Journal of Drug Delivery Science and Technology 67 (2022) 102922

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Journal of Drug Delivery Science and Technology

journal homepage: www.elsevier.com/locate/jddst

Opinion Paper

Intranasal micellar curcumin for the treatment of chronic asthma

Ruchi Chawla a, *, Bhupendra Sahu a, Mohini Mishra a, Varsha Rani a, Rashmi Singh b
a
Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005, India
b
Department of Zoology, MMV, Banaras Hindu University, Varanasi, 221005, India

A R T I C L E I N F O A B S T R A C T

Keywords: Curcumin is a natural phytoconstituent obtained from the rhizomes of Curcuma longa (turmeric) and is known for
Chronic asthma its diverse anti-oxidant and anti-inflammatory benefits, but its clinical utility is limited by its poor aqueous
Curcumin solubility and rapid metabolism, which ultimately affects its bioavailability. The present study is focused on the
Micelles
formulation of curcumin loaded micellar dispersion for intranasal delivery for the treatment of chronic asthma.
Intranasal
Micellar dispersion was prepared by film formation method using poly-(ethylene oxide)-block-distearoyl
Dispersion
Anti-oxidant phosphatidyl-ethanolamine (mPEG5000-DSPE) as lipid surfactant and characterized for its physic-chemical
Anti-inflammatory properties. The curcumin micelles were evaluated for their anti-asthmatic action in ovalbumin (OVA)-induced
allergic asthma model in male wistar rats against standard drug dexamethasone. The micelles showed mean
particle size in the range of 20.03 nm–26.48 nm. The micellar dispersion exhibited negative zeta potential in the
range of − 26.22 to − 25.52 mV. Incorporation of curcumin into micelles helped in enhancing the solubility of
curcumin besides providing it protection against degradation. In vitro studies showed sustained relase of cur­
cumin up to 36 h. A 14- fold increase in the bioaviability of the curcumin was measured when administered as
micelles. In addition, 4.4- fold higher concentration of drug was measured in the lungs for micellar curcumin.
Comparable reduction in the levels of intracellular ROS was observed for i.n. curcumin-micelles (approx.57.6%)
and dexamethasone (59.3%). Also, micellar curcumin produced significant supression (p < 0.05) in the release of
nitric oxide. Through the present study, we can suggest the potential application of curcumin micelles in the
treatment of asthma.

1. Introduction hydroxyeicosatetranoeic acid (HETE) and leukotriene B3 [3].

Amongst the inflammatory diseases, asthma is highly prevalent and
is marked by varying respiratory signs and airflow limitations like
hyper-responsiveness with repeated occurrence of wheezing, tightness
of chest, coughing, difficulty in breathing, and airway remodeling.
Asthma is triggered by allergic stimuli (mites, cockroach residue, pol­
lens, mould, and animal scurf) and non-allergic stimuli (viral contagion,
subjection to tobacco smoke, exercise, cool air), which induce a series of
episodes leading to chronic airway inflammation (swelling) [1].
The mast cell is the progenitor of inflammatory response to all sorts
of stimuli in the airways [2]. A very small proportion of the mast cells is
present in intra-luminal region, but activation and degranulation of
these mast cells causes progressive mucosal permeability, with subse­
quent activation of tissue mast cells [3]. The mast cells generate a va­
riety of pro-inflammatory mediators like histamine, serotonin,
eicosanoides and platelet activating factors, cytokines and chemokines
[4], chemotactic factors for neutrophils and eosinophils, such as
Drugs to manage chronic signs of asthma include inhaled cortico­
steroids (ICs), leukotriene modifiers, long-acting beta-agonists (LABAs),
theophylline and combination inhalers containing corticosteroids and
LABA [2]. Despite effective therapeutic effects, toxicity associated with
inhaled corticosteroid acts as a limiting factor.
Different inflammatory signaling mechanisms involved in asthma
include Src family kinases (SFKs), protein kinase C (PKC), growth factor
tyrosine kinase receptors, nicotinamide adenine dinucleotide phosphate
(NADPH)/reactive oxygen species (ROS), mitogen activated protein ki­
nase (MAPKs), nuclear factor-kappa B (NF-KB), and activator protein-1
(AP-1). MAPK pathway significantly influences and regulates cell
growth and proliferation, chemotaxis, degranulation, and other secre­
tary processes [5]. Airway remodeling is the resultant consequence of
long term inflammation, so use of anti-inflammatory agents in the
treatment of asthma can help diminish the risk of aggravation of asthma
and its effects. Among the natural phytochemicals, curcumin consider­
ably controls inflammation, apoptosis and cell growth, making it

* Corresponding author.
E-mail address: rchawla.phe@iitbhu.ac.in (R. Chawla).

https://doi.org/10.1016/j.jddst.2021.102922
Received 18 March 2020; Received in revised form 13 September 2020; Accepted 14 October 2021
Available online 27 October 2021
1773-2247/© 2021 Elsevier B.V. All rights reserved.
R. Chawla et al.
beneficial for the prevention and treatment of inflammatory diseases.
Curcumin is a vital curcuminoid present in Curcuma longa (a rhizome)
and exhibits considerable antioxidant and anti-inflammatory properties
[6]. In one of our previous studies we have proposed the use of intra­
nasal curcumin as a complementary medication for prevention of airway
inflammations and bronchoconstrictions in asthma without any side
effect [7]. Studies have shown that intranasal delivery of drugs results in
fascinating results especially for treatment of respiratory tract disorders
like asthma, chronic obstructive pulmonary disease, pulmonary infec­
tion, pulmonary hypertension, lung cancer, and cystic fibrosis. The
intra-nasal region provides highly vascularized surface area for ab­
sorption of lipophilic drugs and its eminence lies in sidestepping of
first-pass metabolism by liver which in turn improves the bioavailability
of the drug [4]. The radical scavenging property of
polymer-glycerosomes of curcumin prepared using sodium hyaluronate
and trimethyl chitosan was observed in A549 cells stressed with
hydrogen peroxide, in addition to suppressed production of cytokine IL6
and IL8. Nebulized glycerosomes delivered to rats showed significant
accumulation of curcumin in lungs [8].
Curcumin, though a wonder drug, with multi-targeting pharmaco­
logical potential suffers from poor aqueous solubility and low systemic
bioavailability [9]. In this present study, we have tried to improve the
pharmaceutical properties of curcumin by loading them into micelles for
treatment of asthma. Using coprecipitation rehydration (solvent evap­
oration) method, micelles were prepared using poly-(ethylene oxide)-­
block-distearoyl phosphatidyl-ethanolamine (mPEG5000-DSPE)/MW
5000 Da (mPEG5000-DSPE) [10]. The hydrophobic core of DSPE pro­
vides a suitable environment for incorporation of lipophilic drug (cur­
cumin) protecting it against degradation. Also, PEG5000-DSPE micelles
are effective drug carriers especially for poorly water soluble drugs
which show non-specific biodistribution and targeting, and poor oral
bioavailability [11]. Moreover, PEGylation results in increase in the
molecular weight of carriers leading to increased half-life and further,
higher retention in the lungs [12]. Studies have shown that PEG mole­
cules larger than 5ooo daltons stay longer in the lungs [12]. Further, use
of PEG5000-DSPE forms micelles with longer circulation time. Also, these
structure escape complement activation and scavenging through opso­
nisation leading to better biodistribution [13]. The complement is
further weakened, if the PEG-conjugated nanocarriers possess a negative
zeta potential [14].
2. Materials and methods
2.1. Materials
Curcumin, Ovalbumin, aluminium hydroxide were generously pro­
vided by Sisco Research Laboratory Pvt. Ltd, India and mPEG5000-DSPE
was provided by Lipoid, Germany as a gift sample. Potassium dihy­
drogen orthophosphate, disodium hydrogen phosphate and sodium
hydroxide were purchased from Merck Life Science Pvt. Ltd, Mumbai.
Analytical grade chemicals were used in the study.
2.2. Formulation of curcumin loaded micellar dispersion
Curcumin-loaded micelles (C-mic) were prepared by co-precipitation
rehydration (solvent evaporation) method. Varying ratios (1:2, 1:4, 1:6)
of curcumin (drug) and mPEG5000-DSPE as shown in Table 1 were
Table 1
Formulation batches prepared using different drug-polymer ratio.
Formulation (Drug: Polymer Amount of drug (in Amount of polymer (in
ratio) mg) mg)
F1 (1:2) 20 40
F2 (1:4) 20 80
F3 (1:6) 20 120
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Journal of Drug Delivery Science and Technology 67 (2022) 102922
dissolved in 10 ml of ethanol. The mixture was sonicated for 2 min using
probe sonicator and then vortex mixed thoroughly. The solvent was
eliminated using vacuum rotary evaporator operated at 100 rpm at
45 ◦ C under vacuum (200 psig), to form a thin film of drug-containing
lipid. Traces of remaining solvent were removed by applying vacuum
overnight. The dried film was rehydrated with 10 ml of phosphate buffer
saline [10] alongwith gentle shaking at 40 ◦ C for 10 min. The unsolu­
bilized drug was removed by filtration using 0.22 μm filter [15].
3. Characterization of curcumin loaded micellar dispersion
3.1. UV–Vis spectrophotometric method
Curcumin was analyzed using UV–Vis spectrophotometric at 427 nm
λmax according to the method described elsewhere with modification
[16].
3.2. Particle size, polydispersity and zeta potential
The particle size, polydispersity, and zeta potential of micelles was
measured using DELSA™ NANO (Beckman Coulter, USA) based on dy­
namic light scattering technique. After suitably diluting the samples in
ultra purified water and filtration through 0.22μ, size of the micelles was
measured at 25 ◦ C. For the measurement of zeta potential, samples (in
triplicate) were diluted suitably in 1 mM NaCl solution and using elec­
trophoretic light scattering technique the potential was recorded at
25 ◦ C under applied electric field of 16 V/cm [17].
3.3. Determination of entrapment efficiency
The amount of drug entrapped in micelles provides an estimate of the
percent entrapment efficiency (%EE). The micellar dispersion was ultra-
centrifuged at 10000 rpm at 4 ◦ C for 15 min. The clear supernatant was
discarded and dispersion was centrifuged again at 15000 rpm at 4 ◦ C for
30 min. The pellet free from unentrapped drug was soaked in 10 ml of
acetonitrile and sonicated for 10 min. The drug released from micelles
was determined spectrophotometrically against similarly digested pla­
cebo blank (without curcumin) [17]. The entrapment efficiency was
calculated using the formula:
%EE = (Drug entrapped in micelles / total drug added) x 100.
3.4. Solid state characterization studies
Surface morphology of prepared micelles was visualized using
scanning electron microscope (ZEISS Supra 40, Germany) under normal
atmospheric conditions. For analysis, suitably diluted samples were
placed on a glass slide, spread evenly and allowed to dry. The dried
samples were then gold coated with the help of QUORUM (Q150RES)
sputter coater for 90 s at 20 mA, after which they were placed in the
sample holder and analyzed at 20 kV.
Interaction between the drug and the excipients can be studied using
Attenuated Total Reflectance-Infrared (ATR-IR). The properties of a
drug are governed by the type of functional groups present in it, and in
case of any interaction with the excipients, it leads to alteration of the
functional groups; thereby leading to appearance or disappearance of
characteristic peaks. In the present study, drug-excipient compatibility
was studied by obtaining ATR spectra (using infrared spectrophotom­
eter; Bruker ALPHA ECO-ATR, USA) of curcumin, PEG5000-DSPE,
physical mixture of curcumin and PEG5000-DSPE (in 1:1 ratio), and
lyophilized C-mic. The spectra was analyzed and interpreted for the
presence of characteristic peaks of the functional groups.
R. Chawla et al.
3.5. Stability study
The prepared formulation samples were stored and sealed in USP
Type-1 amber colored glass vials and subjected to stability studies at 30
± 2 ◦ C/65% RH. The samples were evaluated at different time intervals
(15, 30, 45 days) and the stability was evaluated on the basis of changes
in particle size, zeta potential and drug content [18].
3.6. In-vitro drug release study
In-vitro drug release studies were performed using dialysis membrane
technique. After hydrating the dialysis bag (cellulose membrane mol.
wt. cutoff 12–14000 Da, HIMEDIA, India) overnight in dissolution
media, it was filled with 2 ml of micellar dispersion and both its ends
were tied and firmly clamped. The sealed dialysis bag was incubated in
150 ml of release medium, (phosphate buffer; pH 7.4), under continuous
stirring at 100 rpm at 37 ± 0.5 ◦ C. 5 ml aliquots (in triplicate) were
withdrawn at pre-determined time intervals with simultaneous
replacement of equivalent volume of fresh media to maintain sink
condition. After suitably diluting the samples, they were analyzed
spectrophotometrically at 427 nm to determine the extent of release of
curcumin [15].
3.7. In-vivo studies
Male Wistar rats (160 ± 30 g) bred at Central Animal House (CAH),
Institute of Medical Science, Banaras Hindu University (IMS BHU), were
used for the study. The animals were housed under standard laboratory
conditions (25 ± 2 ◦ C, 60–70% humidity), with alternate 12-h dark and
light cycle, with free access to food and water. The animals were accli­
matized to laboratory conditions before the study. The experimental
protocol was approved by the Institutional Animal Ethical Committee
(IAEC), IMS, BHU with registration number 542/GO/ReBi//S/02/
CPCSEA.
3.8. Pharmacokinetic study of curcumin
Animals were fasted overnight before the study. The animal was
divided into two groups with 6 animals each; Group I was treated with
curcumin loaded micellar dispersion (C-mic) and Group II with curcu­
min suspension (C-susp). Each group was administered 5 mg/kg of body
weight of curcumin intranasally. Blood samples (500 μl) were collected
from the femoral vein at designated time intervals (0, 0.5, 1, 2, 4, 6, 8, 12
and 24 h) and stored in Eppendorf tubes containing heparin (0.2 mg/
ml). The samples were centrifuged at 1500 rpm for 15 min and the
supenatant collected was deproteinized with acetonitrile. The depro­
teinzed samples were vortexed for 2 min and then centrifuged at 14000
rpm for 5 min. The supernatant was then filtered through 0.22-μm sy­
ringe filter and subjected to chromatographaic analysis [7,15,14]. Iso­
cratic elution was performed on Waters XTerra RP18, 5 μm, 250 mm ×
4.6 mm i.d C18 analytical column, coupled with a PDA detector at a flow
rate of 1 ml/min, using ACN: Water (60:40) as mobile phase at 425 nm
[19].
3.8.1. Measurement of the concentration of curcumin in bronchoalveolar
fluid
At the end of the study, rats were sacrificed, and bronchoalveolar
fluid was collected by the tracheal cannulation method [20]. 1 ml of
chilled BPS was injected into the lungs through cannula and rinsed
twice. The broncioalveolar fluid was mixed with acetonitrile in the ratio
1:3, vortex mixed. After centrifugation at 3000 rpm for 10 min, super­
natant (organic phase) was collected and filtered through a 0.22-μm
syringe filter and analyzed by chromatographic method to determine the
concentration of curcumin.
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Journal of Drug Delivery Science and Technology 67 (2022) 102922
3.9. In-vivo antiasthmatic (anti-inflammatory) activity of curcumin in
OVA-induced asthma model
Reactive oxygen species serve as signaling molecules or indicators of
inflammation. Asthma is an inflammatory condition of the respiratory
tract and associated with increased generation of ROS and free-radical
mediated reactions resulting in oxidative stress in the respiratory tract
[21]. Increased levels of ROS cause oxidation of proteins, DNA, and
lipids and may also evoke secondary cellular reactive species. Curcumin
acts as a scavenger of ROS by enhancing the activity of antioxidant
enzymes like catalase, superoxide dismutase [22], glutathione perox­
idise and heme oxygenase-1 [22,23]. The extent to which curcumin can
regulate the generation of ROS was evaluated by measuring the level of
ROS in BALF cells [24].
3.10. Animal studies
3.10.1. Sensitization and challenge protocol
The animals were allowed to acclimatize for 5 days prior to treat­
ment. The animals were randomly divided into six groups (n = 6/group)
as: control group (Group I), vehicle (saline) treated (Group II), blank
micelles treated (Group III), curcumin suspension treated (Group IV),
curcumin micelles treated (Group V), and dexamethasone treated
(Group VI). All groups except control were sensitized intraperitoneally
(I.P.) with 1 mg OVA adsorbed on 20 mg Al(OH)3 gelatinous on days 0,
7, and 14. All the animal groups were challenged with 1.1% OVA so­
lution in 200 μL normal saline by intratracheal instillation from day
15–20 as shown in Fig. 1 [25].
3.10.2. Treatment protocol
OVA sensitized and challenged animals were treated from days
15–20 as follows: Group I and II were treated with normal saline i.n.;
group III was treated with i.n. blank micelles, group IV was treated with
C-susp (5 mg/kg i.n.), group V was treated with prepared C-mic (5 mg/
kg i.n.) and group VI was treated with dexamethasone (1 mg/kg i.p.)
(shown in Fig. 1) [26].
3.10.3. Collection of BAL-Fluid
After euthanization (pentobarbital sodium 200 mg/kg, i.p.) the an­
imals were sacrificed on day21. After intratracheal cannulation, 1 ml of
chilled BPS was injected into the lungs and applying gentle aspiration
BAL fluid was collected. The procedure was repeated twice and the fluid
was centrifuged at 3000 rpm for 10 min at 4 ◦ C and the cell pellet was
collected.
3.10.4. Measurement of intracellular reactive oxygen species (iROS)
Total number of viable cells present in BALF were counted and then,
the cells were washed with chilled phosphate buffer saline. For labelling
of iROS, cells at a concentration of 1 X 105 were incubated in 100 μl of
20 mM dichlorofluorescein diacetate (DCFH-DA) for 45 min at 37 ◦ C in
dark. Further, using fluorescence spectrometer (λex: 490 nm; λem: 515
nm), the fluorescence intensity was measured with the values being
normalized against the fluoresence intensity of normal cells [5].
3.10.5. Detection of nitrite level in serum
Nitric oxide is one of the potential makers of inflammation and
higher levels of nitric oxide are produced in asthamatic condition. Three
isoforms (two constitutive and one inducible) of nitric oxide enzyme are
responsible for production of neuronal or type I (nNOS), inducible iso­
form or type II (iNOS), and the endothelial isoform or type 3 (eNOS)
[26–31]. Studies have shown that curcumin acts as a scavenger of nitric
oxide and modulates signaling pathways mediated via (NF-KB)and
mitogen-activated protein kinase, which prevent bronchial inflamma­
tion in asthmatic patients [30]. Further, studies have also shown that
curcumin decreases the nitric oxide synthase activity [32–34]. Studies
have shown that curcumin may be used as an adjuvant in the treatment
R. Chawla et al. Journal of Drug Delivery Science and Technology 67 (2022) 102922

Fig. 1. Study protocol for OVA-induced asthma model.

of asthma as it suppresses iNOS and NO. In the present study, the effect 3.02%, 83.4 ± 3.82% and 88.8 ± 4.14% respectively. With increase in
of intranasally delivered curcumin micelles on the relase of nitric oxide the proportion of lipid, an increase in % entrapment efficiency was
was studied. At room temperature, 100 μl of serum was mixed with 100 observed, probably due to availability of larger micellar core for the
μl of vanadium chloride, followed by addition of 100 μl of Griess reagent incorporation of drug.
to the mixture. The 96 microwell plate was incubated for 45 min at
37 ◦ C, and the absorbance was measured at 540 nm using microplate 4.1.3. Solid state characterization studies
reader. The concentration in samples was estimated against standard The scanning electron micrographs of curcumin-loaded micelles
curve of sodium nitrite [35]. have been shown in Fig. 2. Spherical micelles with a core-shell structure
were observed within size range of 50 nm as a result of minimization of
4. Result and discussion interfacial free energy of block copolymers, which tends towards the
hydrophobic part i.e. the core of micelles [38].
4.1. Evaluation of curcumin loaded micellar dispersion IR spectra of curcumin, PEG5000-DSPE, physical mixture of curcumin
and PEG5000-DSPE, and C-mic (batch F2; data for batches F1 and F3 not
4.1.1. Particle size, polydispersity and zeta potential mentioned) was taken (Fig. 3). Characteristic peaks of curcumin corre­
Table 2, shows the mean particle size, and polydispersity of curcumin sponding to –OH stretching was observed at 3588.23 cm− 1. Peaks for
loaded micellar dispersion. The mean particle size of formulations F1, stretching of aromatic C-H and aliphatic C-H in curcumin were observed
F2, and F3 were found to be 20.03 nm, 24.92 nm, and 26.48 nm at 3115.78 cm− 1 and 2879.01 cm− 1 respectively. Peak corresponding to
respectively with a zeta potential of − 26.22, − 24.68, and − 25.52 C=O was found at 1697.37 cm− 1 and that due to C-O stretching was
respectively. Increase in lipid amount resulted in an increase of particle found between 1288.89 cm− 1 and 1394.33 cm− 1. All the characteristic
size. Sub-micron sized particles with diameter less than 100 nm stay peaks of curcumin were retained and no extra peaks were observed in
dissolve in the alveolar fluid and evade phagocytosis and mucociliary the physical mixture and the formulation, indicating absence of any
clearance thereby increasing the time for which micelles stay in the physical or chemical incompatibility between drug and excipients used
lungs [17]. The carbamate linkage by which PEG is conjugated to dis­ in formulation of micelles.
tearoyl phosphatidylethanolamine (DSPE) imparts a net negative charge
to the phosphate moiety resulting in negative zeta potential of the 4.1.4. Stability study
formulation. A large negative charge provides stability against aggre­ The curcumin-loaded micelles were subjected to stability testing and
gation and as per already published reports retention of negatively the results were compared with freshly prepared batches corresponding
charged entities is significantly higher in the lungs in comparison to to the formulations F1, F2 and F3. No significant difference in particle
their positively charged counterparts [35,36]. The dispersion exhibited size, zeta potential, and drug content were observed between the sta­
as narrow size distribution indicating a homogenous formulation bility batches and fresh batch. Polyethyleneglycol (PEG) conjugated
(Table 2). Monodisperse lower-sized particles are absorbed highly micelles have lower critical micellar concentration (CMS) in comparison
through the nasal mucosa resulting in higher bioavailability. to conventional micelles. Studies have shown CMC of PEG conjugated
micelles to be around 10− 7 M, while in conventional micelles it is about
4.1.2. Entrapment efficiency 10− 3 to 10− 4 M. So, conjugation with PEG result in the formation of a
Percentage ratio of the amount of drug loaded in micelles to the stable micellar system, which shows slow dissociation and allows
theoretical quantity of the drug added during preparation of micelles incorporation and retention of drugs for longer duration providing
can be indicated as Percentage drug entrapment efficiency (%EE). The higher drug concentration in the target tissues [39].
drug entrapment efficiency of F1, F2 and F3 was found to be 73.1 ±
4.2. In-vitro drug release study
Table 2
Fig. 4, shows the cumulative percentage release of curcumin from the
Particle size, polydispersity, zeta potential, and %entrapment efficiency of
formulation batches. prepared micellar dispersion (F1, F2, and F3) in phosphate buffered
saline (pH 7.4). The study performed by the dialysis membrane method
Formulation Drug: Average PDI Zeta Entrapment
at 37◦ ± 0.5 ◦ C with continuous stirring at 100 rpm, showed slow and
Lipid Particle Size Potential Efficiency
ratio (nm) (mV) sustained release for more than 36 h with a burst release during the first
hour. Surface bound drug resulted in burst release followed by diffusion
FI 1:2 20.03 ± 1.92 0.238 − 26.22 ± 73.1 ±
2.02 3.02% controlled sustained release of curcumin from the core to the surface.
F2 1:4 24.92 ± 2.19 0.194 − 24.68 ± 83.4 ± Approximately, 60.38%, 73.19%, and 58.04% of curcumin was released
1.89 3.82% from formulations F1, F2 and F3 respectively after 36 h of study. The
F3 1:6 26.48 ± 1.51 0.141 − 25.52 ± 88.8 ± t50% (time at which 50% of the drug was released in medium) of the
2.11 4.14%
formulations were about 14h, 10h, and 12h for the F1, F2, and F3,

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R. Chawla et al. Journal of Drug Delivery Science and Technology 67 (2022) 102922

Fig. 2. Scanning electron micrographs of formulation batches of C-mic F1, F2 and F3.

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R. Chawla et al. Journal of Drug Delivery Science and Technology 67 (2022) 102922

Fig. 3. ATR-IR spectra of curcumin, PEG5000-DSPE, physical mixture of curcumin and PEG5000-DSPE (1:1), and C-mic formulation batch F2.

Fig. 4. Drug release profile of curcumin from formulation batches F1, F2 and F3.

respectively. The slow and sustained release of the drug could be evi­ coefficient (R2) of around 0.99. The pharmacokinetic parameters of
denced by the values of t50% [37]. intranasally administered curcumin suspension and curcumin loaded
micellar dispersion have been shown in Table 3. Micelles produced
almost double concentration of curcumin at Cmax in comparison to
4.3. In-vivo studies
suspension. The reference chromatograms have been shown in Figs. 5
and 6.
4.3.1. Pharmacokinetic study of curcumin in rat
Formulation batch F2 with highest entrapment efficiency (83.4%)
and in vitro drug release (73.012%) was used for further pharmacoki­
Table 3
netic evaluation. The concentration of curcumin in biological samples
Pharmacokinetic parameters of curcumin.
was determined by RP-HPLC method [40]. For preparation of standard
Product Cmax AUC0–24 h Tmax Ke (1/ T1/2 MRT
calibration curve, working stock solutions were prepared by spiking
(μg/ml) (μg⋅h/mL) (h) h) (h) (h)
blank plasma/BALF with known amounts of drugs in the concentration
range of 2 μg/ml to 50 μg/ml. The calibration curve showed linear C-susp 6.94 66.79 2 0.0958 7.232 7.889
C-mic 13.28 954.33 4 0.0797 8.688 10.266
correlation between drug concentration and peak area with a correlation

6
R. Chawla et al.
Fig. 5. HPLC Chromatogram of (a) blank and (b) pure curcumin (30 μg) analyzed at 425 nm.
Entrapment of curcumin within the micellar core protected it against
degradation and resulted in its increased availability, as can be corre­
lated with a 14-fold increase in the bioavailability of curcumin. The
plasma concentration versus time profile has been shown in Fig. 7.
Further, slower rate of elimination from micelles resulted in increased
residence (MRT) of curcumin in body.
4.3.2. Measurement of the concentration of curcumin in BALF
4.3.2.1. In-vivo antiasthmatic (anti-inflammatory) activity of curcumin in
OVA-induced asthma model. After 24 h of i.n. administration of curcu­
min formulations (micelles and suspension), the concentration of cur­
cumin was measured in BALF (Fig. 8). A significantly higher
concentration of curcumin 2.02 μg/ml was measured in BALF of the
animals to which micelles were administered. However, for the other
group, concentration of curcumin in the BALF was found to be 0.456 μg/
ml. Approximately, 4.4 fold higher concentration of curcumin was
measured in the lungs indicating the efficacy of micelles in carrying the
drug to the lungs and also protecting it against degradation accounting
for its higher concentration in the lungs.
4.3.2.2. Effect of curcumin on the generation of intracellular reactive ox­
ygen species. ROS serves as a signaling molecule that could be directly
linked to multiple cell functions and inflammation. The extent of regu­
lation of ROS on intranasal administration of curcumin-loaded micelles
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Journal of Drug Delivery Science and Technology 67 (2022) 102922
was examined in the experimental groups by applying repeated measure
One Way ANOVA with Newman-Keuls Multiple Comparision Test.
Fig. 9 shows significant difference in level of ROS between control
and treatment groups (p < 0.05). Production of ROS increased after OVA
sensitization and challenge in groups II and III as compared to the
Control group. Results has showed decrease level of intracellular ROS
(iROS) that is 57.6% and 59.3% in curcumin-micelle i.n administred
group V, and i.p. dexamethasone administred group VI animals
respectively. The levels of iROS decreased to 46.5% and 44.2% respec­
tively in group II and III (Table 4). These study shows that intranasally
administered curcumin micelles exhibit higher anti-oxidant effect as
compared to curcumin suspension and exert a comparatively similar
effect in reference to standard drug dexamethasone (Fig. 7).
4.3.2.3. Effect of curcumin on nitrite level. The nitrite levels are indica­
tive of the extent of nitric oxide present in the serum. The effect of
intranasally administrated curcumin-loaded micelles in regulating ni­
trite levels was examined and statistically analyzed by applying
repeated measure one way ANOVA with Newman-Keuls Multiple
Comparision Test. Significantly higher nitrite levels were observed in
Group II and III asthmatic groups as compared to Control (group I).
Significant supression (p < 0.05) in the ] nitric oxide release to the
extent of 15% and 37.7% after intransally administration of curcumin
suspension (Group IV) and curcumin micelles (Group V) respectively.
However, comparable decrease in the nitrite levels were observed for
R. Chawla et al. Journal of Drug Delivery Science and Technology 67 (2022) 102922

Fig. 6. HPLC Chromatogram of curcumin released at 12 h from (a) cur-suspension (retention time: 1.295 min) and (b) curcumin micelles (retention time: 1.285 min)
analyzed at 425 nm.

Fig. 7. Plasma concentration-time profile of curcumin loaded micelles and cur-suspension.

Groups V and VI (37.7% and 40.7% respectively) (Fig. 10 and Table 4).
The ability of drug-loaded polymeric micelles to penetrate across the
epithelial cells is further strengthened by the presence of PEG which
facilitates penetration across biological barriers tissue extracellular
matrix, cellular barriers, and biological fluids such as mucus to reach the
inflamed site and decrease inflammation (as compared to non-
PEGylated formulations) [41,42].
5. Conclusion
Curcumin is a pleiotropic herb which interacts with several targets
and controls the progression of disease. Of the various reported effects,
curcumin exhibits potent antioxidant and anti-inflammatory properties
and helps control inflammation especially in inflammatory diseases like
asthma. The therapeutic potential of curcumin is limited by its poor

8
R. Chawla et al. Journal of Drug Delivery Science and Technology 67 (2022) 102922

Fig. 8. HPLC Chromatogram of curcumin analyzed in bronchoalveolar fluid on administration of (a) curcumin suspension (retention time: 1.285 min) and (b)
curcumin micelles (retention time: 1.285 min) at 425 nm.

Table 4
Percentage decrease in level of ROS and nitrite in different treatment groups.
S. Treatment % decrease in ROS level % decrease in Nitrite level
N. Group compared to the asthmatic compared to the asthmatic
group group

1. C-Susp 29.84 15
2. C-Mic 46.51 37.7
3. Dexamethasone 59.37 40.7

Fig. 9. Effect of different treatments on levels of ROS in BALF of rat. Exposure

to OVA increased ROS level in the BALF cells, and reduction of which was
observed on intranasal administration of curcumin micelles. The results are
shown as mean ± SEMs (ap<0.05 as compared to Control, b p < 0.05 as
compared to Group II, cp < 0.05 as compared to Group III, d p < 0.05 as
compared to Group IV and e p < 0.05 as compared to Group IV).

aqueous solubility, rapid metabolism, and quick degradation at acidic

pH. In one of our previous studies we have reported the effectiveness of
intranasally administered curcumin in asthma [7]. Loading of curcumin
into micelles enhances the biopharmaceutical properties of curcumin.
Besides protecting it against degradation, the solubility of curcumin is
increased up to 40% due to micellar solubilization [43,44]. Also, mi­
celles provide sustained release of the drug. The study shows that
Fig. 10. Effect of different treatments on nitrite level in serum of rat. The
micelles produced effects similar to that of standard drug dexamethasone. The
results are shown as mean ± SEMs (ap<0.05 as compared to Control, b p < 0.05
as compared to Group II, cp < 0.05 as compared to Group III, and d p < 0.05 as
compared to Group IV).

9
R. Chawla et al.
micellar curcumin shows significantly better attenuation of
OVA-induced chronic asthma in comparison to curcumin administered
as suspension. Also, curcumin micelles showed significant attenuation in
generation of ROS and nitric oxide, similar to that obtained on treatment
of dexamethasone. Significantly higher concentration of curcumin was
observed in the BAL fluid when administered as micelles in comparison
to curcumin suspension. Overall, the micelles provide an efficient sys­
tem for delivery of drug via intranasal route.
Author declaration
The authors are aware of the modifications made in the revised
manuscript.
Declaration of interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper. The author(s) received no financial
support for the research.
References
[1] K.J.H. Jaclyn Quirt, Asthma. J Allergy Clin Immunol. 14 (Suppl 2) (2018) 57–70.
[2] Chung KF. Role of Inflammation in the Hyperreactivity of the Airways in Asthma.
Vol. vol. 41, Thorax. BMJ Publishing Group; 1986. p. 657–662.
[3] J. Bienenstock, Immunology of the Lung and Upper Respiratory Tract, McGraw-
Hill, 1984, p. 414.
[4] D. Proud, J. Sweet, P. Stein, R.A. Settipane, A. Kagey-Sobotka, M.H. Friedlaender,
L.M. Lichtenstein, Inflammatory mediator release on conjunctival provocation of
allergic subjects with allergen, J. Allergy Clin. Immunol. 85 (5) (1990 May)
896–905.
[5] P.S. Chauhan, D.K. Singh, D. Dash, R. Singh, Intranasal curcumin regulates chronic
asthma in mice by modulating NF-ĸB activation and MAPK signaling,
Phytomedicine 51 (2018) 1–16.
[6] I. Stankovic, Curcumin, Chem Tech Assess 1 (8) (2004) 1–8.
[7] Subhashini, P.S. Chauhan, S. Kumari, J.P. Kumar, R. Chawla, D. Dash, M. Singh,
R. Singh, Intranasal curcumin and its evaluation in murine model of asthma, Int.
Immunopharm. 17 (3) (2013) 733–743.
[8] M.L. Manca, J.E. Peris, V. Melis, D. Valenti, M.C. Cardia, D. Lattuada, E. Escribano-
Ferrer, A.M. Fadda, M. Manconi, Nanoincorporation of curcumin in polymer-
glycerosomes and evaluation of their in vitro-in vivo suitability as pulmonary
delivery systems, RSC Adv. 5 (127) (2015 Dec) 105149–105159.
[9] L. Zhao, J. Du, Y. Duan, Y. Zang, H. Zhang, C. Yang, F. Cao, G. Zhai, Curcumin
loaded mixed micelles composed of Pluronic P123 and F68: preparation,
optimization and in vitro characterization, Colloids Surf. B Biointerfaces 97 (2012)
101–108.
[10] Masoud J, Abdulla A, Tze-Fung Tan Y, Darwis Y. Rehydrated lyophilized
rifampicin-loaded mPEG-DSPE formulations for nebulization.
[11] K.K. Gill, A. Kaddoumi, S. Nazzal, PEG–lipid micelles as drug carriers:
physiochemical attributes, formulation principles and biological implication. https
://doi.org/10.3109/1061186x.2014.997735, 2015.
[12] H. Gursahani, J. Riggs-Sauthier, J. Pfeiffer, D. Lechuga-Ballesteros, C.S. Fishburn,
Absorption of polyethylene glycol (PEG) polymers: the effect of PEG size on
permeability, J Pharm Sci (2009 Aug) [cited 2020 Feb 26];98(8):2847–56.
Available from: http://www.ncbi.nlm.nih.gov/pubmed/19408293.
[13] M.N. Sibata, A.C. Tedesco, J.M. Marchetti, Photophysicals and photochemicals
studies of zinc(II) phthalocyanine in long time circulation micelles for
photodynamic therapy use, Eur. J. Pharmaceut. Sci. 23 (2) (2004 Oct) 131–138.
[14] A. Vonarbourg, C. Passirani, P. Saulnier, P. Simard, J.C. Leroux, J.P. Benoit,
Evaluation of pegylated lipid nanocapsules versus complement system activation
and macrophage uptake, J. Biomed. Mater. Res. Part A [Internet] (2006 Sep 1)
[cited 2020 Feb 26];78(3):620–8. Available from: http://www.ncbi.nlm.nih.gov/p
ubmed/16779767.
[15] M.N. Sahib, S. Abdalwahed, Abdulameer, Y. Darwis, K.K. Peh, Y.T.F. Tan,
Solubilization of beclomethasone dipropionate in sterically stabilized phospholipid
nanomicelles (SSMs): physicochemical and in vitro evaluations, Drug Des. Dev.
Ther. 6 (2012) 29–42.
[16] S. Warule Pooja, P.G.S.A.D. Patel Vipul, Development and validation of UV
spectrophotometric method for the estimation of curcumin in an ayurvedic
formulation haridrakhand, Int J Pharm DRUG Anal 5 (5) (2017) 193–197.
[17] K.K. Gill, S. Nazzal, A. Kaddoumi, Paclitaxel loaded PEG5000-DSPE micelles as
pulmonary delivery platform: formulation characterization, tissue distribution,
plasma pharmacokinetics, and toxicological evaluation, Eur. J. Pharm. Biopharm.
79 (2) (2011 Oct) 276–284.
[18] S. Ugwu, A. Zhang, M. Parmar, B. Miller, T. Sardone, V. Peikov, I. Ahmad,
Preparation, characterization, and stability of liposome-based formulations of
mitoxantrone, Drug Dev. Ind. Pharm. 31 (2) (2005) 223–229.
10
Journal of Drug Delivery Science and Technology 67 (2022) 102922
[19] L. Ji, J. Yunyun, W. Jun, F. Guorong, W. Yutian, Z. Chuan, A rapid and simple
HPLC method for the determination of curcumin in rat plasma: assay development,
validation and application to a pharmacokinetic study of curcumin liposome,
Biomed Chromatogr (2009 Nov) [cited 2020 Feb 26];23(11):1201–7. Available
from: http://www.ncbi.nlm.nih.gov/pubmed/19488971.
[20] W. Qamar, Technical considerations and precautions in in situ bronchoalveolar
lavage and alveolar infiltrating cells isolation in rats, Toxicol. Mech. Methods 25
(7) (2015 Sep) 547–551.
[21] R. Dworski, Oxidant stress in asthma, in: Thorax, BMJ Publishing Group, 2000,
p. S51.
[22] A.C. Pulla Reddy, B.R. Lokesh, Effect of dietary turmeric (curcuma longa) on iron-
induced lipid peroxidation in the rat liver, Food Chem. Toxicol. 32 (3) (1994)
279–283.
[23] G.S. Jeong, G.S. Oh, H.O. Pae, S.O. Jeong, Y.C. Kim, M.K. Shin, Y.S. Byeong, Y.
H. Sang, S.L. Ho, J.G. Jeong, J.S. Koh, H.T. Chung, Comparative effects of
curcuminoids on endothelial heme oxygenase-1 expression: ortho-methoxy groups
are essential to enhance heme oxygenase activity and protection, Exp. Mol. Med.
38 (4) (2006 Aug) 393–400.
[24] D. Paola Rosanna, C. Salvatore, Reactive oxygen species, inflammation, and lung
diseases, Curr. Pharmaceut. Des. 18 (26) (2012 Jul) 3889–3900.
[25] F. Dong, C. Wang, J. Duan, W. Zhang, D. Xiang, M. Li, Puerarin attenuates
ovalbumin-induced lung inflammation and hemostatic unbalance in rat asthma
model, Evidence-Based Complement Altern Med. 2014 (2014) 1–9.
[26] T. Hamaguchi, Y. Matsumura, M. Suzuki, K. Shimizu, R. Goda, I. Nakamura,
I. Nakatomi, M. Yokoyama, K. Kataoka, T. Kakizoe, NK105, a paclitaxel-
incorporating micellar nanoparticle formulation, can extend in vivo antitumour
activity and reduce the neurotoxicity of paclitaxel, Br. J. Cancer 92 (7) (2005 Apr)
1240–1246.
[27] D.S. Bredt, P.M. Hwang, S.H. Snyder, Localization of nitric oxide synthase
indicating a neural role for nitric oxide, Nature 347 (6295) (1990) 768–770.
[28] Q.W. Xie, H.J. Cho, J. Calaycay, R.A. Mumford, K.M. Swiderek, T.D. Lee, A. Ding,
T. Troso, C. Nathan, Cloning and characterization of inducible nitric oxide synthase
from mouse macrophages, Science (80- ) 256 (5054) (1992 Apr) 225–228.
[29] Q. Hamid, Springall, J. Polak, V. Riveros-Moreno, P. Chanez, J. Bousquet,
P. Godard, S. Holgate, P. Howarth, A. Redington, Induction of nitric oxide synthase
in asthma, Lancet 342 (8886–8887) (1993 Dec) 1510–1513.
[30] C.M. Prado, M.A. Martins, I.F.L.C. Tibério, Nitric oxide in asthma physiopathology,
ISRN Allergy 2011 (2011) 1–13.
[31] S. Lamas, P.A. Marsden, G.K. Li, P. Tempst, T. Michel, Endothelial nitric oxide
synthase: molecular cloning and characterization of a distinct constitutive enzyme
isoform, Proc. Natl. Acad. Sci. U. S. A. 89 (14) (1992 Jul) 6348–6352.
[32] D.O. Moon, M.O. Kim, H.J. Lee, Y.H. Choi, Y.M. Park, M.S. Heo, G.Y. Kim,
Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric
oxide, Biochem. Biophys. Res. Commun. 375 (2) (2008 Oct) 275–279.
[33] S. Biswas, I. Rahman, Modulation of steroid activity in chronic inflammation: a
novel anti-inflammatory role for curcumin, in: Molecular Nutrition and Food
Research, vol. 52, Wiley-VCH Verlag, 2008, pp. 987–994.
[34] P. Nilani, N. Kasthuribai, B. Duraisamy, P. Dhamodaran, S. Ravichandran,
K. Ilango, B. Suresh, Invitro antioxidant activity of selected antiasthmatic herbal
constituents, Ancient Sci. Life 28 (4) (2009 Apr) 3–6.
[35] K.M. Miranda, M.G. Espey, D.A. Wink, A rapid, simple spectrophotometric method
for simultaneous detection of nitrate and nitrite, Nitric Oxide - Biol Chem. 5 (1)
(2001) 62–71.
[36] I.J. Fidler, A. Raz, W.E. Fogler, R. Kirsh, P. Bugelski, G. Poste, Design of liposomes
to improve delivery of macrophage-augmenting agents to alveolar macrophages,
Cancer Res. 40 (12) (1980 Dec) 4460–4466.
[37] M.N. Sahib, S. Abdalwahed, S.A. Abdulameer, Y. Darwis, K.K. Peh, Y.T.F. Tan,
Solubilization of beclomethasone dipropionate in sterically stabilized phospholipid
nanomicelles (SSMs): physicochemical and in vitro evaluations, Drug Des. Dev.
Ther. 6 (2012) 29–42.
[38] N. Nishiyama, H. Takemoto, Polymeric micelles, Encycl Polym Nanomater 1–7
(2014).
[39] K. Kazunori, S.K. Glenn, Y. Masayuki, O. Teruo, S. Yasuhisa, Block copolymer
micelles as vehicles for drug delivery, J. Contr. Release 24 (1–3) (1993 May)
119–132.
[40] J. Li, Y. Jiang, J. Wen, G. Fan, Y. Wu, C. Zhang, A rapid and simple HPLC method
for the determination of curcumin in rat plasma: assay development, validation
and application to a pharmacokinetic study of curcumin liposome, Biomed
Chromatogr (2009 Nov), https://doi.org/10.1002/bmc.1244 [cited 2020 Feb 26];
23(11):1201–7. Available from:.
[41] J.S. Suk, Q. Xu, N. Kim, J. Hanes, L.M. Ensign, PEGylation as a strategy for
improving nanoparticle-based drug and gene delivery, in: Advanced Drug Delivery
Reviews, vol. 99, Elsevier B.V., 2016, pp. 28–51.
[42] I.M. El-Sherbiny, N.M. El-Baz, M.H. Yacoub, Inhaled nano- and microparticles for
drug delivery, Glob Cardiol Sci Pract 2015 (1) (2015 Jan) 2.
[43] S.M.H. Rahman, T.C. Telny, T.K. Ravi, S. Kuppusamy, Role of Surfactant and pH in
Dissolution of Curcumin, Indian J Pharm Sci [Internet, 2009 [cited 2020 Sep 5];71
(2):139–42. Available from: www.ijpsonline.com.
[44] S. Rani, S. Mishra, M. Sharma, A. Nandy, S. Mozumdar, Solubility and stability
enhancement of curcumin in Soluplus® polymeric micelles: a spectroscopic study,
J Dispers Sci Technol (2020 Mar 20) [cited 2020 Sep 5];41(4):523–36. Available
from: https://www.tandfonline.com/doi/abs/10.1080/01932691.2019.1592687.