Lightfoot Vidal Et Al 2016

635839
research-article2016
JBC0010.1177/0883911516635839Journal of Bioactive and Compatible PolymersLightfoot Vidal et al.
JOURNAL OF
Bioactive
and
Compatible
Polymers
Original Article
Synthesis and characterization

of polyhydroxybutyrate-cohydroxyvalerate nanoparticles
for encapsulation of quercetin
Journal of Bioactive and

Compatible Polymers
114
The Author(s) 2016
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DOI: 10.1177/0883911516635839
jbc.sagepub.com
Sarah Lightfoot Vidal1,2, Claudio Rojas2,

Rebeca Bouza Padn3, Mnica Prez Rivera4,
Astrid Haensgen5, Marcelo Gonzlez5,6
and Saddys Rodrguez-Llamazares2
Abstract
Polyhydroxybutyrate-co-hydroxyvalerate has been identified as a useful polymer for biomedical
application due to its biocompatibility and processability. Polyhydroxybutyrate-co-hydroxyvalerate
nanoparticles loaded with quercetin, an antimicrobial, anti-inflammatory, and antiviral polyphenol
with limited solubility, were obtained using a high-speed double-emulsion technique. The
nanoparticle size and the dissolution of quercetin were controlled simultaneously through highspeed stirring (15,000r/min) in the emulsification process. The size range of quercetin-loaded
polyhydroxybutyrate-co-hydroxyvalerate nanoparticles was between 250 and 650nm. Spherical
shape with no aggregation of nanoparticles was confirmed by electron microscopy. Loaded
nanoparticles showed less thermal degradation than unloaded nanoparticles. An encapsulation
efficiency of 51% was found. Most of the quercetin was released from the nanoparticles within
the first 5h of water immersion. A biocompatibility analysis of the nanoparticles showed no
cytotoxicity and no significant difference between loaded and unloaded nanoparticles.
1Department
of Biomedical Engineering, Tufts University, Medford, MA, USA

de Investigacin de Polmeros Avanzados (CIPA), Concepcin, Chile
3Grupo de Polmeros, Departamento de Fsica, E.U.P. Ferrol, Universidad de A Corua, Ferrol, Spain
4Department of Polymers, Faculty of Chemical Science, Universidad de Concepcin, Concepcin, Chile
5Laboratorio de Fisiologa Vascular, Departamento de Fisiologa, Facultad de Ciencias Biolgicas, Universidad de
Concepcin, Concepcin, Chile
6Group of Research and Innovation in Vascular Health (GRIVAS Health), Chilln, Chile.
2Centro
Corresponding author:
Saddys Rodrguez-Llamazares, Centro de Investigacin de Polmeros Avanzados (CIPA), Edificio de Laboratorio CIPA,
Av. Collao 1202, Concepcin 4130000, Chile.
Email: s.rodriguez@cipachile.cl
Downloaded from jbc.sagepub.com at UNIVERSIDAD DE CONCEPCION on August 19, 2016
Journal of Bioactive and Compatible Polymers
Keywords
Polyhydroxybutyrate-co-hydroxyvalerate, quercetin, nanoparticles, double emulsion, high-speed
homogenization, biological polyester
Introduction
Polyhydroxyalkanoates (PHA) are polyesters that are synthesized in the cytoplasm of bacteria in
response to nutrient-limiting conditions and excess carbon. The intracellular polymer becomes an
energy source for the bacteria and is degraded in carbon-limited conditions for cell metabolism.1,2
Recent research has shown that the PHA family of polymers, such as polyhydroxybutyrate (PHB)
and its copolymer polyhydroxybutyrate-co-hydroxyvalerate (PHBV), are viable for biomedical
applications due to their natural source, biodegradability, biocompatibility, and tunable mechanical
properties.25 In addition, the degradation product, 3-hydroxybutyric acid, is a metabolite in human
blood at concentrations of 1.3mmol/L.1
PHB copolymers are commonly used as functional matrices and particles, among others, due
to their higher malleability, low crystallinity compared to homopolymer, and broader material
properties (a function of carbon chain length). The PHB homopolymer is less used due to its
brittle behavior.6 PHBV has been used to prepare nanocapsules as enzyme carriers,3 electrospun
films with nanocrystals of hydroxyapatite for mimicking the natural bone tissue,7 and micro and
nanoparticles.811 Thus, for example, PHBV nanocapsules were developed to act as enzyme carriers with model enzymes l-asparaginase, catalase, and glucose oxidase for cancer therapy.12
Ellipticine and paclitaxel, antineoplastic drugs for cancer therapy, were both encapsulated with
PHBV nanoparticles.10,11
The use of nanoparticles provides greater surface area and potentially higher bioavailability and
interfacial absorption of encapsulated compounds, controlled release profiles, and higher degrees
of targeting.8,13 The degree of crystallinity of PHBV copolymers favors its slow degradation rate;
hence, drug encapsulation with PHBV has more potential as a depot device than similar polymers
such as polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA).5,9,14,15
PHBV nanoparticles are most commonly formed via emulsion.3,6,811,16,17 Double-emulsion
techniques are attractive because they protect the encapsulated material, increasing the bioactivity,
stability, and bioavailability of the molecule for targeted delivery.3,13 Furthermore, due to the limited solubility of PHBV in most solvents except for chloroform or dichloromethane,9 PHBV would
be well suited to an emulsion-solvent evaporation technique to encapsulate a hydrophobic drug or
molecule with complementary solubility limitations such as polyphenols.
Polyphenols are natural molecules which exhibit interactions with proteins or ions and have
radical scavenging activity. The scavenging activity limits free radical damage which has been
linked to antioxidant activity.18 Flavanoids are a class of dietary polyphenols, found in fruits, vegetables, and tea, among others. They are thought to reduce incidence of diseases in part caused by
oxidative stresses, including cardiovascular disease, diabetes, neuro-degeneration, and cancer.13
Quercetin is a flavanoid, classified as a flavonol due to its biosynthetic origin.1921 It has one
of the highest antioxidant activities, surpassing trolox, due to the number and position of free
hydroxyl groups in their structure.19 In addition, quercetin has anti-inflammatory and antimicrobial properties.21,22 It has been suggested that the antimicrobial activity of quercetin relates to the
inhibition of DNA gyrase.21 Furthermore, it has been reported that quercetin is inhibitory to at
least seven viruses, including herpes simplex,23,24 and also inhibits HIV-1 integrase.25,26 However,
quercetin use has not been extended in the pharmaceutical industry due to its limited solubility as
previously mentioned.18,19,27
Lightfoot Vidal et al.
PHBV can be utilized to encapsulate quercetin via a double emulsification-solvent evaporation

stage using a mixture of chloroform and ethanol. PHBV is soluble in chloroform and quercetin in
ethanol. The latter also decreases the surface tension of the organic phase.9 In this work, we adapt
an emulsification/diffusion technique for high-speed homogenization to create quercetin-loaded
PHBV nanoparticles. High-speed stirring has a positive impact on particle size and polydispersity
and has shown to increase capacity for drug loading.16
To our knowledge, encapsulation of quercetin with PHBV micro or nanoparticles has not been
studied before. Currently, there is a significant need to create novel antimicrobial drugs to which
polyphenols could provide a solution; in the United States, 70% of bacterial infections in patients
acquired in hospitals are resistant to at least one antibiotic drug.21 Furthermore, 2.6% of surgeries
performed in the United States annually result in a surgical site infection.28 Quercetin-loaded nanoparticles could be used to prevent internal infections following surgery, where the highest risk of
infection is in the month following the procedure.28
The aim of this study was to develop and characterize consistently sized PHBV nanoparticles
with encapsulated quercetin for potential use in the medical field. The size, morphology, crystallinity, thermal properties, polyphenol encapsulation, release profile of the quercetin, and biocompatibility of the PHBV nanoparticles were explored.
Materials and methods

Materials
PHBV 12% biopolymer granule was purchased from Goodfellow, England. Polyvinyl alcohol
(PVA), 89% hydrolyzed, 85,000124,000 molecular weight, and quercetin, >95% (highperformance liquid chromatography (HPLC)) solid, were purchased from Sigma Aldrich. All
chemicals (including chloroform and ethanol) were used without further purification.
Nanoparticle preparation
The protocol of nanoparticle preparation was adapted from Poletto etal.9 and Kumari etal.19
Briefly, 20mg of PHBV was dissolved in 4mL of 80:20 chloroform:ethanol (v:v). The polymer
was dissolved in a closed container under gentle mixing for 4h. Encapsulated (loaded) samples
were prepared by adding 5mg of quercetin to 4mL of the polymer solution, protected from light
during mixing. The PHBV solution was emulsified in 80mL of ultrapure type I Millipore water
containing 20mg of PVA with an UltraTurrax (Digital, IKA) at 15,000r/min for 5min.
The primary emulsion was added to 200mL of aqueous phase containing 20mg of PVA, under
moderate magnetic stirring for 40min at 40C to evaporate any remaining organic solvent.
Concentrated samples of approximately 10mL were obtained under reduced pressure at 40C.
Final solutions were kept tightly closed and protected from light at ambient temperature prior to
analysis. For dry tests (thermogravimetric analysis (TGA), differential scanning calorimetry (DSC),
and X-ray diffraction (XRD)), samples were prepared by evaporating excess water from the nanoparticle solutions in an incubator at 40C. The PHBV bulk sample was analyzed in pellets unless otherwise noted, and for the control sample of quercetin, the lyophilized powder was used as received.
Nanoparticle characterization
Dynamic light scattering. Two milliliters of nanoparticles suspended in ultrapure type I Millipore
water were placed into a plastic cuvette (Corning), cleaned with ethanol prior to use. Samples were
analyzed with a Brookhaven 90 Plus Particle Size Analyzer (USA); diameter was measured as a
function of number of particles.
Scanning electron microscopy. Droplets of concentrated sample were placed on a carbon grid and
dried in a 40C incubator. Dried samples were sputter-coated with gold for 4min (s5150 equipment), or 40nm thickness, prior to imaging. Images were collected with a JSM 6300 LY microscope (JEOL, Japan).
Transmission electron microscopy. A copper grid (G200 Hex) was coated with droplets of concentrated sample and air-dried. Images were collected with a JEM 1200 EX II microscope (JEOL,
Japan) operating at 120kV. Statistical analysis of nanoparticle size was performed with OriginPro
software (version 8.6) where p<0.01 was considered to be statistically significant.
TGA.Approximately 10mg of sample was used for TGA measurements with a Netzsch thermobalance, model TG 209F1 Iris, Germany. TGA curves were taken from 0 to 600C, at 10C/
min in inert conditions. Control samples were taken in triplicate.
XRD. XRD patterns were recorded over a range of 2: 280 with step of 0.02 and time per step
of 0.02 per second. Scans were recorded with a D4/max-B diffractometer (Bruker Endeavour,
USA) using Cu K radiation, 40kV and 20mA. PHBV bulk was melted into a dog-bone and analyzed; nanoparticles were analyzed dry by evaporating excess water in an incubator at 40C.
DSC. Between 5 and 10mg of sample was used per experiment; the sample was placed in an aluminum pan with pierced lid and measured with a Netzsch calorimeter Phoenix 204F1, Germany.
Two cycles were performed under nitrogen environment, from 50C to 200C, at 10C/min with
a 5-min pause between cycles; the first cycle was performed to eliminate material thermal history
and was not presented. Samples were taken in triplicate.
Release profile of quercetin-loaded nanoparticles. The amount of free quercetin and its release profile
was quantified by ultraviolet (UV) measurement using a UV-2600 spectrophotometer (Shimadzu,
Japan). Loaded and unloaded nanoparticles were previously washed by centrifugation at 5000 g for
15min.
The calibration curve was prepared using a 0.177mg/mL quercetin stock solution dissolved in
1:4 (v/v) ethanol:water, and the measurements were taken in the range of 1.825g/mL. The initial
quercetin added into the emulsion was measured at 369nm, which is the specific absorbance for
the quercetin molecule.29 Free quercetin measurements were made in the supernatant of the
decanted nanoparticles. The encapsulation efficiency (EE) was calculated according to the
equation:
Quercetin trapped inthe NPs after 360 h ( mg ) +
EE ( % ) =
Amount of quercetin released ( mg )
Initial quercetincontent ( mg )
100
To measure quercetin release from loaded NPs, washed loaded and unloaded NPs were re-suspended in 25mL of deionized water and allowed to release quercetin at room temperature under
gentle agitation. At selected time intervals, samples were centrifuged, the supernatant was separated, and the amount of quercetin was measured as previously described.
The amount of quercetin trapped in PHBV nanoparticles was measured after 360h of immersion
in aqueous environment. The nanoparticles were dissolved in 4mL of an 80:20 chloroform:ethanol
(v:v) solution for 1h and then was diluted with absolute ethanol up to 50mL. An aliquot (1mL) of
the resulting solution was diluted in deionized water and the concentration was determined using the
calibration curve.
Cell culture and cytotoxicity assay. Human umbilical vein endothelial cells (HUVECs) were freshly
isolated from human umbilical vein (samples obtained were conforming to the principles outlined
in the Declaration of Helsinki and patient-informed written consent forms were obtained 1224h
before delivery) using 0.25mg/mL Clostridium histolyticum Type II Collagenase (Boehringer,
Mannheim, FRG) digestion and cultured up to confluence in medium 199 (M199) plus 20% sera
and antibiotics (penicillin/streptomycin) at 37C in a CO2 incubator (5% CO2 and 95% relative
humidity).30,31 In order to evaluate cytotoxic effect of the loaded and unloaded nanoparticles,
HUVECs were harvested in the exponential phase growth, seeded into 96-well tissue culture plates,
and allowed to adhere for 24h. Thereafter, nanoparticles were added to a final concentration of
0.014mg/mL. After 24h of incubation, 10L of M199 medium containing 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5mg/mL) was added to each well and incubated
for 4h. The medium was replaced with 100L of sodium dodecyl sulfate (SDS) dissolved in
hydrogen chloride (0.01 M) by 12h. Optical densities were determined at 570nm. The MTT assay
was performed in triplicate for each experiment.32 Data were expressed as the meanstandard
deviation. The MannWhitney test was used for paired comparisons with the control. A p <0.05
was accepted as statistically significant.
Results and discussion

Nanoparticle synthesis, size, and morphology
The method proposed in this study combines an emulsion-diffusion technique for preparing nanoparticles using ethanol for controlling surface tension and particle size,9 with a method to encapsulate the polyphenol quercetin.19 The limited solubility of quercetin, the principal obstacle to its
encapsulation, is overcome using ethanol, which dissolves quercetin.19 With this procedure, particles were obtained at a consistent size for both unloaded and loaded PHBV nanoparticles (Figure 1).
A bimodal size distribution was obtained via dynamic light scattering (DLS) for both PHBV nanoparticles. The mean size of loaded particles without filtration was 659nm, which was significantly
different from unloaded particles of 611nm (p0.01). In the filtered suspension (data not presented), the resultant particle size was 249nm (loaded) and 234nm (unloaded) which were significantly smaller than their non-filtered counterparts (p0.001), but there was no statistical difference
between filtered nanoparticle size (loaded vs unloaded). This range was consistent with that of
Poletto etal.9 (78042nm at an 90:10 chloroform:ethanol PHBV dissolution condition and
54035nm at 70:30 chloroform:ethanol).
Image analysis with transmission electron microscopy (TEM) performed on non-filtered samples showed a distribution size not consistent with DLS results (Figure 2). No aggregation behavior
was observed and the average size of nanoparticle, both unloaded and loaded, was less than 100nm.
The distribution curve histogram collected with TEM images demonstrated lognormal behavior for
unloaded nanoparticles. However, the loaded nanoparticles showed a positively skewed size distribution, increasing the percentage of larger-sized nanoparticles.
Differences between particle size distribution observed by TEM and DLS are due to polydispersity of the sample. The larger nanoparticles, even though in low amount, dominate the dispersion
Figure 1. Dynamic light scattering analysis of particle size. Unloaded versus loaded nanoparticles were
analyzed with respect to number (count) and diameter. A bimodal distribution for both samples was
observed.
Figure 2. Transmission electron microscopy and representative histogram of n=5. Unloaded

nanoparticles (top) and loaded nanoparticles (bottom) both demonstrate lognormal distribution of sizes,
with an average diameter of approximately 80nm (loaded) and 70nm (unloaded) by count.
Figure 3. Thermogravimetric analysis. Weight loss (%) and differential (DTG) versus temperature. PHBV
bulk, quercetin as received, loaded, and unloaded nanoparticles were observed; n=3.
signal of light scattering and mask the presence of the smaller nanoparticles. Differences also result
due to the measurement of hydrodynamic size (DLS) rather than physical size (TEM). The scanning electron microscopy (SEM) images demonstrated spherical nanoparticles with a smooth surface (data not presented), which is in agreement with literature on PHA nanoparticles.6,10
Morphology differences were not noted between unloaded and loaded particles.
Thermal and structural characterization of nanoparticles

TGA curves and corresponding data of PHBV bulk, loaded and unloaded nanoparticles, and
quercetin are shown in Figure 3 and Table 1, respectively. The PHBV bulk presented two degradation steps at 284C and 351C. The PHBV degradation is associated with a random chain scission
reaction.26 The second peak is characteristic of degradation in the PHA with terminal double bonds
in their side chains.33 The thermal degradation of nanoparticles showed four degradation steps; the
first one is associated with the loss of residual organic solvent employed in the preparation of nanoparticles. The temperature where the maximum degradation rate occurs, compared to the other
three steps, is higher for loaded nanoparticles than unloaded nanoparticles, suggesting that the
addition of quercetin had a protective effect on the degradation of the nanoparticles.26,34,35 However,
the onset degradation temperature of nanoparticles (second step) is lower compared to that of
PHBV bulk. The low thermal stability of nanoparticles is due to the disruption of part of the
Table 1. TGA data of bulk PHBV, loaded and unloaded nanoparticles, and quercetin.
Samples
Tmax-1
(C)
WL-1
(%)
Tmax-2
(C)
WL-2
(%)
Tmax-3
(C)
WL-3
(%)
Tmax-4
(C)
WL-4
(%)
Residue at
550C (%)
PHBV bulk
Quercetin
Loaded NPs
Unloaded NPs
284
96
45
41
88.7
3.2
2.2
6.1
351
347
269
217
8.6
32.4
14.2
11.4
338
321
48.6
50.3
411
439
9.7
19.7
44.8
16.6
6.4
Tmax: temperature at which maximum rate of decomposition occurs in the each step (number subscript represents
decomposition step); WL: weight loss associated at each decomposition step; PHBV: polyhydroxybutyrate-cohydroxyvalerate; NP: nanoparticle.
Figure 4. X-ray diffraction. Intensity versus 2 for PHBV bulk, loaded, and unloaded nanoparticles. The
arrows represent the crystalline peaks for quercetin.
crystalline structure of the polymer bulk. In the TGA curve of quercetin, two degradation steps
were observed, the first one corresponds to water loss.34
PHBV is a copolymer with a high degree of crystallinity (ranging from 35% to 60%), which
depends in part on the 3-hydroxyvalerate content.5,33,36,37 The main characteristic peaks of PHBV
bulk are located at 2=13.4, 16.9, 19.9, 25.3, and 27.0 (Figure 4). These peaks are assigned to
(020), (110), (021), (121), and (040) crystallographic planes, respectively.38,39
The XRD patterns of unloaded and loaded nanoparticles showed a lower crystallinity compared
to PHBV bulk: two broad peaks, one between 10 and 29 and other centered at 40. In the unloaded
nanoparticles, emerging peaks at 22.3 and 26.5 as well as peaks corresponding to PHBV bulk
were observed. Loaded nanoparticles had slight representative peaks for quercetin which the
unloaded nanoparticles did not have, at 2=10.7, 12.4, and 27.3, suggesting that the quercetin
was crystalline.34,35 A similar phenomenon was reported for quercetin encapsulated in poly(3-caprolactone) (PCL) nanoparticles prepared by the nano-precipitation method. The XRD pattern of
polyphenol-loaded PCL nanoparticles showed that the drug was in a crystalline state, even though
its peak intensities were low.40
DSC analysis, reported in Figure 5, demonstrated a loss in structural crystallinity of PHBV
nanoparticles in agreement with results obtained with XRD. PHBV bulk displayed lower melting
Figure 5. Differential scanning calorimetry. Secondary heating curve (continuous line) and first cooling
(discontinuous line) of PHBV bulk, loaded, and unloaded nanoparticles.
Table 2. Thermal parameters of PHBV bulk, loaded, and unloaded nanoparticles obtained from the first
cooling and the second heating scans of DSC.
Sample
Second heating scan
First cooling scan
Tm-1 (C)
Tm-2 (C)
Hm (J/g)
c (%)
Tc (C)
PHBV bulk
Unloaded NPs
Loaded NPs
154.9
151.9
162.4
186.0
182.2
63.9
13.2
11.4
43.6
9.0
7.8
104.9
156.6
150.3
PHBV: polyhydroxybutyrate-co-hydroxyvalerate; NP: nanoparticle.
and crystallization temperatures compared to both nanoparticles types with more defined peaks
and a higher value for the enthalpy of fusion. PHBV bulk and unloaded nanoparticles had a split
melting peak due to (1) melting, recrystallization, and re-melting during heating (defective or
smaller crystals melted, then recrystallized forming less imperfect crystals which melted at higher
temperature); (2) different crystalline phases; and (3) different molecular weight species.26,41,42
DSC and XRD revealed no notable differences of the structural characteristics of loaded and
unloaded nanoparticles. The melting enthalpy of loaded nanoparticles was slightly lower, indicating a decrease in the degree of crystallinity (c) as shown in Table 2. The c was calculated as
H m / H m 100, where Hm is the measured melting enthalpy and H is 146.6J/g for heat of
fusion of 100% crystalline PHBV.37 These findings suggest that the interaction between quercetin
and PHBV modified the crystallization through a disturbance of crystal packing of the bio-based
polyesters. In addition, the melting and crystallization temperatures for nanoparticles containing
quercetin decreased compared with the unloaded nanoparticles.
m
Release profile of quercetin

To enhance the understanding of the release of quercetin from the nanoparticles, investigation via
UVvis spectroscopy was performed on separated, washed nanoparticles over 320h and quercetin
10
Figure 6. Release profile of quercetin from loaded PHBV nanoparticles. Washed, loaded nanoparticles
were observed with respect to time up to 360h.
over 2weeks (Figure 6, Supplementary Figure 1). Through this methodology, of the 5.0mg of quercetin added, only 2.55mg was encapsulated corresponding to an EE of 51.1%. Similar encapsulation
efficiencies for quercetin-loaded PCL nanoparticles have been reported by Dinesh Kumar etal.40
Most of the quercetin was released from the particles after 5h of water immersion. At 360h, the
absorbance signal of the supernatant from the loaded particles was equivalent to that of the unloaded
particles, suggesting a complete release at this time, according to the detection limit of the UV
spectrophotometer. The total amount of quercetin released during 360h was 0.42mg which is
related to the poor water solubility of quercetin, as mentioned above. However, loaded nanoparticles remained vibrant yellow (like quercetin) over the 2weeks, compared to the opaque white
unloaded nanoparticles. The polyphenol was still trapped in the nanoparticle, or physically entangled in the PHBV. The amount of quercetin trapped within the nanoparticles was 2.13mg after
360h in an aqueous environment.
Due to the potency of quercetin, the encapsulated amount of 2.55mg could be efficacious in an in
vivo system.40 The quercetin molecule can be detected in the plasma at 0.75M; many fresh fruit and
vegetables have free quercetin heterosides.19,43 However, the behavior of polyphenols in vivo is not
well understood compared to in vitro studies and thus the ideal range has yet to be discovered; the
bioavailability of the molecule is thought to be of high importance.13 Generally, the range of quercetin
concentrations in vivo is actually much lower than required for efficacious in vitro analyses.18
Cytotoxicity assay
The HUVEC is a good model to determine the effect of drugs on human macrovasculature.44
HUVECs are maintained as primary cultures (non-immortalized cells), so they are very sensitive
to toxic agents present in the culture medium, and due to the physiological role of the endothelium
of HUVECs, it is possible to extrapolate these results to the effect of PHBV nanoparticles in
human plasma. The MTT assay was performed on washed and unwashed, loaded and unloaded
nanoparticles (Figure 7). There was no significant difference noted in the cell viability in HUVECs
exposed to different nanoparticles compared to the control. Importantly, there was no significant
11
Figure 7. Biocompatibility of PHBV nanoparticles. Washed (w) and unwashed loaded and unloaded
nanoparticles were analyzed via an MTT assay for cytocompatibility.
difference between the washed and unwashed particles in terms of cytotoxicity. However, a tendency of reduction in viability of cells incubated with loaded nanoparticles without purification
was observed. In the washing process, free quercetin as well as remaining organic solvent is
eliminated. The remaining solvent could exert a toxic effect on HUVECs; correspondingly, TGA
results showed a higher amount of weight loss (more than double) in the first step of decomposition for loaded NPs compared to unloaded NPs. As mentioned previously, the first step at 40C is
associated to evaporation of organic solvents. According to literature, PHBV1 and quercetin43 do
not show a toxic effect at the concentrations studied. For potential medical applications of quercetin-loaded nanoparticles, the purification step is important to minimize the possibility of toxic
effects on live cells.
Conclusion
Quercetin was successfully encapsulated into PHBV nanoparticles via a double-emulsion method
with high-speed homogenization. The consistent size range of quercetin-loaded PHBV nanoparticles was between 50 and 450nm. Both loaded and unloaded nanoparticles demonstrated smooth
and spherical morphology and biocompatibility at the evaluated concentrations. Thermal properties and crystal structure of nanoparticles showed differences due to the addition of quercetin and
the processing via double emulsion. The favorable biological properties of PHBV as well as the
therapeutic potential of quercetin make the use of quercetin-loaded PHBV nanoparticles attractive
in many biomedical applications. Furthermore, the encapsulation method presented could be
applied to numerous other small molecules where the solubility is of particular concern, in particular certain vegetable extracts with therapeutic properties.
Acknowledgements
The authors thank the anonymous reviewers for their insightful comments to improve the quality of the paper.
S.E. Lightfoot Vidal would like to thank the Fulbright US Student Program and the Fulbright Commission of
12
Chile for their support, as well as the Microscopy Laboratory of the University of Concepcin for assistance
with imaging. We also thank Ms. Francisca Saavedra for helping in sample preparation and testing.
Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
This project was supported by a Fulbright Program grant sponsored by the Bureau of Educational and Cultural
Affairs of the United States Department of State and administered by the Institute of International Education.
This work has been financed by CONICYT-REGIONAL R08C1002, Programa de Financiamiento Basal para
Centros Cientficos y Tecnolgicos de Excelencia PFB-27 and Ministerio de Economa y Competitividad
(Project MAT2013-41892-R), and Project Innova Corfo 13IDL2-23120.
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JBC0010.1177/0883911516635839Journal of Bioactive and Compatible PolymersLightfoot Vidal et al.

Synthesis and characterization

Journal of Bioactive and

Sarah Lightfoot Vidal1,2, Claudio Rojas2,

of Biomedical Engineering, Tufts University, Medford, MA, USA

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Journal of Bioactive and Compatible Polymers

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