Crosslinked Chitosan Nanoparticle Formulations for Delivery From Pressurized

8/17/2019 Crosslinked Chitosan Nanoparticle Formulations for Delivery From Pressurized

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Research paper

Crosslinked chitosan nanoparticle formulations for delivery from pressurized

metered dose inhalers

Ketan Sharma a, Satyanarayana Somavarapu a, Agnes Colombani b, Nayna Govind b, Kevin M.G. Taylor a,⇑

a UCL School of Pharmacy, London, UK b AstraZeneca R&D Charnwood, Loughborough, UK

a r t i c l e i n f o

Article history:

Received 5 August 2011

Accepted in revised form 22 December 2011

Available online 9 January 2012

Keywords:

Chitosan

Inhalation

Nanocarrier

Nanoparticle

Polyethylene glycol

Pressurized metered dose inhaler

a b s t r a c t

Crosslinked chitosan nanoparticles, prepared using ionic gelation, have been successfully formulated into

pressurized metered dose inhalers (pMDIs) with potential for deep lung delivery of therapeutic agents.

Nanoparticles were prepared from crosslinked chitosan alone and incorporating PEG 600, PEG 1000 and

PEG 5000 for dispersion in aerosol propellant, hydrofuoroalkane (HFA) 227. Spherical, smooth-surfaced,

cationic particles of mean size less than 230 nm were produced. Nanoparticles were positively charged

and non-aggregated at the pH of the airways. Crosslinked chitosan–PEG 1000 nanoparticles demonstrated

greatest dispersibility and physical stability in HFA-227, whereas other formulations readily either

creamed or sedimented. Following actuation from pMDIs, the fine particle fraction (FPF) for crosslinked

chitosan–PEG 1000 nanoparticles, determined using a next generation impactor, was 34.0 ± 1.4% with a

mass median aerodynamic diameter of 4.92 ± 0.3lm. The FPFs of crosslinked chitosan, crosslinked chito-

san–PEG 600 and crosslinked chitosan–PEG 5000 nanoparticles were 5.7 ± 0.9%, 11.8 ± 2.7% and

17.0 ± 2.1%, respectively. These results indicate that crosslinked chitosan–PEG 1000-based nanoparticles

are promising candidates for delivering therapeutic agents, particularly biopharmaceuticals, using pMDIs.

2011 Elsevier B.V. All rights reserved.

1. Introduction

Pulmonary drug delivery may be employed for therapeutic

agents having local or systemic activity. It provides advantages

over other delivery routes as it is non-invasive, avoid first-pass

metabolism and the lung offers a highly vascularized, large surface

area for drug absorption [1]. Pressurized metered dose inhalers

(pMDIs) are widely used inhalation devices, being convenient to

use and offering a sealed environment, providing protection from

air, light, moisture and microbial degradation. These medical de-

vices comprise a therapeutic agent either suspended or dissolved

in a hydrofluoroalkane (HFA) propellant. To achieve deep lung

deposition of particles, one successful approach has been to formu-

late low density hollow particles, which have relatively large phys-

ical diameters, corresponding to a much smaller aerodynamic

diameter [2]. An alternative approach is the use of nanoparticles

that are particularly attractive for pulmonary delivery, as their size

not only permits access to the peripheral airways [3] but also en-

sures that they escape both phagocytic and mucociliary clearance

mechanisms [4]. Incorporating drugs into, or onto, nanoparticles

potentially provides protection against intracellular and extracel-

lular barriers, degradation and may overcome formulation chal-

lenges, such as delivery of poorly aqueous soluble and unstable

drugs without compromising the native conformation of these

molecules [5,6]. The small size of dry nanoparticles leads to high

inter-particle cohesive forces that negatively impact on their

aggregation behaviour, which is particularly problematic for dry

powder inhaler (DPI) formulation. However, in a pMDI formula-

tion, the presence of propellants such as HFAs offers potential for

deaggregating the nanoparticles, though other excipients may be

necessary [7]. A number of groups have studied pMDI nanoparticle

delivery, though adequate dispersion of such small particles in liq-

uefied aerosol propellants is a major formulation challenge [3,8,9].

Oleic acid, sorbitan trioleate, dipalmitoylphosphatidylcholine and

volatile oils have been used within HFA propellants, permitting

the successful dispersion of protein nanoparticles [10] which

maintained protein integrity, and were successfully delivered in

an aerosol having appropriate aerodynamic characteristics for

therapeutic activity.

Chitosan has gained considerable interest as a polymer for pre-

paring nanoparticles because of its biodegradable, biocompatible,

non-toxic and mucoadhesive properties [11,12]. Chitosan has been

reported to increase the uptake of macromolecules through open-

ing of tight junctions of epithelial cells [13] and has also been for-

mulated as nanoparticles designed to improve the delivery of

therapeutically active molecules across mucosal surfaces [14].

The successful application of chitosan for in vitro and in vivo gene

delivery has demonstrated its potential for pharmaceutical and

0939-6411/$ - see front matter 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.ejpb.2011.12.014

⇑ Corresponding author. UCL School of Pharmacy, 29-39 Brunswick Square,

London WC1N 1AX, UK. Tel.: +44 207753 5853; fax: +44 207753 5942.

E-mail address: [email protected] (K.M.G. Taylor).

European Journal of Pharmaceutics and Biopharmaceutics 81 (2012) 74–81

Contents lists available at SciVerse ScienceDirect

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j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e j p b

http://dx.doi.org/10.1016/j.ejpb.2011.12.014mailto:[email protected]://dx.doi.org/10.1016/j.ejpb.2011.12.014http://www.sciencedirect.com/science/journal/09396411http://www.elsevier.com/locate/ejpbhttp://www.elsevier.com/locate/ejpbhttp://www.sciencedirect.com/science/journal/09396411http://dx.doi.org/10.1016/j.ejpb.2011.12.014mailto:[email protected]://dx.doi.org/10.1016/j.ejpb.2011.12.014


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biomedical applications [15]. The potential for pulmonary delivery

has been recognized, and chitosan nanoparticles encapsulated in

mannitol microspheres have been demonstrated to be biocompat-

ible with Calu-3 and A549 human respiratory epithelial cell lines

for up to 48 h [16]. Crosslinked chitosan microparticulates contain-

ing the bronchodilator, salbutamol sulphate prepared using spray

drying, were able to achieve controlled release of the drug [17]

and could be formulated into DPIs, with good aerosolization prop-

erties. Chitosan has not been extensively studied as a carrier for

delivery from pMDI systems; hence, in this study, the formulation

and characterization of crosslinked chitosan nanoparticles for

delivery using pMDIs are explored. Previous studies [9] suggested

that tripolyphosphate (TPP)-crosslinked chitosan microspheres

were not suitable for HFA-134a-based pMDI systems, because of

the density difference between the particles and propellant. The

dispersibility of such particles within a non-polar medium, such

as HFA-227 (1,1,1,2,3,3,3-heptafluoropropane, which has higher

density than HFA-134a), can be improved by controlling the steric

repulsive forces between particles [18], for instance by inclusion of

hydrophilic polymers, such as polyethylene glycol (PEG) and poly-

vinylpyrrolidone (PVP) [19,20]. A recent study [19] has demon-

strated that PEG acts as a polymeric surfactant and helps to

reduce the cohesive interactive forces between drug particles that

are suspended in a fluorinated solvent (2H,3H-perfluoropentane)

used as a non-volatile model for HFA propellants. PEG was chosen

in the formulation of crosslinked chitosan nanoparticles investi-

gated in this study, since it has appreciable solubility in HFAs

[21] and is approved by the FDA as an excipient for use in pMDIs

[22]. In a previous study [23], inclusion of PEG300 within particles

of salbutamol sulphate prevented cohesive interactions when par-

ticles were dispersed in propellant HFAs (HFA-227 and HFA-134a).

Together, these studies indicate that polymers such as PEGs can

adsorb at an interface in fluorinated liquid solvents, including HFA

propellants, and that crosslinked chitosan–based microparticles

can be formulated into pMDIs. The objective of the current study

was to prepare and characterize crosslinked chitosan–based nano-

particles, which might have future application in the delivery of small molecules and biopharmaceuticals to the peripheral airways.

Dispersion of nano-sized particles is a considerable challenge, and

the study is the first to explore the applicability of PEG of different

molecular weights as stabilizers for such particles dispersed in

HFA-227. The study describes the use of combinations of FDA-ap-

proved propellant and PEG to produce a functional and potentially

therapeutically useful nanoparticle formulation for pulmonary

delivery, via a pMDI.

2. Materials and methods

2.1. Materials

Chitosan (Protasan UP G 113, m.w 150–200 kDa, degree of

deacetylation 75–90%) was purchased from Novamatrix (Norway).

Fluorescein 5-isothiocyanate (FITC) with P90% purity, glycerol

(P99% purity), polyethylene glycol 1000 and sodium tripolyphos-

phate 85% were purchased from Sigma–Aldrich (Germany). Poly-

ethylene glycol 5000 monomethyl ether was obtained from Fluka

(US). Polyethylene glycol 600 and HPLC grade water were pur-

chased from Fischer Scientific (UK). HFA-227 (1,1,1,2,3,3,3-hepta-

fluoropropane) was obtained from INEOS Fluor (UK).

2.2. Preparation of nanoparticles

Nanoparticles were produced using an ionic gelation method

[24], whereby sodium tripolyphosphate (TPP) solution (0.5 mg/ml) was added drop-wise to a chitosan solution (1 mg/ml) in the

ratio of 1:5 (w/w) using a peristaltic pump (Gilson, France) under

constant stirring. Three molecular weights of PEG (600/1000/

5000) were used, and the same protocol was followed to prepare

chitosan–TPP–PEG (5:1:30) nanoparticles by dissolving PEG with

TPP prior to its addition to chitosan. The ratio of chitosan/TPP/

PEG was established in preliminary experiments (data not shown),

which demonstrated that this ratio was optimal for preparation of

non-aggregated, nano-sized particles.

2.3. Preparation of fluorescent crosslinked chitosan nanoparticles

Chitosan was labelled with FITC by conjugating the primary

amine group of the chitosan with the isothiocyanate group of FITC

[25,26]. Briefly, 1% (w/v) of chitosan was dissolved using 0.1 M ace-

tic acid, followed by an equal volume of methanol (16 ml). To this,

2.5 ml of FITC in methanol (2 mg/ml) was added drop-wise with

constant stirring for 3 h. The solution was adjusted to pH 10.0 by

the addition of 0.5 M sodium hydroxide solution, which resulted

in a precipitate, and then centrifuged at 25,000 g for 10 min.

The FITC-labelled chitosan precipitate was washed with metha-

nol/water (70:30) mixture and centrifuged until no fluorescence

was detected in the supernatant at excitation and emission max-

ima of 492 and 518 nm, respectively (LS 55 Fluorescence Spec-

trometer, Perkin–Elmer, UK). The precipitate was freeze-dried

(Virtis Advantage, SP Scientific, USA) to obtain a dry powder. Label-

ling efficiency (percent weight of FITC to weight of the FITC-chito-

san) was calculated by measuring the fluorescence intensity of the

FITC-labelled chitosan solution against a standard solution of FITC.

Chitosan-conjugated FITC was used to prepare fluorescent nano-

particles as described in Section 2.2, and these were used in

in vitro aerosolization studies.

2.4. Size and surface charge of nanoparticles

The hydrodynamic diameter and zeta potential of nanoparticles

were determined using photon correlation spectroscopy (PCS) and

laser Doppler electrophoresis (LDE), respectively, using a Zetasizer(Nano ZS, Malvern Instruments, UK). The instrument parameters,

such as refractive index (1.333) and viscosity (0.8872 cP), were

set for the dispersion medium (water) in which the nanoparticles

were dispersed at 25 C. Data are presented as the mean of three

independent experiments.

2.5. Morphology of nanoparticles

Each formulation was freeze-dried, and a small sample of the

dried particles was placed on a scanning electron microscopy

(SEM) stub. Samples were sputter-coated with gold and examined

by SEM (FEI XL30 TMP, Philips, the Netherlands). The morphology

and integrity of nanoparticles post-aerosolization were deter-

mined by actuating pMDI formulations of nanoparticles from a dis-tance of approximately 15 cm onto a glass slide, which was cut into

pieces, mounted on an SEM stub and visualized by SEM as de-

scribed above.

2.6. Effect of pH on nanoparticle properties

The effect of pH on the size and zeta potential of nanoparticles

was studied using a Zetasizer (Nano ZS, Malvern Instruments, UK)

equipped with an auto-titration unit (MPT-2, Malvern Instruments,

UK). The aqueous dispersion of nanoparticles (12 ml) was titrated

with 0.1 M sodium hydroxide solution (NaOH) under constant stir-

ring over a range of pH (5.5–8). The titrated dispersion was trans-

ferred to a measuring capillary cell by a spinning disc, and changes

in the properties of the nanoparticles were measured as a functionof pH.

K. Sharma et al. / European Journal of Pharmaceutics and Biopharmaceutics 81 (2012) 74–81 75


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2.7. Preparation of nanoparticle dispersions in HFA propellant

A uniform dispersion of nanoparticles in propellant HFA-227

was prepared. Briefly, 7 mg of freeze-dried nanoparticles was

added to a clear transparent polyethylene terephthalate (PET) vial.

Vials were crimped with a continuous valve (Valois, France) usinga

pneumatic crimper (P2002/20, Pamasol, Switzerland). 0.5 g of pro-

pellant was manually added through the valve. The mixture was

vortexed for 30 s and sonicated for 5 min at 20 C. The process

was repeated twice, and at each step, additional propellant was

added until the desired weight of 8.4 g was achieved. These PET

vials were used for studies of nanoparticle dispersion. A similar

process was followed to manufacture pMDI batches containing

FITC-labelled nanoparticle formulations. In this case, coated alu-

minium canisters (supplied by AstraZeneca, UK) were employed.

After filling with nanoparticle formulation (7 mg), the canisters

were crimped with metering valves (Valois, France) having a deliv-

ery volume of 50 lL per actuation. Additional propellant HFA-227

was added through the valve in a step-wise manner using auto-

matic pressure filling equipment (P2011, Pamasol, Switzerland)

to achieve a total content weight of 8.4 g. This corresponds to a

dose of 59lg nanoparticles per 50lL actuation. All pMDI canisters

were vigorously shaken for 10 s; stored inverted for one month at

ambient temperature, and later, after insertion into a plastic actu-

ator (supplied by AstraZeneca, UK), investigated for ex-actuator

aerosol particle size distribution and fine particle fraction (FPF).

2.8. Analysis of nanoparticle suspension stability

PET vials containing the HFA-227 nanoparticle suspensions

were vigorously hand-shaken and then placed in a light box for vi-

sual inspection to analyse their physical characteristics such as

sedimentation, creaming, flocculation and coalescence. The stabil-

ity and dispersion behaviour were monitored by naked eye obser-

vation after manual shaking ceased, until sedimentation or

creaming was apparent. Photographic images were taken at 10 s

and 1 min time points.HFA-based formulations in PET vials were further investigated

using an optical analyzer; Turbiscan (MA2000, Formulaction,

France) [27]. The content of each vial was transferred, via the valve,

into a pressure-sealed glass tube, using purpose-built apparatus

(courtesy of AstraZeneca, UK), then studied using the Turbiscan.

After shaking, the entire length of the glass tube was scanned four

times at 1-min intervals using a light source (NIR, k = 850 nm) and

the backscattered light measured.

2.9. Aerosol particle size analysis

Aerosol particle size analysis was performed using a Sympatec

particle size analyzer (Sympatec GmbH System-Partikel-Technik,

Germany). pMDI suspensions were shaken 10 times and actuated

via a sealed central adapter into the Sympatec at a flow rate of

60 L/min for 10 s. The aerosol cloud generated passed through

the laser beam. An optical lens (0.45–87.5lm size range) was used

to collect the diffracted light for calculationof size distribution. The

first ten doses of each new pMDI were fired to waste and a time

interval of 60 s elapsed between each actuation to prevent exces-

sive cooling of the pMDI metering chamber. The data generated

are presented as 10th (D10), 50th (D50, volume median diameter;

VMD) and 90th (D90) percentile of the cumulative particle under-

size frequency distribution.

2.10. Determination of aerosol parameters using the next generation

impactor

The aerosol performance of FITC-labelled pMDI formulationswas determined using a next generation impactor (NGI; Copley

Scientific, UK), operated in accordance with the specifications for

pMDIs described in the European Pharmacopoeia [28]. The collec-

tion plates of the NGI were uniformly coated with glycerol prior to

the measurement, with stage 8 comprising a micro-orifice filter, to

collect very fine particles. The NGI was operated at an air flow rate

of 30 L/min for preset 10 s intervals following pMDI actuation, to

allow particle deposition on the plates. The pMDI was vigorously

shaken for 10 s before actuation. Following actuation of five shots

to waste, the pMDI was actuated into the NGI setup, with a 60-s

interval between each actuation. Twenty actuations were cumula-

tively collected on glycerol-coated plates for each pMDI canister.

The collection plates, micro-orifice filter, throat and actuator were

rinsed with water and washings collected and made up to volume.

The amount of FITC-labelled crosslinked chitosan nanoparticles

collected from each stage was determined using a fluorescence

spectrophotometer as described previously. The parameters calcu-

lated for the aerosols produced were fine particle fraction (FPF;

stage 3 to filter, i.e. 0.05) in zeta potential between

the four formulations, whether FITC-labelled or unlabelled. A sig-nificant difference ( p < 0.05) in nanoparticle size was observed.

Post hoc Nemenyis test showed that crosslinked chitosan–PEG

5000 nanoparticles (both FITC-labelled and unlabelled) were sig-

nificantly larger than crosslinked chitosan particles without PEG.

This indicates that PEG (molecular weights 600–5000) had no

influence on nanoparticle surface charge, but that the highest

molecular weight PEG 5000 increased particle size, presumably

due to the presence of the large chain polymer on the particle sur-

face. Labelling of particles had no significant effect on size or sur-

face charge characteristics ( p > 0.05) in comparison to unlabelled

nanoparticles, and hence, both would be predicted to behave in a

similar manner when incorporated into pMDI formulations. The

polydispersity index (PDI) was slightly higher for unlabelled nano-

particles compared to labelled nanoparticles. For all formulations,the PDI was less than 0.3, which is an indication of a narrow parti-

76 K. Sharma et al. / European Journal of Pharmaceutics and Biopharmaceutics 81 (2012) 74–81


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cle size distribution in all instances. A relatively high proportion of

PEG was employed during particle manufacture to ensure that it

was trapped within the crosslinked chitosan nanoparticles during

ionic gelation, permitting modification of their surface properties.

3.2. Morphology of nanoparticles

The nanoparticles produced by the described methodology

were spherical and of approximately uniform size with a smooth

surface. Fig. 1a, for example, shows smooth, spherical crosslinked

chitosan–PEG 1000 nanoparticles having a narrow size distribu-

tion. Similar SEM images were obtained for non-PEG, PEG 600

and PEG 5000 based crosslinked chitosan nanoparticles. Fig. 1b

shows that crosslinked chitosan–PEG 1000 nanoparticles collected

post-actuation from a pMDI canister, via the metering valve, were

similar morphologically to freeze-dried nanoparticles prior to

pMDI manufacture.

3.3. Effect of pH on nanoparticle properties

In order to explore the potential of chitosan-based nanoparti-

cles for lung delivery, it is important to consider their behaviour

in different physiological conditions, especially at lung pH; 6.5

[33]. The change in zeta potential of nanoparticles over a pH range

5.5–8.0 is shown in Fig. 2a. The measured surface charge for all four

formulations was very similar at any single measured pH. The high

positive surface charge density for crosslinked chitosan at lower

pH is due to the free surface amine groups of chitosan. As the pH

of the nanoparticle suspension was increased, a greater proportion

of amine groups were deprotonated resulting in a decrease in the

measured positive zeta potential for the particles.

The influence of pH on nanoparticle size is shown in Fig. 2b. At a

pH range of 5.5–6.5, the mean nanoparticle size was constant. The

positive charge of chitosan in acidic medium results in repulsion

between nanoparticles [34]. However, as the pH was increased,

the mean measured particle size increased, which suggests the

occurrence of aggregation. Such increases were more marked for

non-PEG-based formulations, compared to those with PEG, sug-

gesting that the association of PEG with crosslinked chitosan nano-

particles provides steric hindrance, preventing nanoparticles from

aggregating. At pH 7.5 and greater, the measured particle size in-

creased due to decreased surface charge (Fig. 2a), leading to aggre-

gation of all formulations, and both PEG- and non-PEG-based

dispersions became turbid in appearance. This agrees with another,

parallel study [35], where chitosan and enoxaparin complexes

showed physicochemical stability at a pH range of 3–6.5, but

aggregated at higher pH.

From these studies, it was observed that the physical stability of

crosslinked chitosan nanoparticles is pH dependent, but significant

aggregation does not occur at lung pH; 6.5 [33]. Further, these find-

Table 1

Size and zeta potential of FITC-labelled and unlabelled chitosan and chitosan–PEG nanoparticles, (mean ± S.D., n = 3).

Formulation Unlabelled nanoparticles FITC-labelled nanoparticles

Hydrodynamic diameter

(nm ± S.D) (PDI ± S.D)

Zeta potential

(mV ± S.D)

Hydrodynamic diameter

(nm ± S.D) (PDI ± S.D)

Zeta potential

(mV ± S.D)

Chitosan 169.6 ± 5.9 (0.259 ± 0.025) +25.8 ± 3.3 166.0 ± 3.0 (0.194 ± 0.015) +30.0 ± 1.7

Chitosan–PEG 600 174.7 ± 4.5 (0.267 ± 0.002) +23.7 ± 0.8 183.0 ± 8.2 (0.213 ± 0.015) +25.0 ± 3.6

Chitosan–PEG 1000 193.9 ± 7.2 (0.248 ± 0.003) +28.2 ± 3.7 203.0 ± 8.5 (0.141 ± 0.021) +24.0 ± 5.6

Chitosan–PEG 5000 210.1 ± 9.0 (0.255 ± 0.018) +25.0 ± 3.4 221.0 ± 10.6 (0.217 ± 0.009) +27.0 ± 6.1

Fig. 1. Scanning electron micrographs of chitosan–PEG 1000 nanoparticles; (a)freeze-dried and (b) post-actuation from a pMDI.

a

b

Fig. 2. pH-induced variation in physicochemical properties of nanoparticles

(mean± S.D., n = 4); (a) zeta potential and (b) hydrodynamic diameter. (For

interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)



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ings suggest that PEG-based crosslinked chitosan nanoparticles are

more resistant to aggregation as a result of changing pH than non-

PEG-based formulations.

3.4. Visual analysis of nanoparticle dispersion stability

The physical stability of the nanoparticles following dispersion

in propellant HFA-227 is shown in Fig. 3. Preliminary experiments

suggested that nanoparticle dispersions in HFA-227 had greater

physical stability than in HFA-134a (data not shown), possibly

due to the greater density of HFA-227, and hence, this propellant

was used in all further studies.

Chitosan nanoparticles without PEG aggregated readily, and

phase separation occurred with clearly visible aggregates settling

rapidly (Fig. 3A). The inclusion of PEG 600 within the nanoparticles

did not result in any apparent change in their dispersion properties

(Fig. 3B). Both formulations were physically unstable, resulting in

aggregation in less than one minute. Previously, the inability of

PEG 600 to stabilize silica particles dispersed in a fluorinated sol-

vent (2H,3H-perfluoropentane) has been reported [20]. Chitosan–

PEG 1000 nanoparticles produced a homogeneous translucent dis-

persion in HFA-227, which exhibited no phase separation for up to10 min (Fig. 3C). In addition to exhibiting the slowest rate of sedi-

mentation, these nanoparticles readily re-dispersed upon shaking.

This property is essential for acceptable dose reproducibility froma

pMDI. A good stabilization excipient for pMDI formulation should

be well solvated and block particle–particle interaction [19]. PEG

1000 is able to solvate well within HFA-227 [36], though PEG sol-

ubility is likely to decrease with increasing molecular weight [37].

Chitosan–PEG 5000 nanoparticles produced an opaque, milky dis-

persion in HFA-227, which separated into two phases (Fig. 3D),

with particles creaming to the surface of the liquid propellant, pre-

sumably as the density of these particles is less than the density of

the propellant. Although this formulation creamed in less than

1 min, it was re-dispersible on gentle shaking.

These PEG and non-PEG crosslinked chitosan nanoparticles did

not visibly adhere to the walls of PET vials, even after one month’s

storage. Chitosan–PEG 1000 composition greatly improves the

nanoparticle physicochemical characteristics. From these findings,

the polymeric chain length of PEG 1000 seems optimal to provide

steric stabilization and to minimize particle–particle interaction

between crosslinked chitosan nanoparticles in HFA-227. Previous

studies have revealed the stabilizing effect of PEG 1000 and poly-

vinylpyrrolidone (PVP) within the model propellant 2H, 3H-per-

fluoropentane, resulting in reduced particle–particle and

particle–canister wall surface interactions [38]. Long-term stabil-

ity, dispersibility and ease of redispersion of HFA-based pMDI for-

mulations are key parameters for the quality of an inhalation

product. These findings, undertaken using pressurized apparatus

to investigate properties in the clinically relevant propellant

HFA-227, indicate that PEG 1000, incorporated into crosslinked

chitosan nanoparticles, is an effective formulation strategy to pro-

duce viable nanoparticle dispersions for delivery from a pMDI.

3.5. Optical analysis of nanoparticle dispersion stability

The experimental data obtained using the Turbiscan are a qual-

itative or semi-quantitative indication of nanoparticle suspension

behaviour, in a pressurized system, with respect to time. A formu-

lation is deemed to be unstable if the variation in its scan intensity

on a time scan graph is >10% [39]. The acquired scans for the per-

centage of backscattered light (%BS) obtained for each formulation

as a function of time and sample height are shown in Fig. 4. Turbi-

scan results obtained with the nanoparticle/HFA suspension

showed a sharp increase in backscattering (>10%) between the first

scan (time = 0) and all the consecutive scans with greatest increase

at the lower regions of the tube (Fig. 4a). This is due to particle size

variation and particle migration to the bottom of the glass tube,

which causes a variation in light transmission through the glass

tube and an increase in backscattering. The nanoparticle suspen-sion signal was modified in the presence of PEG 600, indicated

by a step-wise increase in BS signal level between each successive

scan (Fig. 4b). This signifies a delay in the separation between two

different phases compared to the nanoparticle/HFA formulation

(without PEG). The variation in the percentage of backscattering

between the first and the final scan was greater than 10%, indicat-

ing that PEG 600 does not prevent aggregation, but only delays the

onset of aggregation.

The scan for PEG 1000–crosslinked chitosan dispersions in HFA-

227 showed a 2% variation in backscattering for the entire scan at

all time points (Fig. 4c), indicating high stability for this formula-

tion, that is, no settling or creaming behaviour within the HFA.

There was no apparent change in particulate size and volume frac-

tion for the dispersion, even after 3-min post-shaking, leaving theproduct homogeneous. It is likely that the nanoparticle-bound PEG

1000 covers the surface of the nanoparticles suspended within HFA

and reduces the interfacial energy between particulates and pro-

pellant improving suspension stability. The dispersion of cross-

linked chitosan–PEG 5000 nanoparticles in HFA-227 was opaque

and milky in appearance. Due to the turbidity of the suspension,

it was difficult to monitor the onset of creaming at the start of

phase separation using the naked eye. The Turbiscan was a useful

technique to identify the appearance of a clear layer at the bottom

of the glass tube and/or creaming at the top. The backscattering

signal increased to 25% between 40 and 45 mm, reflecting the

creaming behaviour of the sample (Fig. 4d), and suggesting that

crosslinked chitosan–PEG 5000 nanoparticles had lower density

than HFA-227 causing them to cream. These results demonstratethat producing crosslinked chitosan nanoparticles that incorporate

Fig. 3. Images of formulations dispersed within pressure-sealed HFA-227 propel-

lant at 10 s and 1 min time points following hand shaking: (A) Chitosan, (B)

Chitosan–PEG 600, (C) Chitosan–PEG 1000 and (D) Chitosan–PEG 5000nanoparticles.



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PEG 1000 was successful in stabilizing the particle dispersion in

HFA-227. However, PEG 600 and PEG 5000 were not able to

achieve the same degree of physical stability when particles con-

taining these polymers were dispersed in the propellant, such that

the particles either sedimented or creamed. These results are in

line with the data obtained by visual analysis of dispersions, as de-

scribed in the previous section.

3.6. Aerosol particle size distribution

The post-actuation size distribution of the emitted aerosol

clouds from pMDIs is shown in Fig. 5, with each data point repre-

senting the mean size following actuation from four canisters con-

taining the same formulation. The nanoparticles showed a wide

size distribution, with some multi-modality and a VMD of

54.24 lm (Table 2). This suggests that the nanoparticles were

highly aggregated within HFA-227 propellant and largely re-

mained aggregated following actuation and subsequent rapid

evaporation of the propellant. This rapid evaporation or ‘flashing’

of the propellant results in dispersion of fine particles into the air

if formulation is appropriate [40]. PEG 600-based formulations

showed a bi-modal distribution, having a VMD of 23.16lm(Table 2). The inability of PEG 600 to stabilize silica particles has

previously been reported [20], which may be related to the rela-

tively short length of the polymer chains, or the physical state of

PEG 600, which is liquid at room temperature, while PEG 1000

and 5000 are solids. There was a small subpopulation of particles

havinga mode less than 3 lm, and similar observations were made

for crosslinked chitosan–PEG 5000 particles, which had a multi-

modal size distribution, having a VMD of 28.43lm (Table 2). These

data suggest that aerosolization of these three formulations using a

pMDI is not appropriate for lung delivery, as their median size

greatly exceed the size (1–5 lm) required for peripheral lung

deposition. In contrast, the crosslinked chitosan–PEG 1000 particle

a

b

c

d

Fig. 4. Back scattering profiles of formulations dispersed within pressure-sealed

HFA 227 as a function of time (1 min) with percentage changeon Y -axis and sample

height on X -axis, (a) Chitosan, (b) Chitosan–PEG 600, (c) Chitosan–PEG 1000and (d)

Chitosan–PEG 5000 nanoparticles. The increase or decrease in scan intensity on a

time scan graph correlating to the sample height below 2.5 mm (convex bottom of

glass tube) and above the meniscus (shown by major change in backscattering of

plots) of the sample is because of multiple light diffractions and has no physical

meaning. Hence, it is not taken into account for stability analysis. (For interpre-

tation of the references to colour in this figure legend, the reader is referred to the

web version of this article.)

Fig. 5. Post-actuation size distribution data for pMDI systems containing chitosan

and chitosan–PEG nanoparticles (mean ± S.D., n = 4).

Table 2

Post-actuation cumulative size distribution for chitosan and chitosan–PEG nanopar-

ticles, (mean ± S.D., n = 4). D10, D50 and D90 represent the 10%, 50% and 90%

cumulative undersize diameter determined by laser diffraction.

Formulation D10 ± S.D (lm) D50 ± S.D (lm) D90 ± S.D (lm)

Chitosan 1.58 ± 0.31 54.24 ± 4.66 78.56 ± 1.57

Chitosan–PEG 600 2.33 ± 1.03 23.16 ± 3 .03 57.81 ± 2.27

Chitosan–PEG 1000 0.72 ± 0.16 1.53 ± 0.07 2.76 ± 0.27

Chitosan–PEG 5000 1.29 ± 0.05 28.43 ± 6 .52 67.69 ± 5.04



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formulation produced a more uniform size distribution, with a

much smaller VMD of 1.53 lm, which is appropriate for deep lung

alveolar delivery [41]. The improvement in dispersibility of

crosslinked chitosan nanoparticles, when incorporating PEG 1000

as observed in previous sections, is thus reflected in an improved

performance in delivery from a pMDI.

3.7. In vitro assessment of aerosolization performance

FITC was used as a fluorescent marker in order to quantify low

concentrations of chitosan following aerosolization and deposition

in the NGI. FITC percentage content was 2.8%. The measured FPF

was 5.7 ± 0.9%, 11.8 ± 2.7% and 17.0 ± 2.1% for crosslinked chitosan

nanoparticles, crosslinked chitosan–PEG 600 and crosslinked

chitosan–PEG 5000 nanoparticles, respectively (Fig. 6). The poor

performance of these formulations as aerosols may be attributed

to aggregation of particles within the liquefied HFA and/or an

inability to disperse during actuation and propellant evaporation

subsequent to actuation, resulting in poor deposition in the lower

stages of the NGI. Chitosan–PEG 1000 nanoparticles showed high-

est ( p < 0.05) FPF of 34.0 ± 1.4%, with MMAD of 4.92 ± 0.29lm and

GSD of 3.35 ± 0.92, indicating this was the most suitable cross-

linked chitosan nanoparticle formulation for lung delivery, using

a pMDI. These results correlate well with the aerosol sizing by laser

diffraction described earlier. Chitosan deposition in all stages of the

NGI and accessories (actuator, mouthpiece and throat) was be-

tween 75% and 125% of the anticipated label claim, which is in

accordance with the criteria of the European Pharmacopoeia [28].

In comparison with the other formulations, the crosslinked chito-

san–PEG 1000 formulation in HFA demonstrated high and consis-

tent aerosolization performance and good dose recovery (mass

balance).

The mass deposition of chitosan at each stage of the NGI is pre-

sented in Fig. 7, for the crosslinked chitosan–PEG 1000 formula-

tion. NGI is an instrument used to assess inhalation aerosol

performance and gives an indication of the likely performance of

an inhalation product in vivo. The FPF of 34% for this formulationis predictive of the proportion of therapeutically useful aerosol

likely to reach the deep lung. This may be considered adequate

to exhibit a therapeutic effect, as commercially available pMDI

products deliver about 30% of the total emitted dose to the lungs

[42,43]. A similar deposition profile, with FPF of 31.5% was re-

ported for semi-interpenetrating polymeric network microspheres

loaded with bovine serum albumin delivered from a DPI [44]. Pre-

vious reports of nanoparticle delivery from HFA-134a based pMDI

systems have reported a FPF of approximately 45% for insulin

loaded nanoparticles, employing a volatile oil as a dispersant [10]

and lysozyme nanoparticles with oleic acid, sorbitan trioleate or

dipalmitoylphosphatidylcholine as dispersants [8]. Lecithin-based

nanoparticles were successfully dispersed in HFA-227 and

achieved a high fine particle fraction (>58%) [7]. Successful pMDIformulation depends on the properties of the dispersed material,

dispersant and propellant. This study has demonstrated the poten-

tial of a novel crosslinked chitosan–PEG 1000 formulation, capable

of delivery from a pMDI, as a nanocarrier for pulmonary drugdelivery.

4. Conclusion

This study has successfully demonstrated a formulation ap-

proach, potentially capable of delivering crosslinked chitosan–

based nanoparticles to the lung using a pMDI. Nanoparticles pre-

pared from crosslinked chitosan alone showed aggregation, and

the preparation was physically unstable when suspended within

an HFA-227 pressurized system. Inclusion of PEG during particle

production modified their properties. Due to the amphiphilic nat-

ure of PEG, it is likely to be inside the nanoparticle and at the par-

ticle surface; this is supported by the increased size of nanoparticles with PEG 5000 and the improved dispersion proper-

ties, of PEG 1000 and 5000 containing particles. The presence of

PEG 1000, in particular, provided steric stabilization when incorpo-

rated into crosslinked chitosan nanoparticles, prior to dispersion in

HFA-227. Dispersibility of these particles and subsequent deposi-

tion from a pMDI into an NGI were greatly improved compared

to crosslinked chitosan particles alone, or those incorporating

PEG 600 and PEG 5000. PEG1000 may have produced the best re-

sults of the three PEGS investigated for a combination of reasons:

PEG 600 is a small liquid molecule, which may not be well retained

in the nanoparticles. PEG 1000 is well solvated in HFA, thus provid-

ing steric stabilization of the particles. PEG 5000 has a much longer

chain length than PEG 1000, which is likely to be less well solvated,

and association of the long PEG chains may result in aggregation.The relatively high FPF and ease of redispersion, combined with a

small primary particle size and positive charge, suggest that the

crosslinked chitosan–PEG 1000 nanoparticles have potential appli-

cation in delivery of drugs and biopharmaceuticals, such as nucleic

acids to the lungs. However, these findings pertain to a system that

does not include an active pharmaceutical ingredient. Further

studies are required to determine whether a nanoparticle formula-

tion of crosslinked chitosan and PEG 1000 is optimal in the pres-

ence of an incorporated therapeutic agent.

This study will help in early phase new system development for

polymeric nanoparticle delivery of therapeutic agents using pMDIs.

Future work will involve the evaluation of dispersion stability and

aerosolization behaviour over long-term storage of nanoparticles

dispersed within pressurized propellant-based systems and theinclusion of therapeutic molecules.

Fig. 6. Fine particle fraction of pMDI formulations of chitosan and chitosan–PEGnanoparticles determined using the NGI (mean ± S.D., n = 3).

Fig. 7. Deposition of chitosan–PEG 1000 nanoparticles delivered from a pMDI into

the NGI, (mean ± S.D., n = 3).



8/8

Acknowledgements

Financial support from AstraZeneca is gratefully acknowledged.

We also thank David McCarthy, UCL School of Pharmacy for assis-

tance with electron microscopy. Special thanks to Hamid Merchant

(University of London) and Varsha Thakoersing (Leiden University,

The Netherlands) for numerous stimulating discussions.

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Crosslinked Chitosan Nanoparticle Formulations for Delivery From Pressurized