International Journal of Pharmaceutics 447 (2013) 115–123

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International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

Systemic heparin delivery by the pulmonary route using chitosan and glycol
chitosan nanoparticles
Adriana Trapani a,1 , Sante Di Gioia b,1 , Nicoletta Ditaranto c , Nicola Cioffi c , Francisco M. Goycoolea d ,
Annalucia Carbone b , Marcos Garcia-Fuentes e , Massimo Conese b , Maria Jose Alonso e,∗
a

Department of Pharmacy-Drug Sciences, University of Bari “Aldo Moro”, Via Orabona, 4, 70125 Bari, Italy
Department of Medical and Surgical Sciences, University of Foggia, Viale L. Pinto 1, 71122 Foggia, Italy
c
Department of Chemistry, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy
d
Institute of Plant Biology and Biotechnology, Westphalian Wilhelms University Münster, Schlossgarten 3, D-48149 Münster, Germany
e
Center for Research in Molecular Medicine 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 Campus Vida, Santiago de Compostela, Spain
b

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 glycol chitosan-nanoparticles
Lung delivery

a b s t r a c t
The aim of this study was to evaluate the performance of chitosan (CS) and glycol chitosan (GCS) nanoparticles 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 conditions using the ionotropic gelation technique. The size and zeta potential of the NPs were affected by the
pH and also the type of polysaccharide (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 of LMWH varied between 100% and 43% for the nanoparticles obtained in acidic
and neutral conditions, respectively. X-ray photoelectron spectroscopy studies indicated that the surfactant Lipoid S100 was localized on the nanoparticle’s surface irrespective of the 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 promising
features of CS-based nanocarriers for pulmonary delivery of LMWH.
© 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 vein thrombosis, pulmonary embolism and other
thromboembolic disorders (Schulman, 2000; Yang et al., 2004).
Unfortunately, LMWH exhibits poor oral bioavailability and, consequently, has to be administered via parenteral route. Due to
poor patient compliance and side effects associated with injections,
alternative routes for non-invasive LMWH administration have
been actively investigated (Motlekar and Youan, 2006). Among
those, 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 available 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: mariaj.alonso@usc.es (M.J. Alonso).
1
These authors contributed equally to this work.
0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2013.02.035

chronic therapy (Hussain et al., 2003; Labiris and Dolovich, 2003;
Craparo et al., 2011; Licciardi et al., 2012). Moreover, it has also been
observed that LMWH itself causes, upon pulmonary administration,
a transient opening of the tight junctions in the lung epithelium,
leading to a rapid onset of action and a Cmax comparable to subcutaneous administration (Qi et al., 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 circulation might be insufficient for an adequate pharmacological
response.
Among the different pulmonary drug delivery vehicles, chitosan
(CS)-based nanoparticles are particularly attractive. 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 characteristics and it is capable of opening the tight junctions of epithelial
cells, thereby improving the 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, GCS has been reported to

NPs were isolated by ultracentrifugation (16. The final CS/TPP mass ratio was 6/1. 2003). The resulting mixture was used to cross-link 8. 2011). We have also prepared Lipoid S100-LMWH GCS NPs upon neutral conditions. glycerol. viscosity = 13 mPa/s according to manufacturer data sheet) was purchased from Pronova Biopolymer (Norway). 2010.2 mL of the dispersion of Lipoid S100.20% (w/v) in NaCl aqueous solution (87 mM) under magnetic stirring (VWR.07%.3. the limit of quantification (LOQ) of LMWH was 0. Measurements were performed in triplicate after dilution 1:1 (v/v) in double distilled water and at 25 ◦ C. Diagnostica Stago.52 mL of LMWH aqueous solution. The resulting mixture was used to crosslink 3 mL of CS solution (0. 5.07%. Lipoid S100 was kindly donated by Lipoid KG (Germany). In addition.e. the aim of the present work was to develop CS and GCS nanoparticles containing LMWH and to evaluate their performance following pulmonary administration. Lipoid S100-LMWH GCS NPs were formulated starting from the anionic bearing species consisting of 0. 2009).. Italy). . Calvo et al. 2004) and therapeutic peptides such as insulin (Al-Qadi et al. v/v). Analytical determination of low molecular weight heparin The heparin amounts were directly measured using two colorimetric assays (i. in NaCl aqueous solution (87 mM)) to 3 mL of CS solution (0. the mean particle size and distribution were determined in double distilled water by photon correlation spectroscopy (PCS) using a Zetasizer NanoZS (ZEN 3600. The corresponding Lipoid S100-LMWH CS NPs were not formulated due to the poor water solubility of CS at pH > 6.. De Giglio et al.9 × 10−1 ␮g/mL. The autocorrelation functions were analyzed using the DTS v. Mw 110 kDa.07%.0 × 10−1 ␮g/mL concentration range.5. There are some previous reports on the potential of CS nanoparticles for the local and systemic delivery of macromolecules following pulmonary administration..1%. w/v) previously dissolved in Tris (10 mM. pH 7). Such mixture was used to cross-link 3 mL of CS solution (0. LMWH loaded NPs Lipoid S100-LMWH CS NPs were formulated starting from the anionic bearing species consisting of 0.2%. Germany) using a glycerol bed in order to facilitate their resuspension in ultrapure water by manual shaking.2. w/v) were mixed with an aliquot of 0. 177 UI/mg). 3 mL of TPP aqueous solution (0. Briefly. as previously reported (Ieva et al. Makhlof et al.20%. we hypothesized that. Trapani et al. For Lipoid S100containing CS (and GCS) NPs. LMWH (average MW 18 kDa. v/v)..52 mL of the dispersion of Lipoid S100 and an aliquot of 0. 2. w/v). Chitosan hydrochloride salt (Protasan. w/v) previously dissolved in acetic acid (0... 2. All other standard chemicals were of reagent grade.. For the determination of zeta-potential values. in 87 mM NaCl aqueous solution). and pentasodium tripolyphospate (TPP) were purchased from Sigma–Aldrich (Milan. In addition. 2005). Lipoid S100 CS NPs were prepared by mixing 0. by combining the mucoadhesive characteristics of the polymers with the nanoscale dimensions and the absorption enhancing properties of the surfactant and polymers.. Ultrapure water was used throughout the study.4 mL of Lipoid S100.2 mL of LMWH solution and 1. 45 min. respectively). 2. 2009)..9/1 (Gan et al. For Azure A colorimetric assay. Ultimately. The final GCS/TPP ratio was 2. a laser Doppler velocimetry was adopted by using Zetasizer NanoZS after dilution with 1 mM KCl (pH 7. VMS C-4.116 A. 2. 2009). Moreover.. Mao et al.1.5 mL of TPP (0. bound sialic acid. The resulting mixture was used to cross-link 3 mL of GCS solution (0. 2012) or calcitonin (Makhlof et al. Unloaded CS (and GCS) NPs CS NPs were spontaneously formed by adding 1 mL of TPP (0. Herrenberg. ∼1%). Materials and methods 2. glycol chitosan (MW 400 kDa according to supplier instructions). being a mixture of natural phospholipids. 2010)..1%.20%.20%. Italy). w/v. w/v) were mixed with an aliquot of 0. we could significantly enhance the pulmonary absorption of LMWH. 2010). w/v) to 3 mL of GCS (0. The results of this work have shown that CS–HA nanoparticles are able to enhance the inherent ability of heparin to block the degranulation of mast cells (Oyarzun-Ampuero et al.1 mL of the dispersion of Lipoid S100.2%. Lipoid S100. we found it critical to incorporate the non ionic surfactant Lipoid S100 to the nanoparticle’s structure. For example. 2012).e.1 software provided by Malvern.. Such concentration of NaCl was previously found to allow the formation of small size CS-based NPs (Goycoolea et al.20%). 3. Preparation of nanoparticles LMWH loaded CS or GCS-based NPs were prepared according to the ionic gelation technique. Eppendorf 5415D. nanoparticles made of CS and hyaluronic acid (HA) have been used for local heparin delivery to the lungs (Oyarzun-Ampuero et al..e. Malvern.. These assays were performed according to the manufacturer’s instructions and linearity was checked in the 140 ␮g/mL to 1. Stachrom® Heparin. w/v) dissolved in acetic acid (0.2 mL of Lipoid S100.20% (w/v) in 87 mM NaCl aqueous solution) under stirring.5 mL of TPP (0. deacetylation degree 86%. the surfactant was dispersed to give a final concentration of 0. mucine from porcine stomach (type II. in 87 mM NaCl aqueous solution). 2012. w/v. / International Journal of Pharmaceutics 447 (2013) 115–123 retain the mucoadhesive properties inherent to CS (Trapani et al. w/v.. The resulting mixture was used to cross-link 3 mL of GCS solution (0. Similarly. 2009) and Azure A colorimetric method (Ramadan et al..07%. NPs made of CS or GCS have been recently described for pulmonary delivery of DNA (Bivas-Benita et al. 0.1% (w/v) in 87 mM NaCl aqueous solution. Italy (Oyarzun-Ampuero et al. 1. Lipoid S100 GCS pH 4..5 mL of TPP aqueous solution (0. Materials The following chemicals were used as received. 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. Germany). For GCS based NPs (i.8 mL of TPP (0. Physicochemical characterization of nanoparticles For all tested NPs.5.0) (Montenegro et al. 2009. UP CL 113. 1997).3). 2.2% (w/v) in 87 mM NaCl aqueous solution) with an aliquot of 0.000 × g.4. 2. Lipoid S100-LMWH GCS NPs were formed as follows.1%. 0. and thus NPs were formed. was thought to improve the biocompatibility of the nanosystems in contact with the alveolar surface.0 mL of GCS solution (0. GCS NPs were obtained by mixing 3 mL of TPP aqueous solution (0.2 mL of LMWH solution and 0... nanoencapsulation was also conceived as a way to protect the anticoagulant drug from possible enzymatic degradation.8 and 4. It should be noted that all the nanosuspensions prepared as above displayed acidic pH values (i. 2009.3 for Lipoid S100LMWH CS and Lipoid S100-LMWH GCS.. Taking this into account.6.

All images are 512 × 512 data points. corresponding to an amount of LMWH in the range 20–70 ␮g/mL.7. A small spatula was used to open the lower jaw of the mouse and blunted forceps were used to help displace the tongue for maximal oropharyngeal exposure. . To achieve high-resolution images of nanoparticles deposited on the substrate. Mice were sacrified 4 h after each treatment. 1. for 1 week at 25 ◦ C and for 4 h at 37 ◦ C. To evaluate the stability of the NPs under different incubation conditions.5 mL of SLF.1. then the sample was processed directly on the AFM scanner. In vivo studies Buffer (Tris–HCl. Anticoagulation study Anticoagulation was studied with activated partial thromboplastin time (APTT). 15 and 24 h) each sample was centrifuged at 16. Philadelphia.4 and an osmolarity of 300 mOsm/kg.1 eV. Italy) using a MicroSprayerTM aerosoliser (IA-1 C.0 mEq/L MgCl2 . attached to a high-pressure syringe (FMJ-250. lungs collected and inflated with 1 mL of 10% neutral buffered formalin and fixed for a minimum period of 24 h.8. Wide scans and detailed spectra were acquired in constant analyser energy (CAE) mode with a pass energy of 150 eV and 100 eV.13. embedded in a paraffin block. a drop of nanoparticle suspension was spin-coated onto a flat gold thin film deposited on silicon substrate. 300 mOsm/kg). The samples were incubated under mechanical agitation (100 rpm) at 37 ◦ C. 2011). LMWH or Lipoid S100-LMWH GCS NPs formulated in neutral conditions were administered to Swiss mice (25 g. The tip was immediately withdrawn and the mouse was taken off the support. The association efficiency was determined in triplicate. XPS analysis The surface chemical composition was studied by means of Xray Photoelectron Spectroscopy (XPS). One milliliter of this suspension was mixed with different volumes of the NP suspension tested at 1 mg/ml. / International Journal of Pharmaceutics 447 (2013) 115–123 2.0 mEq/L NaHCO3 . The supernatant was analyzed for the content of LMWH using Azure A spectrophotometric assay.0 mEq/L KCl. 102. the MicroSprayer tip was endotracheally inserted until it reached the carina (the first bronchial bifurcation) and 100 ␮L of solution (buffer or LMWH) or NP suspension was sprayed. Sigma–Aldrich. 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. Atomic force microscopy (AFM) AFM studies were performed to characterize the morphology of LMWH loaded CS NPs. The mice were anaesthetized by an intraperitoneal injection of 0. This medium had pH 7.10.0 mEq/L KHCO3 .0 mEq/L NaCl.. AE was calculated as follows: %AE = 100 × total 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. For AFM visualization. The resulting mixtures were stirred (100 rpm) at 37 ◦ C for 15 min and then the zeta potential was measured. 2. Citrate-anticoagulated blood was obtained 4 h after administration of buffer.74. Prior to their analysis. Calco. 1.8 ± 0. 2. 2.4) at 1% (w/v) concentration. 2005.A.9. lungs were placed in tissue cassette. Germany) flexible fiber-optics arm was adjusted to provide optimal illumination of the trachea. 2. a low energy flood gun (−1 eV) was used for charge compensation. For each experiment. 1. the NPs formulations (Lipoid S100 LMWH GCS NPs and Lipoid S100 LMWH CS NPs) were converted into powder using freeze-drying. and 1. 2. XPS analyses were performed with a Theta Probe VG Scientific spectrometer equipped with a micro spot monochromatised Al K␣ source (1486. Mucoadhesion tests The mucoadhesive interaction between LMWH loaded NPs and mucin was determined recording the variations of zeta potential according to the method reported in the literature (Takeuchi et al.2.0). At least 3 mice per group were analysed. 117 Briefly. 5. Germany) and were suspended at a 45◦ angle by the upper teeth. LMWH or NPs. Stability study of LMWH loaded NPs in different media Freshly prepared LMWH loaded CS and GCS NPs were resupended at 0. commercially available porcin mucin was firstly suspended.0 mEq/L K2 HPO4 . After a clear view of the trachea was obtained by an otoscope. Data were averaged over at least three points per sample. NPs were freshly prepared and then centrifuged in presence of 10 ␮L of glycerol. Positive control mice were administered with aerosolised Lipopolysaccharide (LPS) from Pseudomonas aeruginosa (20 ␮g/mouse). Helmut Hund. Plasma was combined 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. 2009. pH 7. were put in Eppendorf tubes containing 0. The tip amplitude oscillations were kept constant in attractive forces regimen to prevent the damage of the NPs. At scheduled time intervals (0. Briefly.2. Following fixation.6 eV. These tissues were processed into paraffin. 2.0 mEq/L CaCl2 . Penn Century. 31. Trapani et al.1% (w/v) concentration in normal saline (NaCl. For nonconductive samples.13.000 × g for 45 min. 2.13. The experiment was performed in triplicate. 1979.0 mEq/L sodium acetate. respectively. sectioned on a microtome to a thickness of 5 ␮m. on air in semi-contact mode with standard Si cantilever with 316 kHz resonant frequency.5 mg/g body weight Avertin (2. The scan speed was proportional to the scan size with a typical scanning frequency of 2–3 Hz.0 mEq/L sodium citrate. 6. 0. appropriate volumes of nanodispersions. the systems were incubated in normal saline for 12 weeks at 4 ◦ C.11. Acquisition and data processing were performed using Avantage software v. Histopathological study Mice received the same treatment described above. 2. Penn Century). 2003). spot size: 300 ␮m). USA) suitable for mice. In vitro release studies In vitro release studies from Lipoid S100-LMWH GCS NP formulated in neutral conditions were carried out up to 24 h in simulated lung fluid (SLF) (Moss.12. in PBS (pH 7. 1. The AFM images were obtained using a Ntegra Prima AFM. 1. NSG03 silicon cantilevers with a length of 135 ␮m were used with resonance frequency of about 90 kHz and a nominal force constant around 1. (mean 2–3 lines per second). Davies and Feddah. 4.8 N/m.0 mEq/L MgSO4 . 1.3. Particle size of the samples was determined at scheduled time intervals. Charles River..2-tribromoethanol. pH 7.0 mEq/L Na2 HPO4 . Jintapattanakit et al. at room temperature. The composition of SLF is: 5. Das Neves et al. PA. 7.. Calibration of the binding energy (BE) scale was performed by fixing the C C component at BE values of 284.9%. Scan speed was between 2 and 3 Hz. The light source’s (lamp type FLQ85E.

More precisely.9 (±13.0 166 (±21) 209* (±18) 280** (±35) 156 (±25) 187 (±24) 214* (±22) 385** (±27) 0.e.38 0.118 A. respectively. but not peaks 4 and 6. Mean ± S.04 0.21–0. whereas GCS NPs had an average particle size in the 156–214 nm range.2 (±3.28 0.D.2 (±1.e. The XPS spectrum from Lipoid S100-LMWH GCS NPs (panel c) resembles that from the surfactant: peaks 3 and 5 are found. Carbon. For both types of NPs.3) 88. The association efficiency values of LMWH to all formulations of NPs prepared in acidic conditions were very high (i.4) +17. The surfactant Lipoid S100 was dispersed in NaCl 87 mM prior to its addition to this mixture.0 (±5..1) 70. oxygen. but not Lipoid S100 GCS. The zeta potential values were positive. 4. respectively). The incubation of the particles at 4 ◦ C for 12 weeks showed an increase in particle size at the latest . pH 7.22–0.4) 49. it is evident that peaks 3 and 5 are attributed to specific functional groups both coming from the surfactant. / International Journal of Pharmaceutics 447 (2013) 115–123 Table 1 Physicochemical properties of LMWH loaded CS.35 +15. The overall significance of the differences between groups was analysed by a Kruskall–Wallis test. and CS (data not shown) provided information to better understand the surface chemical composition of Lipoid S100 LMWH GCS and CS NPs (panels c and d). 3.0) – – 100 (±0.8 (±7. placed on a microscope slide.2 mV for CS nanoparticles and +22. As shown in Table 1. PI: polydispersity index.9%. C1s XP spectra for the components and the resulting NPs are reported.0 (±3. Moreover.6 (±1. 99–100%).8 Lipoid S100-LMWH CS pH 3.32–0. Control nanoparticles were unloaded CS and unloaded GCS. Lipoid S100-LMWH GCS NPs were also prepared in neutral media and their size resulted significantly larger than the ones obtained in acidic medium (i. The gelation of CS was induced by adding to the polycation solution a mixture of LMWH. In this figure. Fig. Table 2 shows the relative percentages of elements present in the different constituents of the nanoparticles. showed a further significant size increase when loaded with LMWH (p < 0.1) +29. Moreover. are reported. were dissolved in diluted NaCl (pH = 7) and acetic acid (pH = 4) aqueous solutions.3) 95. 1a and b show the topography and phase contrast images. p < 0. Results 3.3.9) * ** Significantly different from the control (i. while peaks 4 and 6 are related to GCS chemical composition.3) +11. Significantly different from the control (i. XPS studies The surface chemical composition of the resulting NPs formulations was evaluated by means of XPS analyses. In this medium. and around 200 nm in section.05) using GraphPad Prism v.37 0. while sodium and chlorine are residuals of the preparation media.4 (±5. Additionally. Inc. These data suggest that there is a preferential localization of the surfactant Lipoid S100 on the outer surface of the NPs. rather than using organic solutions. the Lipoid S100-LMWH GCS NPs prepared at pH 7 resulted in moderate association efficiency (43%). using a Mann–Whitney test (p < 0. The two polysaccharides. GCS (panel b). 1c and d show a tridimensional image and cross-section of the NPs respectively. CS NPs showed an average particle size from 166 to 280 nm.8 GCS pH 3. However. CS NPs were slightly larger in size than GCS NPs when prepared in acidic conditions. 30–70 nm high. Preparation and characterization of LMWH-loaded CS and GCS nanoparticles LMWH-loaded NPs were prepared by the well-known ionotropic gelation technique (Trapani et al.001).. according to the binding energy (Wagner et al.05). Trapani et al. Statistics Data from different experimental groups were compared by a one-way ANOVA and differences were considered significant at 95% level of confidence (p < 0.0) 59.8) 64. 300 mOsm/kg).38). The same considerations are valid for Lipoid S100-LMWH CS NPs (panel d).8 Lipoid S100-GCS pH 4.1.2) 43 (±2. These images illustrate the appearance of individual NPs. The zeta potential values of Lipoid S100-LMWH GCS NPs prepared in neutral medium were positive but lower than those prepared in acidic conditions.9 (±11. In vivo data were evaluated using the same software. Lipoid S100 and the polyanion TPP.. NPs were exposed to three different temperature conditions (4 ◦ C. respectively (Goycoolea et al.26 0.6 Lipoid S100 CS pH 3.33–0. NPs formulation Size (nm) PI Zeta potential (mV) Yield (%) Association efficiency (%) CS pH 5. In contrast.2 (±2.2 mV for GCS nanoparticles.e. Stained sections were analysed using an Olympus BX41 microscope. and nitrogen are compatible with all the organic molecules used to prepare the NPs.7) 85. 3. The analysis of the carbon spectral region provided some additional information.. phosphorous comes from the surfactant Lipoid S100 and TPP used in the preparation process.38 0. CS.00 computer program (GraphPad Software.e.02–0.2 (±12. Lipoid S100 CS NPs. the relative abundance of peaks 1 and 2 is different in Lipoid S100 and GCS samples. CA. the size values do not perfectly correlate with those obtained by PCS. 3.05). In Fig. 2. USA).5) +16.25–0.or GCS-NPs. 2009). Further analyses were carried out by comparing the control “buffer” group with the other individual groups. varying between +15.8) +21. n = 6. 2009). The particle size distribution of CS and GCS particles was moderate as it is shown by their polydispersity index (PI) values (0. The XPS analysis of the bare constituents Lipoid S100 (panel a).. In particular.6** (±0.31–0. CS. and stained with hematoxylin and eosin stain.. 2009. CS and GCS.02–0.0) – – 99(±0.and GCS-based NPs. the introduction of surfactant Lipoid S100 resulted in a marked size increase. A very low amount of sulfur associated to traces of LMWH was detected on the surface of CS based NPs. 385 nm). 2009). Trapani et al.3 Lipoid S100-LMWH GCS pH 7.. 25 ◦ C and 37 ◦ C). probably due to the adsorption and dehydration of the NPs onto the gold substrate. Bonferroni tests were used for post hoc contrast.. Fig. p < 0. All the spectra have been curve-fitted to attribute each peak to the corresponding chemical environment.0 (±12.. 0.9** (±1. the ratio of peak 1 and peak 2 is closer to the spectrum of Lipoid S100 than to GCS.2.9) +22.3 Lipoid S100-LMWH GCS pH 4. 2. 2003).05). 1). as previously reported (Hafner et al. The morphology of selected LMWH-loaded NPs was examined by AFM (Fig.. 214 vs. it is possible to identify some individually separated NPs. Stability studies of LMWH-loaded NPs The determination of nanoparticle colloidal stability was evaluated in normal saline (NaCl.14.or GCS-NPs.

/ International Journal of Pharmaceutics 447 (2013) 115–123 119 Fig. or collapse of the alveolar spaces which all can be defined as a manifestation of pulmonary toxicity. as shown in Fig. which is able to trigger a strong inflammatory response. 7a shows a tissue section taken from a lung of an animal treated with aerosolized buffer (negative control) and examined by microscopy. However.1 1.4.6. This effect was more intense for Lipoid S100 LMWH GCS NPs prepared in acidic medium.6 65. Trapani et al. Additional experiments were intended to study whether the passage of LMWH to the blood circulation could be due to the leakage through a damaged lung. In vitro release studies In vitro release studies from Lipoid S100-LMWH GCS NPs prepared under neutral conditions were carried out in SLF and showed that these nanocarriers exhibited a progressive LMWH release up to 6 h.4 2. The incubation of the particles at 25 ◦ C and 37 ◦ C displayed retention of particle sizes with the exception of Lipoid S100 LMWH GCS NPs formulated in neutral conditions where after an initial increase the size resulted constant (Fig. followed by a plateau for up to 24 h (Fig. As can be seen in Fig. the animals treated with LPS. 4. 1. 3.6 2. (b) image in phase contrast demonstrating mechanical properties of the surface. the zeta potential values changed after addition of increasing amounts of NPs to the mucin dispersion. 5). 3b and c).9 . 1. The section did not exhibit any abnormalities. The results showed that NPs administered as a bolus and also those prepared in acidic conditions and administered by aerosolization did not lead to a change in coagulation times (data not shown). (c) 3D representation of the same nanoparticles and (d) corresponding cross section.3 20. On the contrary. than for the same formulation prepared in neutral medium and also for Lipoid S100 LMWH CS NPs.A.2 24. flooding. showed a severe cell infiltration predominated by neutrophils accompanied by edema which was Table 2 Surface elemental composition of Lipoid S100-LMWH CS and GCS NPs. 6).5 – 0.8 4.5%.7 3. time points (Fig.4 0. and the administration to the lung was done in the form of liquid instillation and aerosolisation. Fig.8 Lipoid S100-LMWH GCS pH 4. 3.8) nanoparticles. The error associated to each percentage is 0.3 72. 3a). NPs formulation %C %O %N %P %S % Na % Cl Lipoid S100-LMWH CS pH 3.5 ␮m × 1. 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.9 0. 3.5. 6.7 0. (a) Image of Lipoid S100-LMWH CS (pH 3. In vivo studies In vivo preliminary experiments were performed with Lipoid S100 LMWH GCS NPs prepared either in acidic or neutral conditions.5 ␮m topography obtained in resonant mode AFM. Mucoadhesion studies of LMWH loaded NPs 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. The alveolar sacs are well defined with no signs of hemorrhage.

Fig. peak (6) N COOH. Values represent means ± SD (n = 3). peak (3) C OP. peak (2) C OH. more prominent in the alveolar region (Fig. These tissues appear very similar to what was seen in the negative control group. 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 inflammatory infiltration in the lung is induced by these treatments. and (d) Lipoid S100-LMWH CS. . Fig. () Lipoid S100-LMWH CS (pH 3. 2. black bars: acidic Lipoid S100-LMWH GCS NPs. peak (5) COOH. () acidic Lipoid S100-LMWH GCS.8). / International Journal of Pharmaceutics 447 (2013) 115–123 Fig. 4. 7b). Particle size of LMWH loaded NPs upon incubation in NaCl 0. Fig. C N. grey bars: neutral Lipoid S100-LMWH GCS NPs. Trapani et al.9% at different temperatures: 4 ◦ C (a).120 A. peak (4) C O. 7c and d show tissue sections taken from animal treated with either LMWH alone or LMWH loaded NPs at the highest dose (8 mg/kg) respectively. 25 ◦ C (b) and 37 ◦ C (c). (c) Lipoid S100-LMWH GCS. Change of zeta potential of coarse mucin particles when mixed with nanoparticles. In each panel the peak attributions to the corresponding chemical functional group are indicated: peak (1) C C. (ⵦ) neutral Lipoid S100-LMWH GCS NPs. 3. (b) GCS. For a comparison with negative and positive controls. C1s XP spectra of the components and of the resulting NPs formulations: (a) Lipoid S100. White bars: acidic Lipoid S100-LMWH CS NPs. O C O.

Panels are representative of three mice per group. To this end. which has been previously used for the association of heparin to CS (Paliwal et al. which showed Fig. Trapani et al. / International Journal of Pharmaceutics 447 (2013) 115–123 121 Fig. This technique. In acidic conditions polycations (CS and GCS) have a high charge density. the nanoparticles have a small size and a highly compacted structure. 2009). Changes in plasma activated partial thromboplastin time (APTT) values after pulmonary administration of free LMWH and LMWH-loaded Lipoid-GCS NPs.2 in 24 h.and GCS-based NPs were formulated using an adaptation of the ionic gelation technique (Mao et al. 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 addition of Lipoid S100 did not change significantly the positive zeta potential values with respect to the controls except for Lipoid S100-LMWH GCS NPs prepared in acidic conditions. 6. LMWH loaded CS.01 for buffer vs.. 2005). Gan et al. thus leading to an enlargement in their size. In vitro release profile of LMWH from Lipoid S100 loaded GCS NPs in SLF buffer at pH 7. 2012) and CS-hyaluronic acid nanoparticles (Oyarzun-Ampuero et al. 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 pulmonary administration. 2010. free LMWH 8 mg/kg (Mann–Whitney). Lung microscopy of experimental mice. was also found to be appropriate for the formation of GCS NPs. 2009.. the size of the NPs prepared in acidic is smaller than that of NPs prepared in neutral conditions. Mice were aerosolised either with buffer (n = 6). Trapani et al. or GCS loaded with 1–8 mg/kg (n = 3–6 per group). **p < 0.A. and (d) animals treated with LMWH NPs. 5. . 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. In contrast... their interaction with TPP and also with LMWH. 1–8 mg/kg LMWH (n = 3–4 per group). GCS with LMWH 8 mg/kg and vs. On the other hand. a fact that favors Fig. The control group with buffer is represented by a gray column. As indicated. Original magnification: 20×.. As a result. Kruskall–Wallis: p = 0.0003. Hematoxylin-stained lung sections (5-␮m thickness) from the following conditions: (a) animals treated with buffer. (b) animals treated with LPS alone. APTT values are expressed in seconds. 7. 4. (c) animals treated with 8 mg/kg of LMWH alone. 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.

. On the other hand. Grenha. G. Sogias et al. Teresi. 157.. 2011.. M.. C. The limited performance of the nanoparticulate formulation and the lack of effect observed for the rest of the formulations could be related with the limited duration of the study (4 h). V. Hafner.M. F. Pantaleo Bufo (Unit of Pathological Anatomy. A. Di Stefano. Hussain. G. Chitosan-based formulations for delivery of si-RNA and DNA systems for protein therapeutics and antigens.. M. J. Latrofa.. Giammona. G.B. Y. Overall. Bahia.. Carrión-Recio.. B.. 2005. 63. Bondi’. Pulmonary delivery of chitosan-DNA nanoparticles enhances the immunogenicity of DNA vaccine encoding HLA-A+ 0201-restricted T-cell epitopes of Mycobacterium tuberculosis. GCS-based nanocarriers prepared in acidic conditions are more stable than those containing CS. Makhlof. Eng.. Cioffi. further studies are required to elucidate the advantage of using this formulation for controlled release of LMWH as compared to free drug and the therapeutic potential of such formulations for the treatment of pulmonary embolism and other thromboembolic disorders. Thanks are due to Lipoid KG (Germany) for a gift of Lipoid S100.. M. M. L. Pharm. M. 2009. M.. The role of mucoadhesion of trimethyl chitosan and PEGylated-trimethyl chitosan nanocomplexes in insulin uptake. A novel method for assessing dissolution of aerosol inhaler products. 207–215. We thank Dr. formed by ionic gelation technique. 447–449. A. Alonso. M. Craparo.) for his help in the AFM analyses.. J. The absence of a burst effect and a progressive increase of LMWH release were observed for up to 6 h. G. N. 2011. it is known that the mucoadhesion of CS-based positively charged NPs is mainly determined by electrostatic interactions of the cationic polymer with negatively charged mucin (Das Neves et al. E. University of Foggia.. A. is completely absent on the Lipoid S100-LMWH GCS NPs surface and its atomic percentage is very low on the Lipoid S100-LMWH CS NPs surface.. M. 600–612.. Remunan-Lopez.. Modulation of surface charge.M... An additional factor that may account for the better mucoadhesive properties of GCS nanoparticles as compared CS is the higher molecular weight of GCS vs.A. T. S. . 2005. Goycoolea.. The target element for LMWH. Sergey Lemeshko (NT-MDT Europe B. Motlekar. 65–73. 2003.. E. D.. Ottenhoff. 383–390. Maurizio Margaglione and the staff at the Medical Genetics Unit.. particle size and morphological properties of chitosan – TPP nanoparticles intended for gene delivery. 92–95. D. Alonso.. Ceci.B. Control.. these nanocarriers might have a potential for systemic delivery of LMWH.. Smart. Chitosan-alginate blended nanoparticles as carriers for the transmucosal delivery of macromolecules. Feddah. G. N. Junyaprasert. 2012. Release. Mao. 205–213. N. Int. Int. Wang. Borchard.. Moss. as shown in Fig. C. Labiris. Acknowledgments We thank Prof. G. 1085–1104. A. Jintapattanakit. the long polymeric chains may facilitate their interpenetration with mucin macromolecules resulting in consolidated adhesive bonding (Das Neves et al. 2004. Microencapsulated chitosan nanoparticles for pulmonary protein delivery: in vivo evaluation of insulin-loaded formulations.... 113. Italy) for histopathological analysis and imaging. Mattioli-Belmonte. The results of the in vivo study indicate that. Our hypothesis is that due to the prolonged release behaviour of the nanoparticles. 496–504.. Romanelli. / International Journal of Pharmaceutics 447 (2013) 115–123 the highest zeta potential values among all the formulations..122 A.J. Int. M. Trapani et al... Conclusion We have developed a novel nanocarrier consisting of Lipoid S100 and CS or GCS. 1255.F.B. Iatta. in particular. A. 1997. P... Craparo. Pharm. Rev. G.. MGF acknowledges a Parga Pondal Fellowship from Xunta de Galicia. A comparable result was observed for free LMWH. Cavallaro.. J. Control... Pulmonary absorption of insulin mediated by tetradecyl-␤-D-maltoside and dimethyl-␤-cyclodextrin. 161. Nanotechnol. PHEA-graft-polybutylmethacrylate copolymer microparticles for delivery of hydrophobic drugs. Kissel... D. To gain more understanding in this regard.A. within the range of conditions investigated. Sarmento. Nano Biomed. A.. These nanosystems. G. Pharm. Pharmacol. Vila-J. 433. R. Bioanal. 2005).L.L. Takeuchi. 4. 12. Cavallaro. J.. Amiji. F. 56..F. it is important that the NPs maintain their size under different storage temperatures. Das Neves.V. chitosan-polyethylene oxide nanoparticles as protein carriers. fundamental for a good biocompatibility of the nanocarriers. Trapani.. J. Remunán-López. Sci.J. Results from stability studies indicated that. This high positive charge may explain the greater interaction of these nanoparticles with mucin.. The analysis of the detailed C1s spectral regions provided useful information about the outer localization of the surfactant in Lipoid S100-LMWH GCS and CS NPs. K. lecithin/chitosan nanoparticles: physicochemical characterisation and permeability through Caco-2 cell monolayers. we showed that the passage of LMWH to the peripheral circulation upon aerosolization happened in the absence of gross lung damage and inflammatory response that may have increased vascular permeability. Geluk. J. 2003. Ieva. C.. 62..E. Appl. T.L. Nanosci. J. In vitro evaluation on a model of blood brain barrier of idebenone-loaded solid lipid nanoparticles. 397. M. 2004)... 8. J. 125–132. De Giglio. J. Ciprofloxacin-loaded chitosan nanoparticles as titanium coatings: a valuable strategy to prevent implant associated infections.. 2006. van Meijgaarden. suggesting an inner localization of the anticoagulant agent in the nanocarriers. Int.. E. T. Biomacromolecules 10.. Smart. Pharm. 393. B: Biointerfaces 44.. Trapani. These results are in accordance with the finding that LMWH cause opening of the tight junctions.. 175–187. Pharm. A.. A.. 2011. 162–168.. Quaglia. Cafagna.. T. Aerosolization of these formulations indicated that heparin could be delivered to the lung. In view of in vivo application.. ´ J. Pharm. S. 2012. J. McCarron. A. Lollo.. Int.. 2011.. M. Junginger. N. 1979. the formulation of Lipoid S100-LMWH GCS NPs prepared at neutral pH was efficient at delivering LMWH to the blood stream following pulmonary aerosolization. P. Ditaranto..... Mucoadhesive nanomedicines: characterization and modulation of mucoadhesion at the nanoscale. the finding that the positive zeta potential values did not change significantly upon addition of Lipoid and anticoagulant (except for Lipoid S100-LMWH GCS NPs) indicates that the electrical charges located at the surface of these nanocarriers are influenced not only by the net chemical positions but also by the molecular organization of the polyelectrolites and the surfactant at the surface. 2010... In fact. Expert Opin.. As reported. B. Sci. 16–24. Trapani.. Monopoli. References ˜ Al-Qadi. Vaccine 22. ˜ C. Phospholipidpolyaspartamide micelles for pulmonary delivery of corticosteroid. The quest for non-invasive delivery of bioactive macromolecules: a focus on heparins.. T. Licciardi. A.H. Chitosan-based nanocarriers: where do we stand? J. A. S. Release. E. Adv.F. Seijo. Puglisi. Alonso. Sun.. 1551–1557.R. W. Anal. Simulants of lung interstitial fluid. H. A.J. 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