Colloids and Surfaces B: Biointerfaces

Volume 80, Issue 2, 15 October 2010, Pages 184-192

Development of biodegradable nanoparticles for delivery of quercetin

Avnesh Kumari a, Sudesh Kumar Yadav a, Yogesh B. Pakade b, Bikram Singh c, Subhash Chandra Yadav a

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https://doi.org/10.1016/j.colsurfb.2010.06.002 ↗
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Abstract
The antioxidant molecule quercetin has been encapsulated on poly-D,L-lactide (PLA) nanoparticles by
solvent evaporation method for the improvement of its poor aqueous solubility and stability. The surface
morphology and average size of PLA and quercetin loaded PLA nanoparticles are 170 ± 25 and 130 ± 30 nm
respectively. The antioxidant activities of the PLA encapsulated quercetin nanomedicine are identical to free
quercetin. The nanoencapsulation efficiency of quercetin evaluated by HPLC and antioxidant assay is 96.7%.
The in vitro release kinetics under physiological condition show initial burst release followed by slow and
sustained release. The complete release and maximum retention of quercetin is 72 and 96 h respectively.
The less fluorescence quenching efficiency of quercetin–PLA nanoparticles than free quercetin on BSA
confirms the controlled release of quercetin from PLA nanoparticles. These properties of PLA encapsulated
quercetin molecule pave way for encapsulating various therapeutically less useful highly active antioxidant
molecules towards the development of better therapeutic compounds.

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Keywords
Quercetin; PLA; Nanoparticles; Encapsulation efficiency; Antioxidant; In vitro release

1. Introduction
Quercetin, 3,3′,4′,5′-7-pentahydroxy flavone (Fig. 1) is one of the most abundant flavonoid in plants. It is

−1
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 abundantly found in varying concentrations in berries between 53 and 153 mg kg−1of dry weight of plant
material. This molecule is an important constituent of wine and its concentration varies from 1 to 33 µM.
The eating of fried onions (equivalent to 225 µmol quercetin) and apples (equivalent to 325 µmol quercetin)
increases the peak plasma levels of quercetin up to 0.74 and 0.30 µM respectively [12]. The antioxidant
activity of this molecule is higher than well-known antioxidant molecules ascorbyl, trolox and rutin [14].
This is due to the number and position of the free hydroxyl groups in the quercetin molecule [1]. The
flavonoid glycosides are rapidly hydrolysed in the small intestine or by bacterial activity in the colon to
generate the quercetin aglycones, which is further metabolized into the glucuronidated or sulfated form of
quercetin [6]. This molecule is retained in the large intestine for approximately 6 h after oral administration.
However, it is chemically unstable, especially in aqueous alkaline medium, which possibly involves attack of
hydroxyl ions on the C-ring of quercetin [11]. Apart from the antioxidant activity, this molecule shows
anticancer and antiviral activities also [2], [20], [29]. In spite of this wide spectrum of pharmacological
properties, the use of quercetin in pharmaceutical field is limited due to its low aqueous solubility and
instability in physiological medium [16]. These properties of quercetin result in poor bioavailability, poor
permeability, instability and extensive first pass metabolism before reaching the systemic circulation [18].
One-way to circumvent these problems are to entrap/adsorb these molecule into biodegradable polymeric
nanoparticles. Among the biodegradable polymeric nanoparticles [8]. PLA is extensively used for the
encapsulation of many therapeutic agents due to its high hydrophobicity, biodegradability, biocompatibility,
low toxicity, strong mechanical strength and slow drug release [9]. Quercetin (synthetic) molecule has been
successfully encapsulated into liposomes [17] and chitosan nanoparticles [27]. However, the detailed
characterisations of liposome and chitosan nanoencapsulated quercetin molecule are not reported. In this
study, we have investigated the feasibility of encapsulating synthetic quercetin molecule into PLA
nanoparticles. The solvent evaporation method has been used for the encapsulation of this molecule on
polymeric PLA nanoparticles. The quercetin loaded PLA nanoparticles have been characterised by scanning
electron microscope, atomic force microscope, UV–vis spectrophotometer. Effect of quercetin loaded PLA
nanoparticles on fluorescence quenching of BSA protein has also been evaluated. The quantification of
encapsulation efficiency, antioxidant activity and in vitro release was also carried to enhance its application
in pharmaceutical field. PLA encapsulated quercetin molecule shows higher aqueous solubility and
sustained release. Thus it is speculated that the PLA nanoencapsulation may improve the bioavailability and
stability of quercetin and other similar small molecular drugs.

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Fig. 1. Structure of quercetin: showing the molecular structure of quercetin.

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 2. Materials and methods

2.1. Materials
Poly-D,L-lactide (PLA) (MW = 75,000–120,000) and polyvinyl alcohol (PVA) were purchased from Sigma–
Aldrich and used as received. Quercetin was purchased from Merck, and used as received. Dichloromethane
(DCM) was purchased from Merck. HPLC grade acetonitrile (ACN), water, ethanol and trifluoroacetic acid
(TFA) were procured from Sigma. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Fluka.
Solutions were prepared using water filtered through a Milli-Q water system (Millipore, Bedford, MA).

2.2. Methods

2.2.1. Synthesis of quercetin–poly(D,L-lactide) (PLA) nanoparticles

Poly(D,L-lactide) (MW = 75,000–120,000) nanoparticles were prepared by solvent evaporation method with
minor modification [26]. Briefly, PLA (50 mg) and quercetin (5 mg) was sonicated (Sonics Vibra cell) at 40%
amplitude in 2 ml dichloromethane (DCM) for 30 s at room temperature. 4 ml of 3% (w/v) PVA solution was
added and again sonicated similarly to form emulsion. The emulsion was diluted by 0.1% (w/v) PVA solution
to make final 80 ml solution. The organic solvent (DCM) was evaporated under vacuum using rotating
evaporator. The nanoparticles were separated by centrifugation at 16500 rpm for 20 min at 10 °C and
washed five times with distilled water by centrifugation. Blank PLA nanoparticles were prepared according
to the same procedure. The final lyophilized product was stored at 4 °C until further use. This synthesis
procedure was repeated minimum 3–4 times to establish the reproducibility of nanoparticles synthesis.

2.2.2. Surface morphological characterisation of quercetin loaded PLA nanoparticles by SEM

Quercetin loaded PLA and PLA nanoparticles were characterised by scanning electron microscope (S-3400 N,
Hitachi, Japan). The water suspended quercetin loaded PLA and PLA nanoparticles solution were mounted
on an aluminium stub using double-sided carbon tape. The solution was slowly evaporated at room
temperature. The completely dried sample was coated with gold by sputter coating unit at 10 Pa vacuum for
10 s (E1010 ion sputter Hitachi, Japan). The image was captured on SEM mode at desired magnification. PLA
nanoparticles were also characterised using similar procedure.

2.2.3. Morphological characterisation of quercetin loaded PLA nanoparticles by AFM

Atomic force microscope (Nanoscope-III, Veeco Instruments, Singapore) was used for surface
characterisation of both PLA nanoparticles and quercetin loaded PLA nanoparticles. A drop of sample was
deposited on the freshly cleaved clean glass surface, spread and dried with nitrogen flow at room
temperature. The image measurement was performed in tapping mode using silicon probe cantilever of
125 µm length, resonance frequency of 209–286 kHz, spring constant of 20–80 N/m and nominal, 5–10 nm
tip radius of curvature. The scan rate used was 1 Hz. A minimum of 10 images from each sample were
analyzed to assure reproducible results. The particle size range and standard deviation (σ) of evenly
distributed (∼3 µm2 area/∼400 particles) PLA and quercetin loaded PLA nanoparticles were calculated by
software as well as manually.

2.2.4. Spectroscopic characterisation

2.2.4.1. Spectrophotometer
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 UV–vis spectrophotometer, Nanodrop (ND 1000) with path length of 1 mm and 2048 element linear silicon
CCD array detector was used to obtain UV–vis spectra. 2 µl of each quercetin solution, PLA nanoparticles and
quercetin loaded PLA nanoparticles and their supernatant were used for the spectroscopic scan analysis
from 220 to 700 nm.

2.2.4.2. Fluorescence

Intrinsic fluorescence emission spectra between 350 and 650 nm were recorded with Perkin Elmer LS50B
fluorescence spectrophotometer equipped with a xenon lamp source using a 3-D scanning mode. Excitation
and emission bandwidths both were set at 2.5 nm. The excitation wavelength was set at 617 nm.

2.2.4.3. FTIR

The FTIR spectra of lyophilized quercetin, PLA nanoparticles and quercetin loaded PLA nanoparticles were
recorded on KBr plates in the scanning range of 400–4000 cm−1 and at 1 cm−1 resolution. The Fourier
transform infrared (FTIR) spectra of quercetin, PLA nanoparticles, and quercetin loaded PLA nanoparticles
were recorded on a Nicolet 5700 FTIR spectroscopy (Thermo, USA) using a Smart OMNI-sampler accessory.

2.2.5. Interaction of quercetin with proteins (BSA)

The quenching interactions of quercetin with BSA proteins were recorded on Perkin Elmer LS50B
fluorescence spectrophotometer by keeping 4 ml of the working solution in quartz cuvette. The solution was
scanned on emission range 290–450 nm and fluorescent intensity at 340 nm was determined on 280 nm
excitation. The final working concentration of BSA for the quenching experiment was kept as 0.10 µM. The
working solution of BSA (10−6 M) was prepared with Tris–HCl buffer and stored in refrigerator prior to use. A
varying concentration (0.0–90 µM) of quercetin was used for the quenching experiments in methanol–0.1 M
Tris–HCl buffer at pH 7.4 (2:8, v/v). All other reagents and solvents were of analytical grade and used
without further purification using double distilled water.

2.2.6. Antioxidant activity of quercetin–PLA nanoparticles using DPPH assay

The free radical scavenging (antioxidant) activity of the free and nanoencapsulated quercetin was measured
in vitro by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay according to method reported by Kumar et al. [7]
with slight modifications. Briefly, stock solution of DPPH (0.2 mM) was prepared in absolute ethanol and
300 µl of varying amount of quercetin solution (0.6–0.12 µg of quercetin) were added into 100 µl of DPPH
stock solution. The reaction mixture was incubated in dark at room temperature for 30 min and the
absorbance was taken at 517 nm in UV–vis spectrophotometer (ND1000) against control solution. The result
was expressed in average mean with standard deviation (σ) of triplicate experimental setup. The radical
scavenging activity was calculated using the following equation:

(1)

The calibration curve was prepared with amount of quercetin (µg) vs. % scavenging activity. Similarly the
scavenging activities of quercetin loaded PLA nanoparticles, and supernatant solution after synthesis of
nanoparticles were measured by this assay.

2.2.7. Encapsulation efficiency of quercetin–PLA nanoparticles using HPLC

Encapsulation efficiency of quercetin in PLA nanoparticles was analyzed using validated HPLC method and
:
 DPPH assay. The supernatant solution (20 µl) of quercetin loaded PLA nanoparticles were filtered through
0.22 µm filter paper. The solution was directly injected on HPLC (Merck Hitachi Lachrom coupled with diode
array detector L-7450,). The reverse phase C18 column (150 mm × 4.6 mm, 5 µm size) was used for HPLC
separation. Acetonitrile and water (40:60) were used as mobile phase with flow rate of 1 ml/min. The
detection wavelength was selected as 354 nm. HPLC separation was pre-equilibrated with fresh quercetin
for qualitative and quantitative analysis. The calibration curve was drawn by preparing different amount of
quercetin (0.12–2 µg) vs. peak area of eluted peak. The linearity range of calibration curve was found to be
(0.12–2 µg) while correlation coefficient was 0.995. The area of eluted peak of supernatants of quercetin
loaded PLA nanoparticles and PLA nanoparticles was integrated and used for quercetin quantification. The
encapsulation efficiency (EE) and the actual drug loading were calculated using the formula:

(2)

(3)

2.2.8. In vitro release assay of quercetin–PLA nanoparticles

Amount of released quercetin was quantified using validated HPLC method and DPPH assay with the help of
calibration curve. In vitro release studies of a quercetin loaded PLA nanomedicine formulation were
performed by incubating 10 mg quercetin PLA nanoparticles in 10 ml of 0.1 M PBS at pH 7.4, stabilized with
0.1% NaN3 (w/v). The nanoparticles suspension was continuously stirred in a thermostat (50 rpm) at 37 °C.
At pre-selected times (0, 0.5, 6, 24, 72 and 96 h), 1.0 ml of sample was taken and lyophilised. This lyophilised
solution was dissolved in 200 µl of 100% ACN and quickly centrifuged at 8000 rpm for 5 min to remove the
unreleased quercetin loaded PLA nanoparticles. This released quercetin was subjected for quantitative
analysis by validated HPLC and DPPH assay.

3. Results and discussion

3.1. Synthesis and encapsulation of quercetin in PLA nanoparticles

The antioxidant molecule quercetin is hydrophobic in nature. Thus for encapsulation of this molecule PLA
has been found to be the best biodegradable polymer among other known polymeric molecules used for
nanoparticles synthesis. This molecule was encapsulated on PLA by solvent evaporation method in its fully
active forms. This method is based on the emulsification of organic phase (DCM + PLA + quercetin) and
aqueous phase (PVA solution). The emulsion droplets were stabilised by the surfactant molecules. These
droplets were perfectly stable during solvent evaporation stage. Nanoparticles resulted from a single volume
shrinkage of the initial emulsion droplet [5], [25]. Emulsification is a crucial step in the preparation process
and is obtained by sonication. Sonication induces an increase in temperature which can affect the activity of
drugs [26]. Thus, sonication was carried out in pulsed mode to retain the activity of quercetin and to reduce
the temperature increase. The antioxidant activity of quercetin is found to be similar as free quercetin after
the encapsulation on PLA nanoparticles. The nanoparticles synthesis was reproducible and each time
virtually similar type of nanoparticles was synthesized.

3.2. Characterisation of quercetin loaded PLA nanoparticles

Particle size and size distribution is an important parameter towards development of suitable
nanomedicines for therapeutic purposes. It determines in vivo distribution, biological fate, toxicity and the
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 targeting ability of nanoparticle systems. In addition, they can also influence the drug loading, drug release
and stability of drug inside nanoparticles [13]. Polymeric nanoparticles are synthesized according to the
nature of drug, release profile required and mode of administration. Due to high hydrophobicity, quercetin
was successfully loaded on PLA nanoparticles. Quercetin loaded PLA nanoparticles have been characterised
in terms of mean size, morphology, and size distribution. SEM characterisation of PLA (Fig. 2a) and quercetin
loaded PLA nanoparticles (Fig. 2b) reveals that nanoparticles are smooth and spherical with an average size
of 172.7 ± 30 and 136 ± 35 nm respectively. The result was expressed by taking average of three
consecutively synthesized nanoparticles solution by same methods. This was calculated by taking sample
size of 2 µm2 fresh area (∼400 particles) of PLA nanoparticles and quercetin loaded PLA nanoparticles. The
reduction in size of quercetin loaded PLA nanoparticles than PLA nanoparticles is similar to oridonin loaded
PLA nanoparticles [25].

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Fig. 2. Scanning electron micrographs of (a) PLA nanoparticles and (b) quercetin loaded PLA nanoparticles
synthesized simultaneously. These nanoparticles were synthesized by solvent evaporation method as
described in Section 2.

A similar sized nanoparticle with bright and smooth surface has been observed by atomic force microscopy
(Fig. 3a and b). The average particle size range was about 170 ± 25 and 130 ± 30 nm for the PLA and
quercetin loaded PLA nanoparticles respectively. This particle size was calculated by taking average of 10
images of three successively synthesized nanoparticles solution by same methods. The small particle size as
well as uniform size distribution of quercetin loaded PLA nanoparticles is suitable for the development of
nanomedicines.
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 Download : Download full-size image

Fig. 3. AFM images of (a) PLA nanoparticles and (b) quercetin loaded PLA nanoparticles synthesized
simultaneously. The nanoparticles have been characterised using tapping mode.

3.3. Spectroscopy properties

The absorption spectra provide valuable information about the nanoencapsulation of quercetin like
flavonoids on PLA nanoparticles. The PLA polymer, quercetin, PLA nanoparticles and quercetin loaded PLA
nanoparticles have distinct absorption spectrum (Fig. 4a). The absorption spectrum of quercetin is quite
different after encapsulation on PLA nanoparticles. Free quercetin powder showed an absorption peak at
350 nm. The intensity of this peak was decreased significantly after the encapsulation of quercetin. The
absorbance of quercetin loaded PLA nanoparticles was higher than PLA nanoparticles. The UV–vis spectra of
PLA nanoparticles and quercetin loaded PLA nanoparticles represents a characteristic peaks like turbidity
(Fig. 4a). This might be due the presence of nanoparticles.
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 Download : Download full-size image

Fig. 4. (a) UV–vis spectra of PLA polymer and free quercetin, PLA NPs and quercetin PLA nanoparticles. (b)
Fluorescence emission spectra (excited at 617 nm) of equimolar free and nanoencapsulated quercetin
molecule. UV–vis spectra and fluorescence spectra were recorded on Nanodrop spectrophotometer and
Perkin Elmer LS50B fluorescence spectrophotometer respectively.

The fluorescence measurements provide the indirect information about the nanoencapsulation of quercetin
on the PLA nanoparticles. The encapsulation of quercetin into PLA nanoparticles causes increase in the
fluorescence emission intensity (Fig. 4b), which indicates the change in microenvironment of quercetin in
PLA nanoparticles. Similar phenomenon has been observed on the inclusion of fluorophoric reagents into β-
cyclodextrins [30].

FTIR analysis is one of the important tools for the quick and efficient identification of encapsulated chemical
molecules. The major characteristic peaks of quercetin as (1100–1600 cm−1) and –OH phenolic bending
(1200–1400 cm−1) are present in free and PLA encapsulated quercetin (Fig. 5b and c). These peaks are absent
in PLA nanoparticles (Fig. 5a). PLA characteristic peaks of –C O stretching (1618.3 cm−1) and –C–O
stretching (1108.1 cm−1) appeared in the quercetin loaded PLA nanoparticles and PLA nanoparticles (Fig. 5a
and c). The presence of quercetin characteristic peaks on the PLA–quercetin nanomedicine is an indirect
confirmation of quercetin encapsulation on PLA nanoparticles.
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 Download : Download full-size image

Fig. 5. FTIR analysis of (a) PLA, (b) quercetin and (c) quercetin loaded PLA nanoparticles. FTIR spectra of
quercetin loaded PLA nanoparticles show peaks of quercetin associated with aromatic bending and
stretching (1100–1600 cm−1), –OH phenolic bending (1200–1400 cm−1) and PLA characteristic peaks –C O
stretching (1618.3 cm−1) and –C–O stretching (1108.1 cm−1).

3.4. Effect of quercetin loaded PLA nanoparticles on fluorescence spectra of BSA

The flavonoid quercetin has strong quenching effect on the protein fluorophores especially tryptophan and
tyrosine amino acid residue. The fluorescence intensity of BSA was decreased remarkably with the
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 increasing concentration of free as well as quercetin loaded PLA nanoparticles (Fig. 6a and b). Weak blue
shifts of the maximum λem (1–2 nm) were observed for free and nanoencapsulated quercetin. This confirms
the negligible effect of this molecule on the unfolding of BSA molecules. The increase in quenching effect
upon increasing quercetin concentration of free and nanoencapsulated form indicate the interaction of
quercetin to fluorophore (tryptophan) residue of BSA. The quenching effect of quercetin and quercetin PLA
nanoparticles was 45% and 25% respectively (Fig. 6c). The less quenching of quercetin loaded PLA
nanoparticles may be due to controlled release of quercetin from the nanoparticles. This is because less
amount of quercetin molecule is interacted with fluorophore of BSA. This is also an indirect proof of
sustained release formulation of quercetin PLA nanoparticles. Fluorescence quenching could proceed via
different mechanisms, usually classified as dynamic quenching and static quenching. It is concluded that the
quenching of BSA was not initiated by dynamic quenching, but probably partly by static quenching resulting
from the formation of quercetin–BSA complex [19], [21], [23], [24], [28].

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 Fig. 6. Fluorescence quenching effect of (a) quercetin and (b) quercetin loaded PLA nanoparticles on BSA. (c)
Stern–Volmer plot of F/Fo of PLA, quercetin and quercetin loaded PLA nanoparticles as BSA quencher.

3.5. Free radical scavenging activity

DPPH (1,1-diphenyl–2-picrylhydrazyl) assay was used to estimate the scavenging activity of free quercetin
and quercetin loaded PLA nanoparticles. The calibration curve was generated with the different known
amount (µg) of quercetin vs. DPPH scavenging activity (Fig. 7). The activity of free quercetin of similar
amount was taken as 100% activity. The equimolar quercetin loaded PLA nanoparticles and free quercetin
have similar antioxidant activity as free molecules. This confirms the retention of complete functional
architecture of quercetin after nanoencapsulation on PLA nanoparticles.

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Fig. 7. (a) Molecular representation of antioxidant mechanism of DPPH and (b) DPPH calibration curve of
quercetin reaction. This was obtained by plotting various amounts of quercetin (µg) vs. % inhibition of DPPH.

3.6. Encapsulation efficiency of quercetin loaded PLA nanoparticles

Biodegradable nanoparticles like PLA, PLGA, etc. have been reported for the encapsulation of large number
of therapeutic molecules such as taxol and ellagic acid [22] with highest affinity. Drug loading and
entrapment efficiency very much depend on the solid-state drug solubility in matrix material or polymer
(solid dissolution or dispersion). This is related to the polymer composition, molecular weight, drug
polymer interaction and the presence of end functional groups (ester or carboxyl) [3], [4], [15].
Encapsulation efficiency of quercetin in PLA nanoparticles was quantified using the validated HPLC and
DPPH based antioxidant assay [7]. The calibration curve was prepared by analysing different concentration
of free quercetin vs. peak area of eluted peak. The concentration of quercetin present in different sample
was calculated by regression equation generated by calibration curve (Fig. 8b):
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 (4)

Where y is the arbitrary area of quercetin eluted peak in HPLC and x is the amount of quercetin in µg. The
amount of nanoencapsulated quercetin was back calculated by subtracting the amount of quercetin in
nanoparticle synthesis mixture and supernatant after separation of synthesized quercetin loaded PLA
nanoparticles. HPLC analysis reveals that the encapsulation efficiency of quercetin loaded PLA nanoparticles
was 96.7% and actual drug loading was 19.4% (Table 1).

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Fig. 8. (a) Calibration curve of quercetin generated by validated HPLC methods. This was obtained by
plotting various amounts of quercetin (µg) vs. corresponding HPLC eluted peak area. (b) HPLC
chromatograms of equimolar PLA supernatant (upper scan) and quercetin loaded PLA supernatant (lower
scan). Chromatogram of PLA supernatant and quercetin loaded PLA nanoparticles supernatant were
recorded on HPLC after nanoparticles synthesis. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of the article.)

Table 1. Determination of encapsulation efficiency by DPPH and HPLC method.

By DPPH assay By HPLC methods

2 ml of each DPPH Abs. (at % Inhibition DPPH Std. Peak area HPLC Concentration
sample – 517 nm) of DPPH assay calib. HPLC (20 µl) assay (mg/ml) (std
lyophilized & activity calculated Concn. calculated calibration)
dissolved (std. (calculated by amount (mg/ml) amount
procedure) equation 1)a (µg) (µg)
:
 Quercetin initial 0.0434 ± 0.0058 40.5 0.12 0.0625 1184312 ± 5005 1.2 0.0625
NPs reaction
(0.0625 mg/ml)

PLA–quercetin 0.0440 ± 0.0064 39.7 0.116 0.0604 1144312 ± 6235 1.15 0.0598
nanoparticles

PLA–quercetin 0.073 ± 0.0028 0.00 0.004 0.0020 27803 ± 1145 0.05 0.0040
supernatant

OD of control of DPPH solution = 0.073.

a
Calculated by Eq. (1) in Section 2.

The encapsulation efficiency was further validated by DPPH based functional antioxidant activity assay
(Table 1). The antioxidant activity of complete reaction mixture and supernatant after the separation of
synthesized quercetin loaded PLA nanoparticles were determined by DPPH scavenging activity assay. The
encapsulation efficiency was back calculated by subtracting the antioxidant activity of supernatant from
antioxidant activity of original quercetin solution. Further, encapsulation efficiency was also calculated by
taking the quercetin loaded PLA nanoparticles by DPPH assay. The activity of PLA nanoparticles was taken as
blank. The amount of quercetin was calculated from the calibration curve (Fig. 7) generated on the basis of
DPPH scavenging activity for calculating the encapsulation efficiency. The relative scavenging activity (40%)
of quercetin–PLA nanoparticles was further crosschecked indirectly by subtracting the relative antioxidant
activity of supernatant after quercetin–PLA nanoparticles synthesis and initial synthesis solution. This is
because the antioxidant activity of same amount of normal quercetin and quercetin loaded PLA
nanoparticles was almost similar (data not shown). Both the results confirm very high encapsulation
efficiency (96.7%) of quercetin on PLA nanoparticles. Similar results have been reported earlier for the
encapsulation of different drugs onto PLA nanoparticles [22], [27].

3.7. In vitro release assay

The encapsulation efficiency of quercetin loaded PLA nanoparticles is very high (96.7%). This is due to the
incorporation method used for the nanoencapsulation of this molecule. The drug loaded by incorporation
method has a relatively small burst effect and better-sustained release characteristics [10]. The
nanoencapsulation of quercetin into PLA nanoparticles significantly improves the therapeutic efficacy and
bioavailability of this molecule. The in vitro release studies are important to know the adsorption or
encapsulation of quercetin on the PLA nanoparticles (Fig. 9b). An average 40–45% quercetin was released
within 0–0.5 h showing rapid burst release. This was normally attributed to the fraction of quercetin which
was adsorbed close to the surface of the nanoparticles [19]. Upon addition of the nanoparticles to the
release medium, this fraction of quercetin was diffused rapidly into the surrounding liquid. This shows the
rapid initial burst release profiles (Fig. 9a). The maximum release of quercetin was 87.6% after 96 h. The
slower and sustained release of quercetin may be attributed to diffusion of the quercetin entrapped within
the core of the nanoparticles. DPPH activity assay reveals the same trend for the amount of quercetin
released (Fig. 9a). Such controlled release of quercetin favours the development of quercetin nanomedicines.
Being amenable to surface modification, quercetin loaded PLA nanoparticles can also be used for the
targeted delivery.
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 Download : Download full-size image

Fig. 9. (a) Release profile of quercetin from quercetin loaded PLA nanoparticles by HPLC and DPPH method.
Release curve was obtained by plotting % of quercetin released vs. time. (b) HPLC chromatograms of release
solutions after regular intervals recorded on HPLC.

4. Conclusion
Quercetin, a lipophilic drug, was successfully encapsulated on PLA nanoparticles using solvent evaporation
method with encapsulation efficiency of 96.7% and 19.4% actual drug loading. The mean diameter of PLA
nanoparticles and quercetin loaded PLA nanoparticles was ∼170 ± 25 and ∼130 ± 30 nm respectively.
Antioxidant activity assay revealed that the functional activity of quercetin was retained after
nanoencapsulation. The biphasic release profile includes initial burst effect followed by sustained slow
release. High encapsulation efficiency, small size and slow release make quercetin loaded PLA nanoparticles
a suitable candidate for the further development of nanomedicines.

Acknowledgements
We are grateful to Dr. P.S. Ahuja, Director, IHBT for providing necessary facilities for carrying out this work.
The IHBT communication number of this article is 2009. Financial assistance to Avnesh Kumari, from
Council of Scientific and Industrial Research (CSIR) and Women Scientists Scheme (A) (SR/WOS-A/CS-
61/2008), Department of Science and Technology (DST), Government of India is truly acknowledged.
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