, to ensure that the mixing time of reacting fluids is shorter than the
388
time for both nucleation and growth of precipitating solutes, a high mixing rate in the
389
MIVM is essential, as this is critical to achieving not only a homogeneous solution
390
but also a sufficiently high degree of supersaturation within a short timescale for rapid
391
precipitation of nanoparticles (Chen et al., 2005) with a narrow particle size
392
distribution (Stepanyan et al., 2012). Consequently, an increase in Re would decrease
393
the particle size of the nanosuspensions to an essentially constant level, as
394
demonstrated by UA in the present study at Re of around 2,000 (Figure 4). At higher
395
Re, the flow circulation would be augmented, and the streamlines became closer. This
396
would expedite molecular diffusion across the streamlines and enable the attainment
397
of homogeneous mixing prior to solute nucleation and recrystallization. Recent
398
studies have substantiated that the particle size of nanosuspensions prepared with
399
amphiphilic polymers as stabilizer is highly dependent on the competitive kinetics
400
between polymer micellization and solute precipitation (Beck C, 2010; Thorat AA,
401
2012). For effective stabilization of drug nanoparticles, the polymer stabilizer used
402
should be able to diffuse fast enough to the surface of the newly formed particles and
403
be able to adsorb on their surface sufficiently strongly to halt further growth. If such
404
adsorption cannot occur in a timely manner, the particles may grow to an undesirably
405
large size. Thus the time gap between drug nucleation and stabilizer adsorption on the
406
particle surface determines the particle size of the resulting nanosuspensions
- 20 -
407
(Campardelli et al., 2012).
408
In addition to the flow rate of fluids, the feeding solute concentration and the
409
volume ratio of ethanol to the water phase are also important determinants of the
410
particle size of the generated nanosuspensions. A decrease in the ethanol-to-water
411
volume ratio (Figure 2) or an increase in the feeding concentration of UA in the
412
ethanol phase (Figure 3) resulted in an increase in particle size, which could be
413
explained by a longer time required for mass transfer of solute from the organic to the
414
aqueous phase and for completing the mixing process prior to nanoprecipitation
415
(Bally et al., 2012).
416
corresponded to an increase in the supersaturation of the final mixture. According to
417
the classical nucleation theory, a higher supersaturation or driving force for
418
crystallization should afford a higher nucleation rate and smaller particles (Mullin,
419
J.W., 2001), which appears to be discordant with the observed increase in particle size
420
in the present study. Such discrepancy may be explained by the fact that ultrafine
421
particles formed initially from solution under high supersaturation are particularly
422
prone to ensuing Ostwald ripening and/or particle aggregation owing to their
423
extremely high surface energy, resulting in an increase rather than a decrease in
424
particle size.
The above adjustments in the two operating parameters also
425
426
427
4.2 Effect of particle size on in vitro anticancer activity
To determine whether the MIVM-prepared nanosuspensions with different mean
- 21 -
428
particle sizes would exhibit different bioactivities, cell death in MCF-7 cells related to
429
cell cycle arrest, apoptosis and morphology changes was evaluated. As a form of
430
programmed cell death, apoptosis is triggered through activation of the cell’s intrinsic
431
suicide machinery and is the major form of cell death in various physiological events
432
(Zhang et al., 2013b). In the present study, MCF-7 cells treated with the UA
433
nanosuspensions
434
morphological changes (Sun et al., 2013).
developed
nuclear
condensation,
indicative
of
marked
435
The apoptosis induced in MCF-7 cells by treatment with UA nanosuspensions
436
increased in a particle size-dependent manner. An increase in the particle size of UA
437
nanosuspensions from 100 nm to 300 nm increased the cell accumulation in G2/M
438
phase. Compared with the two nanosuspensions, the excipients used did not induce
439
any cell cycle changes or cell apoptosis (data not shown), implying that the inhibition
440
of MCF-7 cell proliferation was solely attributed to the UA nanoparticles. Cell cycle
441
control is the major regulatory mechanism of cell growth and apoptosis induction
442
(Smith and Schnellmann, 2012). Compared with the control group, apoptotic cells
443
resulting from treatment with UA solution increased in the G2/M phase but decreased
444
in the G1 and S phases, suggesting that UA down regulation could induce G2/M
445
phase arrest and apoptosis as well as inhibit tumor cell proliferation (Yu et al., 2012).
446
Compared with the blank UA solution group, the nanosuspension formulations
447
displayed significantly better bioactivity. Similar studies with other bioactive
448
compounds also yielded comparable results (Du et al., 2012; Qi et al., 2012; Zheng et
449
al., 2011). In addition to bioactivity enhancement, our present results also suggest that
- 22 -
450
the particle size of the UA nanosuspensions may govern the degree of in vitro MCF-7
451
cell cycle arrest and apoptosis.
452
The observed enhancement of cell cycle arrest and apoptosis in MCF-7 cells with
453
the UA nanosuspensions (100 nm or 300 nm) could be attributed to a number of
454
factors including improved adhesion as well as enhanced solubility and dissolution of
455
the nanosuspension (Talekar et al., 2013). Similar results were reported by Feng et al.
456
for MCF-7 cells where apoptosis was identified as the primary mechanism for cell
457
death induced by oridonin nanosuspension (Feng et al., 2011). It has been well
458
documented that improved adhesion of nanoparticles to cell surface would increase
459
the area and duration of contact between the drug and cells, thus promoting cellular
460
uptake and internalization of nanoparticles via endocytosis or phagocytosis.
461
Furthermore, the improved solubility and dissolution properties of nanosuspensions
462
also provide sufficient drug concentration around the cells, thereby further enhancing
463
cellular uptake and delivery efficiency (Talekar et al., 2013). Based on these
464
arguments, there should exist an optimal particle size for efficient and enhanced
465
delivery of nanoparticles. If the particle size is too small, the duration of adhesion and
466
exposure time between the nanoparticles and cells or targeting tissues will be
467
substantially decreased, as very small particles take very little time to dissolve
468
completely (Müller et al., 2011). On the other hand, if the particles are too large, their
469
adhesion ability may be significantly compromised despite their much slower
470
dissolution and extended lifespan in aqueous medium (Koegler et al., 2012). This
471
could explain why the 300 nm nanosuspension showed better anticancer activity and
- 23 -
472
possibly higher delivery efficiency than the 100 nm nanosuspension in the present
473
study.
474
475
5. Conclusion
476
The
present
study
has
clearly
demonstrated
the
utility
of
the
477
engineering-designed MIVM for reproducibly fabricating UA nanosuspensions or
478
nanoparticles in a continuous production mode, and with the possibility to tailor their
479
particle size according to the fluid properties inside the mixer. The particle size of the
480
nanosuspensions can be regulated by adjusting the Reynolds number (Re), a
481
parameter which reflects the fluid properties and depends on both flow rate and drug
482
concentration. Particle size has also been shown to exert a significant impact on the in
483
vitro anti-proliferation activity of the UA nanosuspensions against MCF-7 cells. There
484
exists an optimal particle size for the enhanced anticancer activity and possibly more
485
efficient delivery of the formulated nanoparticles. In summary, the MIVM is an
486
effective tool for tailoring the particle size of the UA nanosuspensions for potential
487
cancer treatment.
488
489
Acknowledgments
490
This work was supported by the Macao Science and Technology Development
491
Fund (No: 044/2011/A2) and the National Natural Science Foundation of China - 24 -
492
(No:81302711). The authors would like to thank Dr. Xiuping Chen (University of
493
Macau) for his constructive suggestions on cell-related experiments. Thanks are also
494
due to Prof. Robert K. Prud’homme (Department of Chemical and Biological
495
Engineering, Princeton University) for his kind assistance with the refabrication of an
496
MIVM for the present study at CUHK and the University of Macau.
497 498
- 25 -
499
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626 627 628 629
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630
Figure captions
631 632
633
Figure 1. Schematic of MIVM for nanosuspension preparation.
634
Figure 2. Effect of ethanol:water volume ratio on the mean particle size and size
635
distribution of UA nanosuspensions.
636 637
Figure 3. Effect of UA concentration on the mean particle size and particle size
638
distribution of nanosuspensions prepared at different mixing rates of the solvent and
639
antisolvent phases.
640 641
Figure 4. Effect of mixing rates of the solvent and anti-solvent phases (expressed as
642
ml min-1 or Re) on the mean particle size and particle size distribution of
643
nanosuspensions prepared with the MIVM.
644 645
Figure 5. Short-term physical stability and TEM images of the 100 nm and 300 nm
646
UA nanosuspensions.
647 648
Figure 6. Representative nuclear morphology fluorescence photomicrographs of
649
MCF-7 stained by DAPI. MCF-7 cells were incubated with 5 μM of UA solution or
650
nanosuspensions for the indicated time and were then harvested and stained with
- 32 -
651
DAPI to detect all nuclei and visualized under a fluorescence microscope.
652 653
Figure 7. Representative flow cytometry histograms of cell cycle and apoptosis
654
analysis of MCF-7 cells after incubation with UA nanosuspensions of different
655
particle sizes. A, Control, normal cells without any treatment; B, UA solution; C, UA
656
nanosuspension 100 nm; D, UA nanosuspension 300 nm. The concentration of UA, if
657
used, was 5 μM.
658 659
Table 1. The mean particle size, polydispersity index and zeta potential of UA
660
nanosuspensions with mean particle size of 100 nm. Batches
Mean particle size
Polydispersity index
Zeta potential
(d, nm)
(PDI)
(mV)
Batch 1
99.34 ± 0.48
0.197 ± 0.008
-9.92 ± 1.34
Batch 2
105.0 ± 0.46
0.198 ± 0.007
-9.45 ± 0.203
Batch 3
98.32 ± 0.07
0.220 ± 0.002
-10.0 ± 0.39
Average
101.2 ± 3.53
0.205 ± 0.012
-9.79 ± 0.794
661 662
Fig. 1
Syringe A Water
Syringe C Water
MIVM
Syringe B Water
Syringe D Ethanol
663 664
Figure 1.
- 33 -
665
Fig. 2 1:19
Size Distribution by Intensity
Size distribution by intensity
1:15
14
Intens ity (% )
12 10
1:11
8
1:7
6 4 2 0 10
100
1000
Size (d.nm)
666
Figure 2
667 668 669
Fig. 3 Size Distribution by Intensity
Intensity (%)
20 15
A
3:33 ml min-1
10 5 0 100
1000 Size (d.nm)
Intensity (%)
15
B
10:110 ml min-1
10
5
0 10
100
1000
Size (d.nm) 15 Intensity (%)
C
17:187 ml min-1
10
5
0 10
100
1000
Size (d.nm)
Intensity (%)
15
D
24:264 ml min-1
10
5
0 10
100
1000
Size (d.nm)
1mg ml-1
2mg ml-1
670
Figure 3.
671 672 673 674
Fig. 4
- 34 -
3mg ml-1
4mg ml-1
Size Distribution by Intensity
Intensity (%)
15
10
5
0 10
100
1000
Size (d.nm)
3:33 ml/min
4:44 ml/min
6:66 ml/min
8:88 ml/min
400
Particle size . (P, nm) .
320
240
160
80
0 0
1000
2000
3000
4000 Re
675 676
Figure 4.
677 678 679 680 681 682 683 684 685 686 687 688 689 690 - 35 -
5000
6000
7000
8000
691 692
Fig. 5 Nano. 100 nm
Nano. 300 nm
Nano. 100 nm
Nano. 300 nm Nano. 300 nm
Nano. 100 nm
693
Figure 5.
694 695 696
Fig. 6
- 36 -
Control
Solution
Nano.100 nm
Nano.300 nm
697
Figure 6.
698 699
Fig. 7
G1: 48.67%
G1: 49.96% G2: 7.45%
16 0
100
Number
B
150
2 40
A
200
Debris Debris Aggregates Dip G1 Aggregates Dip G2 S Dip Dip G1 Dip G2 Dip S Number
320 80
G2: 5.86%
42.60%
45.47%
0
0
S:
S: 50
700
0
30
60
90
120
15 0
0
50
G1: 50.56%
250
G1: 46.25%
180
Number
120
Number
200
G2: 15.12%
G2: 9.38% 40.06%
S:
38.64%
0
0
40
60
80
120
S:
150
D
240
160
C
100
Channels (PE-A)
Channels (PE-A)
0
50
100
150
Channels (PE-A)
200
0
250
40
99.8
120
160
UA solution
Control 0.1
80
Channels (PE-A)
0
2.8
26.3
0.1
55.1
15.8
% UA Nano. 100nm
UA Nano. 300nm
1.6
40.1
1.7
44.5
34.7
23.6
25.1
28.7
701 702
Figure 7.
- 37 -
703 704 705 706 707
- 38 -
S0378-5173(15)30149-6 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.08.052 IJP 15135
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
6-5-2015 10-8-2015 18-8-2015
Please cite this article as: Wang, Yancai, Song, Ju, Chow, Shing Fung, Chow, Albert H.L., Zheng, Ying, Particle size tailoring of ursolic acid nanosuspensions for improved anticancer activity by controlled antisolvent precipitation.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.08.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Particle size tailoring of ursolic acid nanosuspensions for improved
2
anticancer activity by controlled antisolvent precipitation
3
Yancai Wang a,b,#, Ju Song a,c,#, Shing Fung Chowd, Albert H. L. Chow d,*, Ying Zheng a,*
4
a
5
Sciences, University of Macau, Macao
6
b
7
250353, China
8
c
Beijing Aohe Pharmaceutical Research Institute Co. Ltd., Beijing, 101113, China
9
d
School of Pharmacy, The Chinese University of Hong Kong, Hong Kong
10
#
These authors contributed equally to this work.
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical
School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology, Jinan
11 12
*To whom correspondence should be addressed:
13
1. Ying Zheng, PhD
14
Institute of Chinese Medical Sciences, University of Macau
15
3/F, Rm 204A, Block 3, Av. Padre Tomás Pereira, S.J. Taipa, Macao
16
Tel: (853) 83974687; Fax: (853) 28841358
17
E-mail: [email protected]
18
2. Prof. Albert H. L. CHOW, BPharm, MSc, PhD, MRPharmS
19
School of Pharmacy, Faculty of Medicine
20
The Chinese University of Hong Kong
21
Tel: (852) 3943 6829; Fax: (852) 2603 5295
22
Email: [email protected]
-1-
23 24
Graphical abstract
25 26
Abstract
27
The present study was aimed at tailoring the particle size of ursolic acid (UA)
28
nanosuspension for improved anticancer activity. UA nanosuspensions were prepared
29
by antisolvent precipitation using a four-stream multi-inlet vortex mixer (MIVM)
30
under defined conditions of varying solvent composition, drug feeding concentration
31
or stream flow rate. The resulting products were characterized for particle size and
32
polydispersity. Two of the UA nanosuspensions with mean particle sizes of 100 and
33
300 nm were further assessed for their in-vitro activity against MCF-7 breast cancer
34
cells using fluorescence microscopy with 4',6-diamidino-2-phenylindole (DAPI)
35
staining, as well as flow cytometry with propidium (PI) staining and with double
36
staining by fluorescein isothiocyanate. It was revealed that the solvent composition,
37
drug feeding concentration and stream flow rate were critical parameters for particle
38
size control of the UA nanosuspensions generated with the MIVM. Specifically,
39
decreasing the UA feeding concentration or increasing the stream flow rate or ethanol
40
content resulted in a reduction of particle size. Excellent reproducibility for
41
nanosuspension production was demonstrated for the 100 and 300 nm UA
42
preparations with a deviation of not more than 5% in particle size from the mean
43
value of three independent batches. Fluorescence microscopy and flow cytometry
44
revealed that these two different sized UA nanosuspensions, particularly the 300 nm
-2-
45
sample, exhibited a higher anti-proliferation activity against the MCF-7 cells and
46
afforded a larger population of these cells in both early and late apoptotic phases. In
47
conclusion, MIVM is a robust and pragmatic tool for tailoring the particle size of the
48
UA nanosuspension. Particle size appears to be a critical determinant of the anticancer
49
activity of the UA nanoparticles.
50
Keywords: nanoparticles; antisolvent nanoprecipitation; multi-inlet vortex mixer
51
(MIVM); ursolic acid; in-vitro anticancer activity; breast cancer
52
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53
1. Introduction
54
Nanotechnology-based formulations, notably nanosuspensions, offer an effective
55
strategy for in vivo delivery of water-insoluble drugs (Ma et al., 2013; Wang et al.,
56
2014). In pharmacy, nanosuspensions are defined as colloidal dispersions of nano- or
57
submicron-sized drug particles (i.e., ≤ 1000 nm) in a liquid medium stabilized by a
58
suitable polymer and/or surfactant (Müller et al., 2011; Müller and Keck, 2012). They
59
may be prepared by both “top-down” and “bottom-up” technologies (Ghosh et al.,
60
2011). The former technology has been most extensively investigated and applied in
61
nanosuspension production, as exemplified by the preparation of silybin (Wang et al.,
62
2010) and ascorbyl palmitate (Teeranachaideekul et al., 2008) nanosuspensions, while
63
the latter technology is still at the exploratory stage (Thorat and Dalvi, 2012) with the
64
evaporative precipitation of nanosuspension (EPN) and sonoprecipitation methods
65
being the most widely studied (Jiang et al., 2012; Kakran et al., 2010). Despite the
66
growing interest in the bottom-up technology in recent years, the top-down method,
67
notably high pressure homogenization, is still the preferred approach for
68
nanosuspension product development in the pharmaceutical industry since it is well
69
validated and relatively free from lengthy regulatory hurdles.
70
The major obstacle that has precluded the widespread utilization of conventional
71
bottom-up techniques in nanosuspension production is their relatively poor
72
reproducibility (Shegokar and Müller, 2010). As proper control of the operating
73
conditions in such processes is often operator-dependent, frequent interbatch
-4-
74
variations of the generated nanoparticles particularly with regard to their particle size
75
have been a major concern in the application of this technology in industry
76
(Sievens-Figueroa et al., 2012). Furthermore, the production is often limited by the
77
relatively small batch size and high energy consumption of the associated equipment
78
(Singare et al., 2010).
79
Flash nanoprecipitation (FNP) utilizing a multi-inlet vortex mixer (MIVM) is a
80
novel bottom-up technique based on antisolvent precipitation for nanoparticle
81
preparation (D'Addio and Prud'homme, 2011; Han et al., 2012). The mixer consists of
82
a central circular chamber with four tangentially positioned peripheral inlets for
83
admitting separate solvent or solution streams. Premised on the vortex mixing
84
principle, FNP is a simple, scalable process that relies on rapid micromixing to create
85
a high supersaturation level for inducing rapid precipitation of a water-insoluble
86
solute together with a stabilizer, normally an amphiphilic copolymer (Pustulka et al.,
87
2013). The technique offers two major advantages for nanosuspension production.
88
Firstly, the process allows tight control of nanoparticle properties. For instance, the
89
particle size of the preparation can be tailored by adjusting the Reynolds number (Re)
90
of the fluid mixture. Re is a parameter reflecting the physical properties of the fluid,
91
and is determined by the viscosity and velocity of the fluid (see Eq. 1) (Lubbersen et
92
al., 2012; Mohseni and Bazargan, 2011), in addition to the internal dimensions of the
93
mixer. The viscosity of the fluid is in turn governed by its composition and solute
94
concentration. Hence, Re can, in principle, be regulated by altering the properties of
95
the fluid (Liu et al., 2008). Secondly, the process offers continuous nanoparticle
-5-
96
production. With a constant infusion of the solvent and anti-solvent phases into the
97
MIVM, nanosuspensions can be produced over a prolonged time period without
98
interruption. This can substantially increase both the manufacturing efficiency and
99
product yield, which is particularly vital for large-scale industrial production (Van
100
Eerdenbrugh et al., 2008).
101
The present study aimed to evaluate the viability of tailoring the particle size of
102
nanosuspensions of organic materials (including drugs) by FNP using an
103
engineering-designed four-stream MIVM. For this purpose, ursolic acid (UA), a
104
naturally occurring pentacyclic triterpenoid with poor water solubility (approximately
105
5.6 μg ml-1 in water) and high lipophilicity (log P = 6.5), was employed as the model
106
compound in the nanosuspension development studies (Zhang et al., 2013a). Our
107
previous work has demonstrated that UA nanocrystals with mean particle size of ~200
108
nm displayed enhanced anticancer activity against MCF-7 cells compared with a
109
solution formulation at equivalent UA concentration (Song et al., 2013), suggesting
110
that nanoparticles of a particular size may be advantageous in improving the delivery
111
and bioactivity of UA. Thus another aim of the current study was to further
112
investigate the impact of particle size on the anticancer activity of the UA
113
nanoparticles.
114
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115
2. Materials & methods
116
2.1 Materials
117
A four-stream multi-inlet vortex mixer was fabricated at the mechanical
118
workshop of the Chinese University of Hong Kong based on the design, geometry and
119
dimension of the mixer reported previously (Liu et al., 2008). Ursolic acid (UA) of
120
purity>98% was purchased from Nanjing Zelang Pharmaceutical Co. Ltd., China.
121
Polyvinylpyrrolidone K 90 (PVP K90) was supplied by Sigma-Aldrich (Saint Louis,
122
USA). SDS and ethanol (analytical grade) were obtained from Merck (Darmstadt,
123
Germany). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum
124
(FBS) were purchased from Life Technologies (New York, USA). Analytical grade
125
dimethyl sulfoxide (DMSO) was supplied by Fisher Scientific (Paris, France). FITC
126
Annexin V/Dead Cell Apoptosis Kit and DAPI (4,6-Diamidino-2-phenylindole) were
127
purchased from Invitrogen (Eugene, USA). All chemicals and solvents were used as
128
received. Water used was from a Millipore Q purification unit.
129
130
2.2 Preparation of UA nanosuspension
131
As depicted in Figure 1, fluids were separately admitted into the mixer via its
132
four inlets from Syringes A, B, C and D. Syringes A, B and C were filled with water
133
containing dissolved stabilizers as the anti-solvent phase while Syringe D was filled
134
with ethanol containing 4 mg ml-1 UA as the solvent phase. The stabilizers consisted
-7-
135
of PVP K90 (0.05% w/v) and SDS (0.05% w/v) in MilliQ water (Song et al., 2013).
136
The fluid flow rate was controlled by means of two precision programmable syringe
137
pumps (PHD2000 Infusion, Harvard Apparatus USA). Prior to mixing, all solutions
138
were filtered through 0.45-μm filters.
139
Figure 1.
140 141 142
For ease of comparison between different mixing systems, Reynolds number
143
(Re), a dimensionless parameter reflecting the flow properties of fluids, was employed
144
and calculated by the following expression:
Re
145
vd
Eq. 1
146
where ρ and μ are the density and the dynamic viscosity of the fluid, respectively. The
147
variable ν is the inlet velocity, and d is the hydraulic diameter of the channel. The Re
148
in Equation 1 refers to the overall Re of the final fluid mixture, which is different
149
from that obtained by summation of Re’s of individual streams. The density and
150
dynamic viscosity of the final mixture were assumed to be 978 kg m3 and 1003 Pa s,
151
respectively in the present study (Quijada-Maldonado E, 2013).
152
To investigate the impact of operating parameters on particle size, UA
153
nanosuspensions were prepared by varying the following conditions while keeping the
154
other variables constant:
155
a) the volume ratio of solvent and antisolvent phases was varied at 1:7, 1:11, 1:15 and
156
1:19 while the UA feeding concentration in the solvent phase was kept at 3 mg ml-1 -8-
157
and the mixing rate of the solvent phase at 10 ml min-1;
158
b) the mixing rates for the solvent/antisolvent phases were varied at 3/33, 10/110,
159
17/187 and 24/264 (ml min-1/ml min-1) (i.e., at a fixed mixing rate or volume ratio of
160
1:11) with the UA feeding concentration maintained at 1, 2, 3 or 4 mg ml-1 ; and
161
c) the mixing rates for the solvent/antisolvent phases were further varied at 2/22, 4/44,
162
6/66, and 8/88 (ml min-1/ml min-1) with the UA feeding solution kept at 3 mg ml-1.
163
Finally, to assess the effect of particle s ize on anticancer activity, two batches of
164
nanoparticles with mean sizes of 100 and 300 nm were prepared and the conditions
165
for preparing them were tested for reproducibility of particle size and other physical
166
properties. Production of two different-sized nanoparticle samples employed the same
167
UA feeding concentration (3 mg ml-1 UA), but different mixing rates for the
168
solvent/antisolvent phases (2:22 ml min-1 and 8:88 ml min-1 for the 300 nm and 100
169
nm samples, respectively). These conditions were selected based on the results from
170
the aforementioned studies with regards to the effect of processing variables on the
171
particle size of the resulting nanosuspensions.
172
Samples were analyzed for particle size, PDI and zeta potential immediately after
173
preparation, and then stored in a refrigerator at 4°C for five weeks. Stability was
174
monitored during the storage period by measuring the particle size and PDI of the
175
samples at defined time intervals.
176
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177
2.3 Determination of particle size, polydispersity and zeta potential
178
The particle size and polydispersity (PDI) of sample were determined by photon
179
correlation spectroscopy (PCS) using a Malvern Zetasizer (Nano ZS system, Malvern
180
UK). The same equipment was also employed to determine the zeta potential of
181
nanoparticles through measurement of the net velocity and electrophoretic mobility of
182
the nanoparticles in the liquid that results when an electric field is applied. For
183
electrical characterization, zeta potential (ζ) was calculated from electrophoretic
184
mobility (μ) using Henry’s equation as follows:
185
2 f (r ) 3 0
Eq. 2
186
where κ is the Debye-Huckel parameter and r is the particle radius. The term f(kr)
187
refers to Henry’s function; this function takes the value of 3/2 (according to the
188
Smoluchowski approximation which generally applies to aqueous media) (Tantra et
189
al., 2010). The values of dielectric constant (or permittivity) (ε) and viscosity (η0)
190
were used as supplied by the instrument computer program. All measurements were
191
reproducible and average values of triplicate measurements for each condition were
192
reported.
193
194
2.4 Transmission electron microscopy (TEM)
195
The particle morphology of representative UA nanosuspensions was examined
196
by a transmission electron microscope (JEOL, JEM-1400). Fresh nanosuspension was
- 10 -
197
diluted 20 fold with water, and a drop of the diluted nanosuspension was then
198
transferred to the surface of carbon grid. After drying, TEM was conducted at
199
magnification between 50,000x and 8,000x under 120-kV accelerating voltage.
200
201
2.5 MCF-7 cell cultures
202
The MCF-7 cell line was obtained from American Type Culture Collection
203
(Rockville, USA) and cultured with DMEM containing FBS (10%), streptomycin
204
(100 μg ml-1) and penicillin G (10 μg ml-1) at 37°C under a humidified atmosphere of
205
5% CO2.
206
207
2.6 MCF-7 cell morphology
208
MCF-7 cells were seeded in 96-well plates for 24 h and then treated with 5 μM
209
of either DMSO or the UA nanosuspensions for 24 h. Following the treatment, the
210
cells were harvested, incubated with DAPI (5 μg ml-1) in DMEM medium, washed six
211
times with PBS at 4°C, and resuspended in PBS prior to testing. The whole
212
experimental procedure was performed in a darkroom. Morphological changes and
213
apoptosis of MCF-7 cells were examined by DAPI staining and IN Cell Analyzer
214
2000 software (GE Healthcare Life Sciences). The DAPI stained the cell nuclei, and
215
the apoptotic cells exhibited a bright blue fluorescence. All experiments were
216
performed in triplicate.
- 11 -
217
218
2.7 Cell cycle determination
219
The effect of UA nanosuspensions on cell cycle phase arrest was studied by flow
220
cytometry (BD FACS CantoTM, BD Biosciences, San Jose, USA). Briefly, MCF-7
221
cells were cultured in six-well plates for 24 h. Then, the cells were treated with either
222
UA nanosuspension or UA solution at a concentration of 5 μM. Following the 24-h
223
treatment, the cells were trypsinized. The harvested cells were washed twice with PBS
224
(4°C) and fixed in 70% ethanol (4°C) overnight. The cells were then resuspended in
225
100 μl of binding buffer, followed by addition of 5 μl of propidium iodide (PI, 100 μg
226
ml-1). Flow cytometric measurements of cellular DNA content were conducted with
227
ethanol-fixed cells using the intercalating DNA fluorochrome PI as described earlier
228
(Bhardwaj et al., 2012). The experiments were performed in triplicate.
229
230
2.8 Apoptosis determination
231
Cell apoptosis was analyzed by flow cytometry using double staining with
232
fluorescein isothiocyanate (FITC Annexin V/Dead Cell Apoptosis Invitrogen, USA)
233
in accordance with the manufacturer’s protocol. Briefly, the MCF-7 cells were seeded
234
in six-well plates and incubated at 37°C. After culturing the cells for 24 h, the cells
235
were exposed to the UA nanosuspension or the UA solution at equivalent UA
236
concentration (i.e., 5 μM) for 24 h. The treated cells were harvested by trypsinization
- 12 -
237
and washed twice with PBS at 4°C. The cells were resuspended in 100 μl of binding
238
buffer, 5 μl FITC Annexin V and 5 μl PI (100 μg ml-1). The cell samples were stained
239
in the dark at room temperature for 15 min. Before analysis, 400 μl of binding buffer
240
was added to each sample, and the samples were analyzed by flow cytometry. The
241
percentages of FITC Annexin V-positive and -negative cells were estimated by
242
applying the appropriate gates and the regional statistical analysis provided in the
243
software. Early apoptotic cells were defined as cells that were positive for FITC
244
Annexin V, but negative for PI staining. The experiments were performed in triplicate.
245
246
247 248
2.9 Statistical analysis
All data were expressed as the means ± SD. A one-way ANOVA was used for the statistical analysis. A p value of <0.05 was considered statistically significant.
249
250
3. Results
251
3.1 Influence of processing variables on particle size and particle size distribution
252 253 254
Figure 2.
255 256
As shown in Figure 2, an increase in the relative amount of water to ethanol in - 13 -
257
the final mixture resulted in an increase in mean particle size. Furthermore, the PDI of
258
all nanosuspensions was less than 0.3, indicative of a narrow particle size distribution
259
of the MIVM-generated nanosuspensions. The PDIs of nanosuspensions prepared at
260
the ethanol: water ratio of 1:7, 1:11, 1:15, 1:19 were 0.18, 0.21, 0.17 and 0.21,
261
respectively. While the nanosuspension prepared at the solvent:antisolvent
262
(ethanol:water) volume ratio of 1:7 displayed the smallest mean particle size (90 nm)
263
among all the samples, its physical stability was the poorest, which could be attributed
264
to the relatively high surface volume ratio and surface free energy of the dispersed
265
particles. Since the 1:11 ethanol:water ratio afforded the most desired mean particle
266
size (~100 nm) for the nanosuspension compared with the other two ethanol:water
267
ratios at 1:15 and 1:19 (with mean particle sizes of around 300 nm and 550 nm
268
respectively), this ethanol:water ratio was employed in all subsequent formulation
269
studies unless indicated otherwise.
270 271 272
Figure 3.
273 274 275
In addition to the solvent-to-antisolvent (ethanol:water) volume ratio, the UA
276
feeding concentration in the ethanol phase can exert a significant impact on particle
277
size via its link to the density and viscosity of the fluid mixture. As revealed in Figure
278
3, an increase in UA feeding concentration from 1 mg ml-1 to 2 mg ml-1 resulted in an
- 14 -
279
increase in particle size irrespective of the mixing rates of the solvent and antisolvent
280
phases. However, no apparent particle size change could be observed on raising the
281
UA feeding concentration further from 2 mg ml-1 to 4 mg ml-1.
282
Figure 4.
283 284 285
The effect of mixing rate on particle size was further investigated at a constant
286
UA feeding concentration of 3 mg ml-1. As shown in Figure 4, increasing the flow
287
rates of solvent and antisolvent streams from 3/33 to 8/88 (ml min-1/ml min-1) resulted
288
in a gradual reduction of particle size, reflecting the dependence of particle size on the
289
flow rates of the liquid streams. Such dependence enabled the tailoring of the particle
290
size of nanosuspension through manipulation of the stream flow rate. To further
291
evaluate the influence of stream flow rate (expressed as Re for relative comparison
292
between different mixing systems) on the mixing efficiency and particle size of
293
nanosuspension, the flowrates of pumps A and B were adjusted in the working ranges
294
of 10 to 130 ml min-1 and 2 to 26 ml min-1, respectively. Figure 4 shows that the
295
particle size of nanosuspension drops substantially when Re is increased to the
296
transition boundary from laminar to turbulent flow at around 2,000. Above this
297
transition limit (Re > 2,000), the flow behaviors of both ethanol and water phases
298
become fully turbulent. In this flow region, the particle size of nanosuspension is
299
independent of both flow rate and Re.
300
- 15 -
301
3.2 Reproducibility of nanoparticle preparation
302
To study the impact of particle size on the anticancer activity of the generated
303
nanosuspensions, two batches of nanosuspensions with mean particle sizes of 100 nm
304
and 300 nm were employed, and the conditions for preparing them were tested for
305
reproducibility of particle size and other physical properties.
306
Presented in Tables 1 and 2 are the particle sizes, PDIs and zeta potentials of the
307
two UA nanosuspensions. Both batches (100 nm and 300 nm) exhibited high particle
308
size reproducibility, with less than 5% deviation from the average value. The PDIs of
309
the two batches were less than 0.25, indicative of a narrow particle size distribution.
310
Interbatch variations of the observed PDI and zeta potential were about 15% and 10 %,
311
respectively.
312
nanoprecipitation using the four-stream MIVM is a simple, pragmatic and consistent
313
technique for fabricating drug nanoparticles in the desired particle size range.
All
these
observations
suggest
that
controlled
antisolvent
314 315
Table 1.
316 317
Table 2.
318
319
3.3 Physical stability and particle morphology of UA nanosuspensions
320
The particle morphology and short-term physical stability of the two UA
321
nanosuspensions are presented in Figure 5. The particles in the 100 nm UA - 16 -
322
nanosuspension displayed a mixture of tabular and somewhat elongated or prismatic
323
morphology, while the particles in the 300 nm UA nanosuspension appeared as
324
irregular, roughly spherical clusters. In addition, the particle sizes observed in the
325
TEM micrographs were slightly smaller than those determined by the Malvern
326
Zetasizer with PCS model. This is perhaps not unexpected, as the intensity diameter
327
obtained by conventional PCS model tends to be an overestimate of the actual particle
328
diameter (Zhu et al., 2010).
329
The particles in both nanosuspensions exhibited a slight increase in size with
330
storage time, which could be brought about by a combination of interdependent events
331
including particle aggregation, solute recrystallization and Ostwald ripening (Zhu,
332
2013). In addition, the residual ethanol present in the final mixture could reduce the
333
adsorption of the amphiphilic stabilizers on the particle surface, thus destabilizing the
334
nanoparticle system (Wang et al., 2013).
335 336
Figure 5.
337
338
3.4 Influence of particle size on cell morphology
339
The morphology changes in cell nuclei were examined by DAPI staining and
340
visualized under a fluorescence microscope. The DAPI reagent would stain the nuclei
341
of apoptotic cells and display a bright blue fluorescence. After the treatment of DAPI
342
agent, the cell chromosomes with nanosuspension samples exhibited a dark-blue or
- 17 -
343
blue-black color (Figure 6). Compared with the control group and the solution group,
344
nucleus fragment and irregular nucleus were observed in the nanosuspensions groups,
345
especially for the 100 nm nanosuspensions. It was worth noting that some apoptosis,
346
featured by cytoplasmic shrinkage, was also observed in some sense (Zhang et al.,
347
2012).
348 349
Figure 6.
350
351
3.5 Influence of particle size on cell cycle and apoptosis
352
The distribution of cell cycle was determined by propidium iodide (PI) staining
353
followed by flow cytometry analysis. The results revealed that the UA
354
nanosuspensions showed only a slight cell cycle arrest at the G2/M checkpoint
355
(Figure 7). Compared with either the UA solution or the nanosuspensions, the
356
excipients (0.05% (w/v) PVP K90 and 0.05% (w/v) SDS) used in the preparation of
357
nanosuspensions did not induce apparent cell cycle arrest (data not shown).
358
The percentages of MCF-7 cells with apoptosis and necrosis (post-apoptosis)
359
induced by treatment (incubation at 37ºC for 24 h) with UA nanosuspensions and
360
solution at equivalent dosing concentration were determined by double staining with
361
FITC-labeled Annexin-V and PI and flow cytometry analysis. Compared with the UA
362
solution-treated cells, the population of MCF-7 cells in the early and late apoptotic
363
phases was increased respectively by 49% and 52% when treated with the 100 nm
- 18 -
364
nanosuspension and 82% and 69% when treated with the 300 nm nanosuspension. No
365
significant changes in cell apoptosis were found between the control and excipient
366
groups (data not shown).
367
Figure 7.
368 369
370
4. Discussion
371
4.1 Particle size control of nanosuspensions prepared by MIVM
372
While current studies on nanoformulations have mainly focused on active tumor
373
targeting based on the ligand-receptor-mediated uptake, passive targeting by enhanced
374
permeation and retention (EPR) effect is also very important in drug delivery. To
375
utilize the EPR effect, the particle size should be greater than 70 nm to prevent rapid
376
clearance through renal filtration but smaller than 300 nm to escape capture by the
377
phagocytic cells located in the reticuloendothelial system (Wang et al., 2011). Thus, a
378
nanoparticle preparation method capable of tailoring particle size in this particular
379
range with good reproducibility would be highly desirable for drug targeting
380
purposes.
381
The MIVM is specifically designed for achieving extremely rapid micromixing
382
of the reacting fluids so as to induce rapid nucleation and growth of nanoparticles
383
(Dong et al., 2010). By controlling the supersaturation level and mixing efficiency, the
384
MIVM is capable of tailoring the particle size of the generated nanosuspensions. In - 19 -
385
the present study, a number of key processing parameters have been investigated to
386
evaluate the quality of mixing, product characteristics and performance of the MIVM.
387
Broadly speaking, to ensure that the mixing time of reacting fluids is shorter than the
388
time for both nucleation and growth of precipitating solutes, a high mixing rate in the
389
MIVM is essential, as this is critical to achieving not only a homogeneous solution
390
but also a sufficiently high degree of supersaturation within a short timescale for rapid
391
precipitation of nanoparticles (Chen et al., 2005) with a narrow particle size
392
distribution (Stepanyan et al., 2012). Consequently, an increase in Re would decrease
393
the particle size of the nanosuspensions to an essentially constant level, as
394
demonstrated by UA in the present study at Re of around 2,000 (Figure 4). At higher
395
Re, the flow circulation would be augmented, and the streamlines became closer. This
396
would expedite molecular diffusion across the streamlines and enable the attainment
397
of homogeneous mixing prior to solute nucleation and recrystallization. Recent
398
studies have substantiated that the particle size of nanosuspensions prepared with
399
amphiphilic polymers as stabilizer is highly dependent on the competitive kinetics
400
between polymer micellization and solute precipitation (Beck C, 2010; Thorat AA,
401
2012). For effective stabilization of drug nanoparticles, the polymer stabilizer used
402
should be able to diffuse fast enough to the surface of the newly formed particles and
403
be able to adsorb on their surface sufficiently strongly to halt further growth. If such
404
adsorption cannot occur in a timely manner, the particles may grow to an undesirably
405
large size. Thus the time gap between drug nucleation and stabilizer adsorption on the
406
particle surface determines the particle size of the resulting nanosuspensions
- 20 -
407
(Campardelli et al., 2012).
408
In addition to the flow rate of fluids, the feeding solute concentration and the
409
volume ratio of ethanol to the water phase are also important determinants of the
410
particle size of the generated nanosuspensions. A decrease in the ethanol-to-water
411
volume ratio (Figure 2) or an increase in the feeding concentration of UA in the
412
ethanol phase (Figure 3) resulted in an increase in particle size, which could be
413
explained by a longer time required for mass transfer of solute from the organic to the
414
aqueous phase and for completing the mixing process prior to nanoprecipitation
415
(Bally et al., 2012).
416
corresponded to an increase in the supersaturation of the final mixture. According to
417
the classical nucleation theory, a higher supersaturation or driving force for
418
crystallization should afford a higher nucleation rate and smaller particles (Mullin,
419
J.W., 2001), which appears to be discordant with the observed increase in particle size
420
in the present study. Such discrepancy may be explained by the fact that ultrafine
421
particles formed initially from solution under high supersaturation are particularly
422
prone to ensuing Ostwald ripening and/or particle aggregation owing to their
423
extremely high surface energy, resulting in an increase rather than a decrease in
424
particle size.
The above adjustments in the two operating parameters also
425
426
427
4.2 Effect of particle size on in vitro anticancer activity
To determine whether the MIVM-prepared nanosuspensions with different mean
- 21 -
428
particle sizes would exhibit different bioactivities, cell death in MCF-7 cells related to
429
cell cycle arrest, apoptosis and morphology changes was evaluated. As a form of
430
programmed cell death, apoptosis is triggered through activation of the cell’s intrinsic
431
suicide machinery and is the major form of cell death in various physiological events
432
(Zhang et al., 2013b). In the present study, MCF-7 cells treated with the UA
433
nanosuspensions
434
morphological changes (Sun et al., 2013).
developed
nuclear
condensation,
indicative
of
marked
435
The apoptosis induced in MCF-7 cells by treatment with UA nanosuspensions
436
increased in a particle size-dependent manner. An increase in the particle size of UA
437
nanosuspensions from 100 nm to 300 nm increased the cell accumulation in G2/M
438
phase. Compared with the two nanosuspensions, the excipients used did not induce
439
any cell cycle changes or cell apoptosis (data not shown), implying that the inhibition
440
of MCF-7 cell proliferation was solely attributed to the UA nanoparticles. Cell cycle
441
control is the major regulatory mechanism of cell growth and apoptosis induction
442
(Smith and Schnellmann, 2012). Compared with the control group, apoptotic cells
443
resulting from treatment with UA solution increased in the G2/M phase but decreased
444
in the G1 and S phases, suggesting that UA down regulation could induce G2/M
445
phase arrest and apoptosis as well as inhibit tumor cell proliferation (Yu et al., 2012).
446
Compared with the blank UA solution group, the nanosuspension formulations
447
displayed significantly better bioactivity. Similar studies with other bioactive
448
compounds also yielded comparable results (Du et al., 2012; Qi et al., 2012; Zheng et
449
al., 2011). In addition to bioactivity enhancement, our present results also suggest that
- 22 -
450
the particle size of the UA nanosuspensions may govern the degree of in vitro MCF-7
451
cell cycle arrest and apoptosis.
452
The observed enhancement of cell cycle arrest and apoptosis in MCF-7 cells with
453
the UA nanosuspensions (100 nm or 300 nm) could be attributed to a number of
454
factors including improved adhesion as well as enhanced solubility and dissolution of
455
the nanosuspension (Talekar et al., 2013). Similar results were reported by Feng et al.
456
for MCF-7 cells where apoptosis was identified as the primary mechanism for cell
457
death induced by oridonin nanosuspension (Feng et al., 2011). It has been well
458
documented that improved adhesion of nanoparticles to cell surface would increase
459
the area and duration of contact between the drug and cells, thus promoting cellular
460
uptake and internalization of nanoparticles via endocytosis or phagocytosis.
461
Furthermore, the improved solubility and dissolution properties of nanosuspensions
462
also provide sufficient drug concentration around the cells, thereby further enhancing
463
cellular uptake and delivery efficiency (Talekar et al., 2013). Based on these
464
arguments, there should exist an optimal particle size for efficient and enhanced
465
delivery of nanoparticles. If the particle size is too small, the duration of adhesion and
466
exposure time between the nanoparticles and cells or targeting tissues will be
467
substantially decreased, as very small particles take very little time to dissolve
468
completely (Müller et al., 2011). On the other hand, if the particles are too large, their
469
adhesion ability may be significantly compromised despite their much slower
470
dissolution and extended lifespan in aqueous medium (Koegler et al., 2012). This
471
could explain why the 300 nm nanosuspension showed better anticancer activity and
- 23 -
472
possibly higher delivery efficiency than the 100 nm nanosuspension in the present
473
study.
474
475
5. Conclusion
476
The
present
study
has
clearly
demonstrated
the
utility
of
the
477
engineering-designed MIVM for reproducibly fabricating UA nanosuspensions or
478
nanoparticles in a continuous production mode, and with the possibility to tailor their
479
particle size according to the fluid properties inside the mixer. The particle size of the
480
nanosuspensions can be regulated by adjusting the Reynolds number (Re), a
481
parameter which reflects the fluid properties and depends on both flow rate and drug
482
concentration. Particle size has also been shown to exert a significant impact on the in
483
vitro anti-proliferation activity of the UA nanosuspensions against MCF-7 cells. There
484
exists an optimal particle size for the enhanced anticancer activity and possibly more
485
efficient delivery of the formulated nanoparticles. In summary, the MIVM is an
486
effective tool for tailoring the particle size of the UA nanosuspensions for potential
487
cancer treatment.
488
489
Acknowledgments
490
This work was supported by the Macao Science and Technology Development
491
Fund (No: 044/2011/A2) and the National Natural Science Foundation of China - 24 -
492
(No:81302711). The authors would like to thank Dr. Xiuping Chen (University of
493
Macau) for his constructive suggestions on cell-related experiments. Thanks are also
494
due to Prof. Robert K. Prud’homme (Department of Chemical and Biological
495
Engineering, Princeton University) for his kind assistance with the refabrication of an
496
MIVM for the present study at CUHK and the University of Macau.
497 498
- 25 -
499
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- 31 -
630
Figure captions
631 632
633
Figure 1. Schematic of MIVM for nanosuspension preparation.
634
Figure 2. Effect of ethanol:water volume ratio on the mean particle size and size
635
distribution of UA nanosuspensions.
636 637
Figure 3. Effect of UA concentration on the mean particle size and particle size
638
distribution of nanosuspensions prepared at different mixing rates of the solvent and
639
antisolvent phases.
640 641
Figure 4. Effect of mixing rates of the solvent and anti-solvent phases (expressed as
642
ml min-1 or Re) on the mean particle size and particle size distribution of
643
nanosuspensions prepared with the MIVM.
644 645
Figure 5. Short-term physical stability and TEM images of the 100 nm and 300 nm
646
UA nanosuspensions.
647 648
Figure 6. Representative nuclear morphology fluorescence photomicrographs of
649
MCF-7 stained by DAPI. MCF-7 cells were incubated with 5 μM of UA solution or
650
nanosuspensions for the indicated time and were then harvested and stained with
- 32 -
651
DAPI to detect all nuclei and visualized under a fluorescence microscope.
652 653
Figure 7. Representative flow cytometry histograms of cell cycle and apoptosis
654
analysis of MCF-7 cells after incubation with UA nanosuspensions of different
655
particle sizes. A, Control, normal cells without any treatment; B, UA solution; C, UA
656
nanosuspension 100 nm; D, UA nanosuspension 300 nm. The concentration of UA, if
657
used, was 5 μM.
658 659
Table 1. The mean particle size, polydispersity index and zeta potential of UA
660
nanosuspensions with mean particle size of 100 nm. Batches
Mean particle size
Polydispersity index
Zeta potential
(d, nm)
(PDI)
(mV)
Batch 1
99.34 ± 0.48
0.197 ± 0.008
-9.92 ± 1.34
Batch 2
105.0 ± 0.46
0.198 ± 0.007
-9.45 ± 0.203
Batch 3
98.32 ± 0.07
0.220 ± 0.002
-10.0 ± 0.39
Average
101.2 ± 3.53
0.205 ± 0.012
-9.79 ± 0.794
661 662
Fig. 1
Syringe A Water
Syringe C Water
MIVM
Syringe B Water
Syringe D Ethanol
663 664
Figure 1.
- 33 -
665
Fig. 2 1:19
Size Distribution by Intensity
Size distribution by intensity
1:15
14
Intens ity (% )
12 10
1:11
8
1:7
6 4 2 0 10
100
1000
Size (d.nm)
666
Figure 2
667 668 669
Fig. 3 Size Distribution by Intensity
Intensity (%)
20 15
A
3:33 ml min-1
10 5 0 100
1000 Size (d.nm)
Intensity (%)
15
B
10:110 ml min-1
10
5
0 10
100
1000
Size (d.nm) 15 Intensity (%)
C
17:187 ml min-1
10
5
0 10
100
1000
Size (d.nm)
Intensity (%)
15
D
24:264 ml min-1
10
5
0 10
100
1000
Size (d.nm)
1mg ml-1
2mg ml-1
670
Figure 3.
671 672 673 674
Fig. 4
- 34 -
3mg ml-1
4mg ml-1
Size Distribution by Intensity
Intensity (%)
15
10
5
0 10
100
1000
Size (d.nm)
3:33 ml/min
4:44 ml/min
6:66 ml/min
8:88 ml/min
400
Particle size . (P, nm) .
320
240
160
80
0 0
1000
2000
3000
4000 Re
675 676
Figure 4.
677 678 679 680 681 682 683 684 685 686 687 688 689 690 - 35 -
5000
6000
7000
8000
691 692
Fig. 5 Nano. 100 nm
Nano. 300 nm
Nano. 100 nm
Nano. 300 nm Nano. 300 nm
Nano. 100 nm
693
Figure 5.
694 695 696
Fig. 6
- 36 -
Control
Solution
Nano.100 nm
Nano.300 nm
697
Figure 6.
698 699
Fig. 7
G1: 48.67%
G1: 49.96% G2: 7.45%
16 0
100
Number
B
150
2 40
A
200
Debris Debris Aggregates Dip G1 Aggregates Dip G2 S Dip Dip G1 Dip G2 Dip S Number
320 80
G2: 5.86%
42.60%
45.47%
0
0
S:
S: 50
700
0
30
60
90
120
15 0
0
50
G1: 50.56%
250
G1: 46.25%
180
Number
120
Number
200
G2: 15.12%
G2: 9.38% 40.06%
S:
38.64%
0
0
40
60
80
120
S:
150
D
240
160
C
100
Channels (PE-A)
Channels (PE-A)
0
50
100
150
Channels (PE-A)
200
0
250
40
99.8
120
160
UA solution
Control 0.1
80
Channels (PE-A)
0
2.8
26.3
0.1
55.1
15.8
% UA Nano. 100nm
UA Nano. 300nm
1.6
40.1
1.7
44.5
34.7
23.6
25.1
28.7
701 702
Figure 7.
- 37 -
703 704 705 706 707
- 38 -