Systemic Heparin Delivery by the Pulmonary Route Using Chitosan and Glycol

8/10/2019 Systemic Heparin Delivery by the Pulmonary Route Using Chitosan and Glycol

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International Journal of Pharmaceutics 447 (2013) 115123

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InternationalJournal ofPharmaceutics

journal homepage: www.elsevier .com/ locate / i jpharm

Pharmaceutical Nanotechnology

Systemic heparin delivery by the pulmonary route using chitosan and glycolchitosan nanoparticles

Adriana Trapani a,1, Sante Di Gioia b,1, Nicoletta Ditaranto c, Nicola Cioffic, Francisco M. Goycoolead,Annalucia Carboneb, Marcos Garcia-Fuentes e, Massimo Coneseb, MariaJose Alonso e,

a Department of Pharmacy-Drug Sciences,University of Bari AldoMoro, ViaOrabona, 4, 70125 Bari, Italyb Department ofMedical and Surgical Sciences, University of Foggia, Viale L. Pinto 1, 71122 Foggia, Italyc Department of Chemistry, University of Bari AldoMoro,Via Orabona 4, 70125 Bari, Italyd Institute of Plant Biology and Biotechnology, Westphalian Wilhelms UniversityMnster, Schlossgarten 3, D-48149Mnster, Germanye Centerfor Research in MolecularMedicine and Chronic Diseases (CIMUS), Health Research Institute of Santiago de Compostela(IDIS), Department of Pharmacy and Pharmaceutical

Technology, School of Pharmacy,University of Santiago de Compostela, 15782 CampusVida, Santiago de Compostela, Spain

a r t i c l e i n f o

Article history:

Received 28 December 2012

Received in revised form 12 February 2013

Accepted 13 February 2013

Available online 20 February 2013

Keywords:

Low molecular weight heparin

Lipoid

Chitosan-and glycolchitosan-nanoparticles

Lung delivery

a b s t r a c t

The aim ofthis study was to evaluate the performance ofchitosan (CS) and glycol chitosan (GCS) nanopar-

ticles containing the surfactant Lipoid S100 for the systemic delivery of low molecular weight heparin

(LMWH) upon pulmonary administration. These nanoparticles were prepared in acidic and neutral con-

ditions using the ionotropic gelation technique. The size andzeta potential ofthe NPs were affected by the

pHand also the type ofpolysaccharide (CS or GCS). The size (between 156 and 385 nm) was smaller and

the zeta potential (from +11 mV to +30 mV) higher for CS nanoparticles prepared in acidic conditions. The

encapsulation efficiency ofLMWH varied between 100% and 43% for the nanoparticles obtained in acidic

and neutral conditions, respectively. X-ray photoelectron spectroscopy studies indicated that the surfac-

tant Lipoid S100 was localized on the nanoparticles surface irrespective ofthe formulation conditions.

In vivo studies showed that systems prepared in acidic conditions did not increase coagulation times

when administered to mice by the pulmonary route. In contrast, Lipoid S100-LMWH GCS NPs prepared

in neutral conditions showed a pharmacological efficacy. Overall, these results illustrate some promisingfeatures ofCS-based nanocarriers for pulmonary delivery ofLMWH.

2013 Elsevier B.V. All rights reserved.

1. Introduction

Low molecular weight heparin (LMWH) is a linear anionic

polysaccharide used as an anticoagulant for the prevention and

treatment of deep veinthrombosis,pulmonary embolismand other

thromboembolic disorders (Schulman, 2000; Yang et al., 2004).

Unfortunately, LMWH exhibits poor oral bioavailability and, con-

sequently, has to be administered via parenteral route. Due to

poorpatientcompliance andside effects associatedwith injections,

alternative routes for non-invasive LMWH administration have

been actively investigated (Motlekar and Youan, 2006). Amongthose, the pulmonary route has attracted notable interest as a

potential strategy to deliver therapeutically useful amounts of the

anticoagulant (Qi et al., 2004). The large alveolar surface area avail-

able for drug absorption, the low thickness of the epithelial barrier,

its extensive vascularization and relatively low proteolytic activity

make pulmonary delivery of drugs of particular interest even for

Corresponding author. Tel.: +34 881815454; fax: +34 881815403.

E-mail address:[email protected](M.J. Alonso).1 These authors contributed equally to this work.

chronic therapy (Hussain et al., 2003; Labiris and Dolovich, 2003;

Craparo et al.,2011; Licciardi et al.,2012). Moreover,it hasalsobeen

observedthat LMWHitself causes, uponpulmonaryadministration,

a transient opening of the tight junctions in the lung epithelium,

leading to a rapid onset of action and a Cmaxcomparable to subcu-

taneous administration (Qietal.,2004). Nevertheless, beyond these

positive aspects, it is believed that, without the use of penetration

enhancers and adequate delivery vehicles, the amount of LMWH

overcoming the pulmonary barriers and reaching the systemic cir-

culation might be insufficient for an adequate pharmacological

response.Among the differentpulmonary drug delivery vehicles,chitosan

(CS)-basednanoparticles areparticularlyattractive.Indeed,besides

promising properties including low toxicity, biocompatibility and

high loading of hydrophilic molecules (Garcia-Fuentes and Alonso,

2012; Ieva et al., 2009), CS shows excellent mucoadhesive charac-

teristicsand it is capable of opening the tight junctions of epithelial

cells, thereby improvingthe uptake of hydrophilic drugs (Mao et al.,

2010). A CS derivative conjugated with ethylene glycol branches,

i.e., glycol chitosan (GCS), which is water soluble at neutral and

acidic pH values, has also been described (Siew et al., 2012). In

addition to its adequate biocompatibility, GCShas been reported to

0378-5173/$ seefrontmatter 2013 Elsevier B.V. All rights reserved.

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116 A. Trapani et al./ International Journal of Pharmaceutics 447 (2013) 115123

retain the mucoadhesive properties inherent to CS (Trapani et al.,

2009; Makhlof et al., 2010).

There are some previous reports on the potentialof CS nanopar-

ticles for the local and systemic delivery of macromolecules

following pulmonary administration. For example, nanoparticles

made of CS and hyaluronic acid (HA) have been used for local

heparin delivery to the lungs (Oyarzun-Ampuero et al., 2009). The

results of this work have shown that CSHA nanoparticles are able

to enhance the inherent ability of heparin to block the degranula-

tion of mast cells (Oyarzun-Ampuero et al., 2009). Similarly, NPs

made of CS or GCS have been recently described for pulmonary

delivery of DNA (Bivas-Benita et al., 2004) and therapeutic pep-

tides such as insulin (Al-Qadi et al., 2012) or calcitonin (Makhlof

et al., 2010).

Taking this into account, the aim of the present work was to

develop CS and GCS nanoparticles containing LMWH and to eval-

uate their performance following pulmonary administration. In

addition, we found it critical to incorporate the non ionic surfac-

tant Lipoid S100 to the nanoparticles structure. Our hypothesis

was that the co-encapsulation of LMWH and the penetration

enhancer Lipoid S100 in CS and GCS nanoparticles would favour

for the pulmonary absorption of macromolecules (Hussain et al.,

2003). Moreover, Lipoid S100, being a mixture of natural phos-

pholipids, was thought to improve the biocompatibility of thenanosystems in contact with the alveolar surface. Ultimately, we

hypothesized that, by combining the mucoadhesive characteristics

of the polymers with the nanoscale dimensions and the absorp-

tion enhancing propertiesof the surfactant and polymers, we could

significantly enhance the pulmonary absorption of LMWH. In addi-

tion, nanoencapsulation was also conceived as a way to protect the

anticoagulant drug from possible enzymatic degradation.

2. Materials and methods

2.1. Materials

The following chemicals were used as received. Chitosanhydrochloride salt(Protasan, UP CL 113,Mw 110kDa, deacetylation

degree 86%, viscosity = 13mPa/s according to manufacturer data

sheet) was purchased from Pronova Biopolymer (Norway). Lipoid

S100 was kindlydonated by Lipoid KG (Germany). LMWH (average

MW 18kDa, 177 UI/mg), glycolchitosan (MW400 kDaaccording to

supplier instructions), mucine fromporcine stomach(type II,bound

sialic acid, 1%), glycerol, and pentasodium tripolyphospate (TPP)

werepurchased from SigmaAldrich (Milan, Italy). Ultrapurewater

was used throughout the study. All other standard chemicals were

of reagent grade.

2.2. Analytical determination of low molecular weight heparin

The heparin amounts were directly measured using two col-orimetric assays (i.e., Stachrom Heparin, Diagnostica Stago, Italy

(Oyarzun-Ampuero et al., 2009) and Azure A colorimetric method

(Ramadan et al., 2011). These assays were performed according to

the manufacturers instructions and linearity was checked in the

140g/mL to 1.0101g/mL concentration range. For Azure Acolorimetric assay, the limit of quantification (LOQ) of LMWH was

0.9101 g/mL.

2.3. Preparation of nanoparticles

LMWH loaded CS or GCS-based NPs were prepared according

to the ionic gelation technique, as previously reported (Ieva et al.,

2009; Mao et al., 2010; Calvo et al., 1997).

2.4. Unloaded CS (and GCS) NPs

CS NPs were spontaneously formed by adding1 mLof TPP (0.1%,

w/v, in NaCl aqueous solution (87 mM)) to 3 mL of CS solution

(0.20% (w/v) in NaCl aqueous solution (87mM) under magnetic

stirring (VWR, VMS C-4, Italy). Such concentration of NaCl was pre-

viously found to allow the formation of small size CS-based NPs

(Goycoolea et al., 2009). The final CS/TPP mass ratio was 6/1. GCS

NPs were obtained by mixing 3 mLof TPP aqueous solution (0.07%,

w/v) to 3mLof GCS (0.2%, w/v) dissolved in acetic acid (0.1%, v/v).

ThefinalGCS/TPP ratio was2.9/1(Ganet al., 2005). For Lipoid S100-

containing CS (and GCS) NPs, the surfactant was dispersed to give a

final concentration of 0.1% (w/v) in 87mM NaCl aqueous solution.

Lipoid S100 CS NPs were prepared by mixing 0.5 mLof TPP (0.2%

(w/v) in 87mM NaCl aqueous solution) with an aliquot of 0.2 mL

of the dispersion of Lipoid S100. The resulting mixture was used to

crosslink 3 mLof CS solution (0.20% (w/v) in 87mM NaCl aqueous

solution) under stirring, and thus NPs were formed.

For GCS based NPs (i.e., Lipoid S100 GCS pH 4.3), 3mLof TPP

aqueous solution (0.07%, w/v) were mixed with an aliquot of 0.1mL

of the dispersion of Lipoid S100. The resulting mixture was used to

cross-link 3 mLof GCS solution (0.20%, w/v) previously dissolvedin

acetic acid (0.1%, v/v).

2.5. LMWH loaded NPs

Lipoid S100-LMWH CS NPs were formulated starting from the

anionic bearing species consisting of 0.2mLof Lipoid S100, 0.2 mL

of LMWH solution and 0.5 mLof TPP (0.2%, w/v, in 87mM NaCl

aqueous solution). Such mixture was used to cross-link 3 mL of CS

solution (0.20%, w/v, in 87mM NaCl aqueous solution).

Lipoid S100-LMWH GCS NPs were formulated starting from

the anionic bearing species consisting of 0.4mL of Lipoid S100,

0.2mL of LMWH solution and 1.8 mL of TPP (0.07%, w/v). The

resulting mixture was used to cross-link 3mL of GCS solution

(0.20%).

It should be noted that all the nanosuspensions prepared as

above displayed acidic pH values (i.e., 3.8 and 4.3 for Lipoid S100-

LMWH CS and Lipoid S100-LMWH GCS, respectively).We have also

prepared Lipoid S100-LMWH GCS NPs uponneutral conditions. The

correspondingLipoid S100-LMWHCS NPs were notformulated due

to the poor water solubility of CS at pH> 6.5. Lipoid S100-LMWH

GCS NPs were formed as follows. Briefly, 1.5mLof TPP aqueous

solution (0.07%, w/v) were mixed with an aliquot of 0.52mLof the

dispersion of Lipoid S100 andan aliquot of 0.52 mLof LMWH aque-

ous solution. The resulting mixture was used to cross-link 8.0 mL

of GCS solution (0.20%, w/v) previously dissolved in Tris (10 mM,

pH 7).

NPs were isolated by ultracentrifugation (16,000g, 45min,

Eppendorf 5415D, Germany) using a glycerol bed in order

to facilitate their resuspension in ultrapure water by manual

shaking.

2.6. Physicochemical characterization of nanoparticles

For all tested NPs, the mean particle size and distribution were

determined in double distilled water by photon correlation spec-

troscopy (PCS) using a Zetasizer NanoZS (ZEN 3600, Malvern,

Herrenberg, Germany). Measurements were performed in trip-

licate after dilution 1:1 (v/v) in double distilled water and at

25 C. The autocorrelation functions were analyzed using the DTS

v. 5.1 software provided by Malvern. For the determination of

zeta-potential values, a laser Doppler velocimetry was adopted

by using Zetasizer NanoZS after dilution with 1mM KCl (pH 7.0)

(Montenegro et al., 2012; De Giglio et al., 2012).


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A. Trapani et al. / International Journal of Pharmaceutics447 (2013) 115123 117

2.7. Atomic force microscopy (AFM)

AFM studies were performed to characterize the morphology

of LMWH loaded CS NPs. The AFM images were obtained using

a Ntegra Prima AFM, on air in semi-contact mode with standard

Si cantilever with 316kHz resonant frequency. Scan speed was

between 2 and 3 Hz, (mean 23 lines per second). All images are

512512 data points. For AFM visualization, a drop of nanoparti-

cle suspension was spin-coated onto a flat gold thin film deposited

on silicon substrate, then the sample was processed directly on the

AFM scanner. To achieve high-resolution images of nanoparticles

deposited on the substrate, NSG03 silicon cantilevers with a length

of 135m were used with resonance frequency of about 90kHzand a nominal force constant around 1.8 N/m. The tip amplitude

oscillations were kept constant in attractive forces regimen to pre-

vent thedamage of theNPs. Thescan speed was proportional to the

scan size with a typical scanning frequency of 23 Hz.

2.8. Association efficiency of NPs

The association efficiency (AE) of LMWH to the particles was

determined after isolation of NPs by centrifugation as described in

Section 2.3.

AE was calculated as follows:

%AE = 100total LMWH free LMWH

total LMWH

where the total amount of LMWH and the unbound amount of

LMWH in the supernatant was determined using the colorimetric

assays above mentioned. The association efficiency was deter-

mined in triplicate.

2.9. XPS analysis

The surface chemical composition was studied by means of X-

ray Photoelectron Spectroscopy (XPS). Prior to their analysis, the

NPs formulations (Lipoid S100 LMWH GCS NPs and Lipoid S100

LMWH CS NPs) were converted into powder using freeze-drying.XPS analyses were performed with a Theta Probe VG Scientific

spectrometer equipped with a micro spot monochromatised Al Ksource (1486.6eV; spot size: 300m). Wide scans and detailedspectra were acquired in constant analyser energy (CAE) mode

with a pass energy of 150eV and 100eV, respectively. For non-

conductive samples, a low energy flood gun (1eV) was used

for charge compensation. Calibration of the binding energy (BE)

scale was performed by fixing the C C component at BE values

of 284.80.1eV. Data were averaged over at least three points

per sample. Acquisition and data processing were performed using

Avantage software v. 5.74.

2.10. Stability study of LMWH loaded NPs in different media

Freshly prepared LMWH loaded CS and GCS NPs were resu-

pended at 0.1% (w/v) concentration in normal saline (NaCl, 0.9%,

pH 7; 300mOsm/kg). To evaluate the stability of the NPs under

differentincubation conditions, the systems wereincubated in nor-

mal saline for 12 weeks at 4 C, for 1 week at 25 C and for 4 h at

37 C. Particle size of the samples was determined at scheduled

time intervals.

2.11. Mucoadhesion tests

The mucoadhesive interaction between LMWH loaded NPs and

mucin was determined recording the variations of zeta poten-

tial according to the method reported in the literature (Takeuchi

et al., 2005; Jintapattanakit et al., 2009; Das Neves et al., 2011).

Briefly, commercially available porcin mucin was firstly suspended,

at room temperature,in PBS(pH 7.4) at 1%(w/v) concentration.One

milliliter of this suspension was mixed with different volumes of

the NP suspension tested at 1 mg/ml. The resulting mixtures were

stirred (100rpm) at 37C for 15 m in and then the zeta potential

was measured.

2.12. In vitro release studies

In vitro release studies from Lipoid S100-LMWH GCS NP for-

mulated in neutral conditions were carried out up to 24 h in

simulated lung fluid (SLF) (Moss, 1979; Davies and Feddah, 2003).

The composition of SLF is: 5.0mEq/L CaCl2, 1.0mEq/L MgCl2,

1.0mEq/L MgSO4 , 1.0mEq/L K2HPO4, 1.0 mEq/L KHCO3, 1.0 mEq/L

KCl, 7.0mEq/L sodiumacetate, 102.0mEq/L NaCl, 1.0mEq/L sodium

citrate, 31.0mEq/L NaHCO3, and 1.0 mEq/L Na2HPO4. This medium

had pH 7.4 and an osmolarity of 300mOsm/kg. Briefly, NPs were

freshly prepared and then centrifuged in presence of 10L of glyc-erol.For eachexperiment,appropriate volumes of nanodispersions,

corresponding to an amount of LMWH in the range 2070g/mL,were put in Eppendorf tubes containing 0.5mL of SLF. The samples

were incubated under mechanical agitation (100rpm) at 37 C. At

scheduledtime intervals(0, 4, 6, 15 and 24h) each samplewas cen-

trifuged at 16,000gfor 45min. The supernatant was analyzed forthecontent of LMWH usingAzure A spectrophotometric assay. The

experiment was performed in triplicate.

2.13. In vivo studies

Buffer (TrisHCl, pH 7.0), LMWH or Lipoid S100-LMWH GCS

NPs formulated in neutral conditions were administered to Swiss

mice (25g, Charles River, Calco, Italy) using a MicroSprayerTM

aerosoliser (IA-1C; Penn Century, Philadelphia, PA, USA) suit-

able for mice, attached to a high-pressure syringe (FMJ-250;

Penn Century). The mice were anaesthetized by an intraperitoneal

injection of 0.5 mg/g body weight Avertin (2,2,2-tribromoethanol;

SigmaAldrich, Germany)and weresuspendedat a 45 anglebythe

upper teeth. The light sources (lamp type FLQ85E; Helmut Hund,Germany)flexible fiber-optics arm was adjusted to provide optimal

illumination of the trachea. A small spatula was used to open the

lower jaw of the mouse and blunted forceps were used to help dis-

place the tongue for maximal oropharyngeal exposure. Aftera clear

view of the trachea was obtained by an otoscope, the MicroSprayer

tip was endotracheally inserted until it reached the carina (the first

bronchial bifurcation) and 100L of solution (buffer or LMWH) orNP suspension was sprayed. The tip was immediately withdrawn

and the mouse was taken off the support.

2.13.1. Anticoagulation study

Anticoagulation was studied with activated partial thrombo-

plastin time (APTT). Citrate-anticoagulated blood was obtained 4 h

after administration of buffer, LMWH or NPs. Plasma was com-bined with activated partial thromboplastin time (APTT) reagent

(Beckman-Coulter/Instrumentation Laboratory)for three min prior

to activation with calcium chloride and mechanical determination

of coagulation time. At least 3 mice per group were analysed.

2.13.2. Histopathological study

Mice received the same treatment described above. Positive

control mice were administered with aerosolised Lipopolysaccha-

ride (LPS) from Pseudomonasaeruginosa (20g/mouse). Mice weresacrified 4 h after each treatment, lungs collected and inflated with

1 mL of 10% neutral buffered formalin and fixed for a minimum

period of 24h. Following fixation, lungs were placed in tissue cas-

sette. These tissues were processed into paraffin, embedded in a

paraffin block, sectioned on a microtome to a thickness of 5m,


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Table 1

Physicochemical properties of LMWH loaded CS- and GCS-based NPs. PI: polydispersity index. MeanS.D. are reported, n= 6. Control nanoparticles were unloaded CS and

unloaded GCS, respectively).

NPs formulation Size (nm) PI Zeta potential (mV) Yield (%) Association efficiency (%)

CS pH 5.6 166 (21) 0.210.26 +15.2 (3.4) 64.8 (7.0)

Lipoid S100 CS pH 3.8 209* (18) 0.310.38 +17.2 (2.5) 59.9 (13.1)

Lipoid S100-LMWH CS pH 3.8 280** (35) 0.330.38 +16.6 (1.9) 70.9 (11.4) 100 (0.0)

GCS pH 3.8 156 (25) 0.020.04 +22.2 (1.8) 49.4 (5.3)

Lipoid S100-GCS pH 4.3 187 (24) 0.320.37 +21.0 (3.1) 88.0 (12.7)

Lipoid S100-LMWH GCS pH 4.3 214*

(22) 0.220.28 +29.9**

(1.3) 85.2 (12.3) 99(0.2)Lipoid S100-LMWH GCS pH 7.0 385** (27) 0.250.35 +11.6** (0.8) 95.0 (5.0) 43 (2.9)

* Significantly different from the control (i.e., CS- or GCS-NPs,p


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Fig. 1. (a) Image of Lipoid S100-LMWHCS (pH3.8) nanoparticles, 1.5m1.5m topography obtained in resonant mode AFM; (b) image in phase contrast demonstrating

mechanical properties of the surface; (c) 3D representation of thesame nanoparticles and (d) corresponding cross section.

time points (Fig. 3a). The incubation of the particles at 25C and

37 C displayed retention of particle sizes with the exception of

Lipoid S100 LMWH GCS NPs formulated in neutral conditionswhere after an initial increase the size resulted constant (Fig. 3b

and c).

3.4. Mucoadhesion studies of LMWHloadedNPs

The degree of interaction between NPs and mucin was studied

by recording the variations in zeta potential of the nanoparticles

upon interaction with a mucin solution. As canbe seenin Fig.4, the

zeta potential values changed after addition of increasing amounts

of NPs to the mucin dispersion. This effect was more intense for

Lipoid S100LMWHGCSNPspreparedin acidicmedium, thanfor the

same formulation prepared in neutral medium and also for Lipoid

S100 LMWH CS NPs.

3.5. In vitro release studies

In vitro release studies from Lipoid S100-LMWH GCS NPs pre-

pared under neutral conditions were carried outin SLFand showed

that these nanocarriers exhibited a progressive LMWH release up

to 6h, followedby a plateaufor upto 24h (Fig. 5).

3.6. In vivo studies

In vivo preliminary experiments were performed with LipoidS100 LMWH GCS NPs prepared either in acidic or neutral condi-

tions, and the administration to the lung was done in the form of

liquid instillation and aerosolisation. The results showed that NPs

administered as a bolus andalso those prepared in acidic conditions

andadministeredby aerosolizationdid notlead to a change in coag-

ulation times (data not shown). However, as shown in Fig. 6, the

aerosol-type administration of free LMWH and Lipoid S100-LMWH

GCS NPs (LMWH dose of 8 mg/kg) prepared in neutral conditions

led to a significant elongation of the coagulation time as compared

to the control (buffer) (Fig. 6).

Additional experiments were intended to study whether the

passage of LMWH to the blood circulation could be due to the leak-

age through a damaged lung. Fig. 7a shows a tissue section taken

from a lung of an animal treated with aerosolized buffer (negativecontrol) and examined by microscopy. The section did not exhibit

any abnormalities. The alveolar sacs are well defined with no signs

of hemorrhage, flooding, or collapse of the alveolar spaces which

all can be defined as a manifestation of pulmonary toxicity. On the

contrary, the animals treated with LPS, which is able to trigger a

strong inflammatory response, showed a severe cell infiltration

predominated by neutrophils accompanied by edema which was

Table 2

Surface elemental composition of Lipoid S100-LMWHCS and GCS NPs. Theerror associated to each percentage is 0.5%.

NPs formulation % C % O % N % P % S % Na % Cl

Lipoid S100-LMWH CS pH 3.8 72.6 20.2 3.6 1.8 0.5 0.4 0.9

Lipoid S100-LMWH GCS pH 4.3 65.3 24.7 2.1 4.4 2.7 0.9


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Fig. 2. C1sXP spectra of thecomponents andof theresulting NPsformulations: (a)

Lipoid S100, (b) GCS, (c) Lipoid S100-LMWH GCS, and (d) Lipoid S100-LMWH CS.

In each panel the peak attributions to the corresponding chemical functional groupare indicated: peak (1) C C; peak (2) C OH, C N; peak (3) C OP; peak (4) C O,

O C O; peak (5) COOH; peak (6) N C OOH.

more prominent in the alveolar region (Fig. 7b). The histological

analysis of the mouse lungs treated with either LMWH alone or

LMWH loaded NPs (at the same LMWH dose range used in the

APTT experiments) suggests that no gross damage and inflamma-

tory infiltration in the lung is induced by these treatments. For a

comparison with negative and positive controls, Fig. 7c and d show

tissue sections taken from animal treated with either LMWH alone

or LMWH loaded NPs at the highest dose (8mg/kg) respectively.

These tissues appear very similar to what was seen in the negative

control group.

Fig. 3. Particle size of LMWH loaded NPs upon incubation in NaCl 0.9% at different

temperatures: 4 C (a); 25C ( b) and 37C (c). Values represent meansSD (n=3).

Whitebars:acidic LipoidS100-LMWH CS NPs;black bars:acidic LipoidS100-LMWH

GCS NPs; grey bars: neutral Lipoid S100-LMWHGCS NPs.

Fig.4. Change of zetapotential of coarse mucinparticles whenmixedwith nanopar-

ticles. () Lipoid S100-LMWH CS (pH 3.8); () acidic Lipoid S100-LMWH GCS; ()

neutral Lipoid S100-LMWHGCS NPs.


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Fig. 5. In vitro release profile of LMWH from Lipoid S100 loaded GCS NPs in SLF

bufferat pH7.2in 24h.

4. Discussion

In the context of anticoagulant therapy, the development of

a non-invasive drug delivery system for LMWH could represent

a useful approach to enhance patient compliance and minimize

adverse effects. The purpose of this work was to evaluate the

potential of CS and GCS NPs containing the pulmonary surfactant

Lipoid S100 for the systemic delivery of heparin upon pulmonaryadministration. To this end, LMWH loaded CS- and GCS-based NPs

were formulated using an adaptation of the ionic gelation tech-

nique (Mao et al., 2010; Trapani et al., 2009; Gan et al., 2005).

This technique, which has been previously used for the associa-

tion of heparin to CS (Paliwal et al., 2012) and CS-hyaluronic acid

nanoparticles (Oyarzun-Ampuero et al., 2009), was also found to

be appropriate for the formation of GCS NPs.

Asindicated,the size ofthe NPs preparedin acidicis smaller than

thatof NPspreparedin neutral conditions. In acidic conditions poly-

cations (CS and GCS) have a high charge density, a fact that favors

Fig. 6. Changes in plasma activated partial thromboplastin time (APTT) values

after pulmonary administration of free LMWH and LMWH-loaded Lipoid-GCS

NPs. Mice were aerosolised either with buffer (n=6), 18mg/kg LMWH (n=34

per group), or GCS loaded with 18mg/kg (n=36 per group). APTT values are

expressedin seconds.The control groupwith buffer is representedby a graycolumn.

KruskallWallis:p= 0.0003. **p< 0.01 forbuffer vs.GCSwith LMWH 8 mg/kg andvs.

free LMWH 8 mg/kg (MannWhitney).

their interaction with TPP and also with LMWH. As a result, the

nanoparticleshave a smallsize anda highly compacted structure.Incontrast, the GCS-based NPs formulated in neutral medium might

have a less dense structure because of the limited protonation of

the primary amino groups of GCS, thus leading to an enlargement

in their size.

On the other hand, the size increase observed upon introducing

the surfactant Lipoid S100 in the formulation might be due to the

interference of the surfactant in the interaction of the counterions.

The additionof Lipoid S100did not change significantlythe positive

zeta potential values with respect to the controls except for Lipoid

S100-LMWH GCS NPs prepared in acidic conditions, which showed

Fig. 7. Lung microscopy of experimental mice. Hematoxylin-stained lung sections (5-m thickness) from the following conditions: (a) animals treated with buffer; (b)

animals treated with LPSalone; (c)animalstreated with 8 mg/kg ofLMWH alone;and (d)animals treated with LMWH NPs. Panelsare representative ofthreemiceper group.

Original magnification: 20.


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Systemic Heparin Delivery by the Pulmonary Route Using Chitosan and Glycol

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