Bettega
email: rodolphe.lartizien@gmail.com

Quantification of the inaccuracy of conventional articulator model surgery in Le Fort 1 osteotomy: evaluation of 30 patients controlled by the Orthopilot TM navigation system

Keywords: orthognathic, surgery, maxillary repositioning, 3D analysis, Le Fort I osteotomy, computer-assisted, surgery, occlusal splints, conventional articulator model surgery

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Orthognathic surgery is classically planned by integrating clinical, cephalometric, and dental data from plaster casts. Acrylic occlusal splints are manufactured to transfer the planning data to the operating room and to set the position of the jaws during the operation. It is well established that the manufacture and use of such splints are the causes of errors and imprecision. [START_REF] Olszewski | Limitations of orthognathic model surgery: theoretical and practical implications[END_REF][START_REF] Schouman | Accuracy evaluation of CAD/CAM generated splints in orthognathic surgery: a cadaveric study[END_REF][START_REF] Quast | Traditional face-bow transfer versus threedimensional virtual reconstruction in orthognathic surgery[END_REF] The recorded position of the maxilla with a facebow may be altered by the approximation of the Frankfort plane, and positioning of the face-bow on soft tissues may be difficult. The recorded position of the maxilla may also be modified when transposing to the semiadjustable articulator (cast weight, disruption of facebow). The articulator is another source of inaccuracy: its rotational axis differs from the patient's bicondylar axis, and its reference horizontal plane may differ from the recorded Frankfort plane. [START_REF] Barbenel | Errors in orthognathic surgery planning: the effect of inaccurate study model orientation[END_REF] Plaster casts, dental impressions, and occlusions may be other sources of discrepancy. Consequently, the accuracy of occlusal splints is related to the care brought to previous steps and their use during operation. We routinely used a navigation system for orthognathic surgery (Orthopilot ® , B Braun Aesculap, Tuttlingen), which includes an optical localiser (Polaris NDI,), controlled by a standard personal computer. The camera locates reflective markers called "rigid bodies," which have a specific geometry, in the space in real time. Those rigid bodies are attached (in a reversible and reproducible manner) on a specific ancillary adapted to orthognathic surgery. Its relevance in condylar repositioning after mandibular sagittal split osteotomy has previously been described. [START_REF] Bettega | Computer-assisted orthognathic surgery: clinical evaluation of a mandibular condyle repositioning system[END_REF][START_REF] Bettega | Computer assisted orthognathic surgery: Condyle repositioning[END_REF] This device can also be used for maxillary navigation after a Le Fort 1 osteotomy. The system's accuracy is in the range of one mm and one o .

Our aim was not to illustrate the inaccuracy of maxillary positioning by conventional articulator model surgery, but to quantify it in three dimensions with the navigation system, and to find out the direction in which this inaccuracy was the most important.

Material and methods

This descriptive, single-centre, observational study involved 30 patients from February 2016 -June 2017 in the Centre Hospitalier d'Annecy Genevois. It was approved by the French Sud-Est IV ethics committee on 26 January 2016 (NCT03357211).

The inclusion criteria were: patients who needed a Le Fort I osteotomy, alone or associated to a mandibular osteotomy, whatever the indication; patients more than 16 years of age; and patients who consented to the collection of personal data for this study (patient or holder of parental authority for patients less than 18 of age). The exclusion criteria were: pregnancy; patients without social security cover; dependent adults; and patients who needed a maxillary disjunction.

The surgical indication was confirmed after clinical examination, sagittal and frontal cephalometry, and a plaster cast study. The operation was simulated with tracing paper on the frontal and sagittal cephalometry.

Manufacturing of the occlusal splints

Plaster casts were mounted on a semi-adjustable articulator (Arcon, MESTRA) by a facebow transfer (Elite facebow) and the diagnostic bite splint. Maxillary repositioning splints were manufactured in acrylic resin (Dentsply, Sirona) according to the planning data. For the occlusal splints, the planning data were characterised by the dental movements from the cephalometric planning.

Planning navigation

For the navigation, movements and references were also defined in the porion-nasion plane from the planning of the cephalometry. The movements were characterised by the translations (mm) and by the rotations (o) of the interincisal point in this plane.

The planning data were manually recorded in the specific Orthopilot ® system software.

At the beginning of the operation, the navigation perspective (left and right porion point and interincisal point), was defined by a calibrated pointer, with a cranial rigid body (fixed on the left superior orbital ridge) as reference. These three points were used to build the direct orthonormal reference required for tracking. The preoperative position of the maxilla was recorded with the occlusal splint holding a rigid body before any bony procedure was begun.

Surgical procedure

We used a standard technique for maxillary osteotomy. [START_REF] Bell | Le Forte I osteotomy for correction of maxillary deformities[END_REF] The maxilla was positioned with the planned occlusal splint and stabilised by maxillomandibular fixation. The osteosynthesis was made with four, L-shaped, titanium miniplates. The final position of the maxilla was then recorded by the navigation system with the initial splint and its rigid body (Fig. 1). If the position was judged unacceptable by the surgeon the osteosynthesis was removed and the maxilla repositioned manually, but the values for position used remained those recorded initially. In case of a bimaxillary procedure, the maxillary osteotomy was done first, followed by mandibular osteotomy, without any consequences for the study.

Statistical analysis

The data collected included: sex, age, Angle's classification, type of dysmorphia (posterior vertical excess, anterior vertical excess, asymmetry), planned movements recorded for the navigation (left/right translation (Tlr), top/bottom translation (Ttb), back/front translation (Tbf), frontal rotation (Rf), axial rotation (Ra), sagittal rotation (Rs), inter-incisal vector). The navigational data were automatically recorded during the procedure in a dedicated file and collected at the end of operation on the Orthopilot ® PC. Discrepancies were calculated from the difference between the planned and measured navigational data.

The norm of the interincisal vector (vector II) was calculated as follow:

The accuracy of positioning was classified in nine distinct classes with three major The secondary outcome was the vector II norm (vector between planned interincisal point and the inter-incisal point used). Conformity was defined by a value strictly < 3 mm, corresponding to the three translation conformity limits (

)).

The qualitative variables were expressed in count and percentage. The quantitative variables were expressed by their mean (SD), or median (range). The significance of the differences in qualitative variables were analysed by a chi-squared or Fisher's exact test. Probabilities of less than 0.05 were accepted as significant. The concordance between measures planned and measures done was assessed with a Bland Altman analysis. [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF] The limits of agreement were calculated by the mean (SD) differences (1.96). This analysis was completed by an enhanced Bland Altman analysis, including a linear regression model and a complementary graphical approach. This enhanced Bland Altman method substitutes a needle plot for the scatter plot and creates a plot between the difference of the readings made by the two methods (diff) or the bias (Y-axis) compared with the actual or standard measurement in the X-axis (mean of the two measurements is substituted by the accurate or standard measurements in the enhanced plot). We add the regression line, its 95% CI band and an additional zero bias reference line to the enhanced plot (see supplemental data for further explanations).

We used SAS software (SAS Institute Inc) for the statistical analyses.

Results

We operated on 30 patients (10 male and 20 female). Their mean (SD) age was 27 (13) and median (range) age 20 (16-66). There were two patients in Angle class I, 10 in class II, and 18 in class III. Twenty-one patients presented with an anterior vertical excess (AVE), two with a posterior vertical excess, and 13 patients with asymmetry of the interincisal middle.

The failure, non-conformity, and conformity classifications are listed in Fig. 3.

The conformity rate was 3/30, 95%CI 2% to 27%, and the failure rate was 22/30, 95%CI 54% to 88%. There were 10 cases of rotation failure: 7 were in failure for Rs The conformity rate according to the vector II norm was 13/30, 95% CI 25% to 63%. There was no significant difference for the primary endpoint (Fisher's exact test), when crossing the failure variable with sex (p= 0.68), Angle's classification (p= 0.99), AVE (p= 0.66), PVE (p=0.47), or asymmetry (p= 0.70). As far as the secondary endpoint was concerned, the results did not differ significantly (Fisher's exact test except for asymmetry) when crossing the failure with sex (p= 0.70), Angle's classification (p= 0.62), AVE (p = 0.44), PVE (p= 0.18), and asymmetry (chi-squared, 1 df; p=0.72).

No statistical test was used for the conformity group because it was too small (n = 3).

Three osteosyntheses were redone because the maxilla was in an unacceptable position, essentially because of a deviation of the inter-incisive point.

The distribution of the discrepancy measurements (norm of the interincisal vector, measurement of rotation, and differential translation) is listed in Fig. 4. The dispersion of the values was greater in the sagittal plane, particularly for sagittal rotations (Rs) and back-front translation (Tbf).

The Bland Altman graphic analysis (Fig. 5) showed no concordance between the planned Vector II measures and those used. The mean of differences for this vector was 3.22. However, the aspect of the graph suggested proportionality between the mean and the observed differences. We then looked for a linear relation between the planned vector II and the discrepancy measurement with the enhanced Bland Altman method.

This model gave significant results (p = 0.003) with an estimated slope of 0.42 -that is, the discrepancy increased by 0.42 every time the vector II increased by 1 mm.

Figure 6 shows the projection of the points as well as the regression line, and its 95% CI.

For the measurements Ra, Rs, Rf, Ttb, Tbf, and Trl, the limits of agreement (d (SD 1.96)) were outside the failure range. There was no concordance between measurements made and planned for these variables.

Discussion

The positioning of the maxilla was in conformity with operative planning in 3/30 (95%CI 2% to 27%) of our Le Fort I osteotomies and in failure in 22/30 (95 %CI 54% to 88%). To our knowledge, this is the first time that the accuracy of maxillary positioning by a manufactured occlusal splint has been evaluated with a navigation system, making a comparison with previous studies difficult. Although a small bias of our study was the accuracy of the navigation system (in the range of 1 mm and 1 o ), it had the advantage of giving an objective and direct evaluation compared with 2dimensional cephalometric analysis, which are also not reliable. [START_REF] Miloro | Is there consistency in cephalometric landmark identification amongst oral and maxillofacial surgeons?[END_REF] The other published studies were based on only a sagittal cephalometric analysis, [START_REF] Ong | Surgical accuracy in Le Fort I maxillary osteotomies[END_REF][START_REF] Semaan | Accuracy of a LeFort I maxillary osteotomy[END_REF][START_REF] Sharifi | How accurate is model planning for orthognathic surgery?[END_REF][START_REF] Choi | Surgical accuracy of maxillary repositioning according to type of surgical movement in two-jaw surgery[END_REF][START_REF] Jacobson | The predictability of maxillary repositioning in LeFort I orthognathic surgery[END_REF][START_REF] Abela | Precision of Le Fort I osteotomy: planning versus outcome. A multi-centre retrospective study[END_REF][START_REF] Kretschmer | Accuracy of maxillary positioning in bimaxillary surgery[END_REF] sometimes associated with frontal cephalometry. [START_REF] Kwon | Accuracy of maxillary repositioning in twojaw surgery with conventional articulator model surgery versus virtual model surgery[END_REF][START_REF] Kwon | Reproducibility of maxillary positioning in Le Fort I osteotomy: a 3-dimensional evaluation[END_REF] A more recent study was based on a 3-dimensional analysis. [START_REF] Dreiseidler | Systematic three-dimensional analysis of wafer-based maxillary repositioning procedures in orthognathic surgery[END_REF] In all these studies the accuracy was assessed by the absolute mean of difference [START_REF] Semaan | Accuracy of a LeFort I maxillary osteotomy[END_REF][START_REF] Jacobson | The predictability of maxillary repositioning in LeFort I orthognathic surgery[END_REF][START_REF] Abela | Precision of Le Fort I osteotomy: planning versus outcome. A multi-centre retrospective study[END_REF][START_REF] Govaerts | Accuracy of Le Fort I osteotomy in bimaxillary splint-based orthognathic surgery: focus on posterior maxillary movements[END_REF] and even mean of the difference [START_REF] Sharifi | How accurate is model planning for orthognathic surgery?[END_REF][START_REF] Choi | Surgical accuracy of maxillary repositioning according to type of surgical movement in two-jaw surgery[END_REF][START_REF] Kretschmer | Accuracy of maxillary positioning in bimaxillary surgery[END_REF] between planning and operation. This assessment was biased because even if the dispersion of the values was important, the mean AQ: please do not use the word "average" because it can apply to mean, median, or mode remained within the chosen range.

In our study, the failure range (2 mm for translations and 4 ° for rotations) was defined by a 3-dimensional extension of the criteria used by Padwa et al [START_REF] Padwa | Occlusal cant in the frontal plane as a reflection of facial asymmetry[END_REF] for the rotation and Proffit et al [START_REF] Proffit | The hierarchy of stability and predictability in orthognathic surgery with rigid fixation: an update and extension[END_REF] for the translation. According to these studies, discrepancies superior to 2 mm in translation and to 4° in rotation were clinically relevant. These criteria were commonly used in previous publications. [START_REF] Schouman | Accuracy evaluation of CAD/CAM generated splints in orthognathic surgery: a cadaveric study[END_REF] We made the choice to lower limits of conformity to approach an optimal level of accuracy. Those limits could be relevant to compare the present results with accuracy obtained with new technology (such as CAD CAM manufactured splints, or patient-specific implants).

The limits in the frontal plane (Trl and Rf) were more stringent, because discrepancies in this plane are clinically more evident. Our goal of 1° and 1 mm in Trl and Rf was certainly too ambitious, because those limits were in the range of the accuracy of the navigation system and it could be a source of error. The present results, according to our practical experience, have emphasised the challenge to achieve this accuracy. This could be explained by the dependence of the maxilla on the mandible during osteosynthesis or, if not, the difficulty in stabilising the maxilla.

The failure rate was particularly high (95% CI 54% to 88%) in our study because we considered the six variables of the three dimensions individually. The failure rate according to vector II was 17/30, 95% CI 37% to 75%. This rate was lower than that obtained with the primary outcome because it took into account only the translation measures. We did not find any correlation between patients' characteristics and failure.

This could be because the sample was so small.

The analysis of the distribution of the discrepancies showed that the dispersion of the variables was more important in the sagittal plane, particularly for sagittal rotation (Rs) and for back front translation (Tbf). This inaccuracy of Rs is probably the result of the lack of control of rotations when preparing the model. [START_REF] Olszewski | Limitations of orthognathic model surgery: theoretical and practical implications[END_REF] In addition, the sagittal inclination of the maxilla can be altered by bony interference or by a failure in positioning or maintenance, or both, of the maxillomandibular complex during osteosynthesis (lack of control in condylar positioning). The sagittal view is also the least visible during operation and so is more difficult for the surgeon to control intraoperatively. Our distribution of translational discrepancies in the sagittal plane was similar to those previously reported. [START_REF] Semaan | Accuracy of a LeFort I maxillary osteotomy[END_REF][START_REF] Jacobson | The predictability of maxillary repositioning in LeFort I orthognathic surgery[END_REF] Dreiseidler et al [START_REF] Dreiseidler | Systematic three-dimensional analysis of wafer-based maxillary repositioning procedures in orthognathic surgery[END_REF] also reported less accuracy in the sagittal plane. The inaccuracy of Tbf can be explained by the lack of reference for the horizontal positioning of the maxilla, making it mainly dependent on the occlusal splint and the stabilisation of the maxillomandibular complex. The absence of control of the position of the condyles during osteosynthesis is another great source of error.

An enhanced Bland Altman graphic analysis showed that the norm of vector II was proportional to that of the differential vector. The linear regression model yielded significant results (p = 0.003) meaning that the larger the value of the planned vector, the greater the risk of discrepancy. In other words, the greater the planned maxillary advancement, the greater the risk of malposition. This observation was reported in one previous study. [START_REF] Dreiseidler | Distance-dependent accuracy in Le Fort I maxillary repositioning procedures[END_REF] One of the explanations given was the increase in the retraction forces of soft tissues during larger displacements.

Conclusion

The Orthopilot ® navigation system confirmed that maxillary positioning with traditional occlusal splints during Le Fort I osteotomy was not very accurate, particularly in the

  criteria (conformity, non-conformity, failure): class 1=conformity in translation and rotation; class 2=conformity in translation only, but with rotation values below the failure range; class 3=conformity in translation only, with rotation values over the failure range; class 4=conformity in rotation only, with non-conformity of translation values but below failure range; class 5=non-conformity of rotation and translation values below failure range; class 6=non-conformity of translation values and rotation values over failure range; class 7=conformity in rotation only and translation values over failure range; class 8=non-conformity of rotation values and translation values over failure range; and class 9=failure in translation and rotation. The conformity, non-conformity, and failure limits are listed in Fig. 2.

(

  sagittal rotation) only, 2 for Ra (axial rotation) only, and 1 for both Rs and Ra. Rf was never in failure. There were 20 cases of translation failure: 4 were in failure for Ttb (vertical translation), 5 for Tbf (sagittal translation), 2 for Trl (transversal translation), 4 for Ttb and Tbf, 4 for Trl and Tbf, and 1 for Trl and Ttb.

Legends to Figures:Figure 1 .

 1 Figure 1. Recording of the maxillary position.

Figure 2 .

 2 Figure 2. Conformity, non-conformity, and failure limits of the primary outcome.

Figure 3 .

 3 Figure 3. Failure, conformity, and non-conformity according to the type of

Figure 4 .

 4 Figure 4. Distribution of measurements of discrepancy.

Figure 5 .

 5 Figure 5. Bland Altman analysis for the vector II norm.

Figure 6 .Fig. 1 .

 61 Figure 6. Enhanced Bland Altman analysis for the vector II norm.

Acknowledgements

We thank Pierre-Emmanuel Colle for providing help with the language.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

This study was the subject of a presentation at the 53th congress of the « Société française de stomatologie, chirurgie

Conflict of interest

We have no conflict of interest.

Ethics statement/confirmation of patient's permission

This descriptive, single-centre, observational study was approved by the French Sud-Est IV ethics committee on 26 January 2016 (NCT03357211). We have obtained the patient's permission to publish his picture.

QUERIES FOR AUTHOR:

Throughout: the journal is published in British English, so any American phraseology or spelling has been corrected. Throughout: the journal does not report simple percentages if n is < 100 (see item 5 on your letter of provisional acceptance). Page 9: please do not use the word "average" because it can apply to mean, median, or mode. Supplemental data, online only: page 7.


Flavia Carton

Yves Chevalier

Letizia Nicoletti

Malgorzata Tarnowska

Barbara Stella

Silvia Arpicco

Manuela Malatesta

Lars Petter Jordheim

Stephanie Briancon

Giovanna Lollo
email: giovanna.lollo@univ-lyon1.fr

Małgorzata Tarnowska

Stéphanie Briançon

Rationally designed hyaluronic acid-based nano-complexes for pentamidine delivery

Keywords: hyaluronic acid, polyarginine, pentamidine, biomaterials, drug delivery systems;

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Delivery of hydrophilic drugs is a challenge owing to their difficult crossing through cells walls that limits their therapeutic activity. Encapsulation is often required in order to enhance drug bioavailability. In case of ionic drugs, complexation with polyelectrolytes of opposite charge is a suitable methodology as it has been extensively studied for transfection of DNA and siRNA [START_REF] Marras | Polyelectrolyte Complexation of Oligonucleotides by Charged Hydrophobic -Neutral Hydrophilic Block Polymers[END_REF]2 .

Upon formation of a charge-stoichiometric complex, the neutral species may undergo precipitation as nanoparticles, resulting in drug encapsulation inside a nanocarrier. Electrostatic interaction is one of the driving forces for such complexation. However, electrostatic binding often occurs by an ion exchange process such that the overall charge balance is null. Non-electrostatic interactions also matter, so that each system deserves a specific investigation aiming at better mastery over the encapsulation process.

Over the last decades, the development of pharmaceutical nanocarriers based on natural polysaccharides aroused a growing interest in drug delivery technologies based on ionic complexation [START_REF] Joshi | Role of biodegradable polymers in drug delivery[END_REF] . Natural polysaccharides have interesting properties as biocompatibility, biodegradability and low toxicity suitable for biomedical applications [START_REF] Goycoolea | Chitosan-alginate blended nanoparticles as carriers for the transmucosal delivery of macromolecules[END_REF][START_REF] Pistone | Formulation of polysaccharidebased nanoparticles for local administration into the oral cavity[END_REF][START_REF] Weber | Polysaccharidebased vaccine delivery systems: Macromolecular assembly, interactions with antigen presenting cells, and in vivo immunomonitoring[END_REF] . Besides, they bear a large variety of reactive groups, a wide range of molar masses (MW), varying chemical compositions and origins, which make them a versatile biomaterial for the preparation of nanometric carriers [START_REF] Liu | Polysaccharides-based nanoparticles as drug delivery systems[END_REF] .

Among the different natural polysaccharides, hyaluronic acid (HA) has been widely used in the pharmaceutical field because of its interesting physicochemical and biological properties [START_REF] Morra | Engineering of biomaterials surfaces by hyaluronan[END_REF][START_REF] Dosio | Hyaluronic acid for anticancer drug and nucleic acid delivery[END_REF] . HA belongs to the class of anionic glycosaminoglycans (GAGs) formed by several identical subunits (D-glucuronic acid and N-acetyl-D-glucosamine disaccharide) bound together by β-1,4 and β-1,3 glycosidic bonds [START_REF] Chen | Hyaluronic acid-based drug conjugates: State-of-the-art and perspectives[END_REF][START_REF] Cadete | Targeting cancer with hyaluronic acid-based nanocarriers: recent advances and translational perspectives[END_REF] . HA is an important component of the extracellular matrix (ECM) highly distributed throughout connective, epithelial, and neural tissues in which plays essential physiological roles [START_REF] Morra | Engineering of biomaterials surfaces by hyaluronan[END_REF] . HA backbone itself is endowed with targeting moieties that specifically recognize and interact with different cell surface proteins like stabilin-2, RHAMM, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and CD44. Also, low molar mass HA regulates Toll Like Receptor (TRL). Among all these receptors, CD44 represents the most studied HAreceptor in inflammation and tumor pathologies [START_REF] Dosio | Hyaluronic acid for anticancer drug and nucleic acid delivery[END_REF][START_REF] Lee | Hyaluronic acid nanoparticles for active targeting atherosclerosis[END_REF][START_REF] Fallacara | Hyaluronic Acid in the Third Millennium[END_REF] . Therefore, specific targeting of these receptors has been exploited as an effective strategy for increasing accumulation of associated drug at the target site [START_REF] Battistini | Hyaluronan as drug carrier. The in vitro efficacy and selectivity of Hyaluronan-Doxorubicin complexes to affect the viability of overexpressing CD44 receptor cells[END_REF][START_REF] Zhong | Hyaluronic acidshelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44overexpressing human breast tumor xenografts in vivo[END_REF] . In addition, HA bears a carboxylic group in each glucuronic unit (pKa 3-4) which provides an acidic polyelectrolyte character enabling interactions with cationic polymers or molecules possessing appropriate basic groups for formation of complexes [START_REF] Battistini | Equilibrium and release properties of hyaluronic acid-drug complexes[END_REF] .

So far, exploitation of HA properties to polymeric conjugates, liposomes, microparticles, and nanoparticles have been attempted. Such dosage forms are generally obtained through formation of self-assembling micelles, chemical conjugation via cross-linking approaches or ionic gelation [START_REF] Battistini | Equilibrium and release properties of hyaluronic acid-drug complexes[END_REF][START_REF] Thomas | Paclitaxel loaded hyaluronic acid nanoparticles for targeted cancer therapy: In vitro and in vivo analysis[END_REF][START_REF] Choi | Selfassembled hyaluronic acid nanoparticles for active tumor targeting[END_REF][START_REF] Lin | Temporal and subcellular distributions of Cy5.5-labeled hyaluronic acid nanoparticles in mouse organs during 28 days as a drug carrier[END_REF] . Among these techniques, polyelectrolyte complexation represents the most widely used method to tailor HA-based drug delivery systems, because of the mild processing conditions, absence of organic solvents and wide range of cationic polymers undergoing interactions with HA [START_REF] Oyarzun-Ampuero | A new drug nanocarrier consisting of polyarginine and hyaluronic acid[END_REF] . Several previous works disclosed instances showing the ability of HA-based polyelectrolyte nanostructures to associate active compounds such as genetic materials or positively charged drugs [START_REF] Dosio | Hyaluronic acid for anticancer drug and nucleic acid delivery[END_REF] . Contreras-Ruiz et al. described nanoparticles made of HA complexed with chitosan (CS) oligomers for pDNA delivery. HA-CS complexes entered cells and yielded significant transfection of pDNA into the corneal and conjunctival cells [START_REF] Contreras-Ruiz | Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells[END_REF] . Recently, a novel ionic metal complex based on HA and an oxaliplatin derivative, dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt), has been patented. Compared to the DACHPt aqueous solution injected intravenously, DACHPt-loaded nano-complexes protected the associated drug from enzymatic degradation, thereby enhancing plasmatic concentration while lowering drug elimination rate [START_REF] Lollo | Drug delivery system[END_REF] . Also, Battistini et al. reported a doxorubicin-HA ionic complex as a tumor targeting drug delivery system. In vitro studies on tumor cells overexpressing CD44 receptors demonstrated the improved internalization of the complexes in comparison to the free drug [START_REF] Battistini | Hyaluronan as drug carrier. The in vitro efficacy and selectivity of Hyaluronan-Doxorubicin complexes to affect the viability of overexpressing CD44 receptor cells[END_REF] .

Based on this background information, the present work deals with an extensive physicochemical characterization of polyelectrolyte complexes (PECs) made of HA and a cationic molecule, pentamidine isethionate (PTM) as a model drug. PTM is well known for its antiprotozoal, antifungal and anticancer activity [START_REF] Jung | Pentamidine reduces expression of hypoxia-inducible factor-1α in DU145 and MDA-MB-231 cancer cells[END_REF][START_REF] Arias | Nanobody conjugated PLGA nanoparticles for active targeting of African Trypanosomiasis[END_REF] . It is a water-soluble molecule with two terminal amidine groups, protonated in a wide pH range (pKa = 12-13) including physiological/neutral conditions. To stabilize PTM-HA PECs and maximize the amount of associated PTM, polyarginine (PArg) was used to crosslink HA and form nanoparticles (NPs). PArg is a biocompatible cationic poly(aminoacid) belonging to the cell-penetrating peptide polymers able to improve intracellular delivery of therapeutic agents [START_REF] Lollo | Polyarginine nanocapsules as a potential oral peptide delivery carrier[END_REF] . At neutral pH, the amine groups of PArg are protonated and interact with carboxylic moieties of HA, leading to the formation of NPs. Also, polyion complexes made of HA and protamine, have been described and developed for the encapsulation of different active compounds [START_REF] Wang | Degradable hyaluronic acid/protamine sulfate interpolyelectrolyte complexes as miRNA-delivery nanocapsules for triple-negative breast cancer therapy[END_REF][START_REF] González-Aramundiz | Protamine-based nanoparticles as new antigen delivery systems[END_REF] . In the current work, the formation of PECs made of HA and PTM and NPs made of HA and PArg were investigated by studying size and zeta potential of NPs and quantifying the amount of isethionate, the PTM counterion, released during the formation process while varying the relative concentrations of the PECs-forming components. Lyophilization studies were also carried out in order to ensure preservation of pentamidine-loaded nanoparticles (PTM-loaded NPs) as a dry form upon long-term storage. The morphology of the formed particles was analyzed by transmission electron microscopy (TEM) and cryogenic-transmission electron microscopy (Cryo-TEM). Finally, in vitro studies were performed on lung and breast human cell lines (A549 and MDA-MB 231) to assess the anticancer activity of encapsulated PTM.

Experimental section

Materials

Sodium hyaluronate (HA) (weight-average molar mass, Mw = 3.9×10 4 g•mol -1 ) was purchased from LifeCore ® Biomedical (Chaska, Minnesota, USA). Poly (L-arginine hydrochloride) (PArg) (weightaverage molar mass, Mw = 5.8×10 3 g•mol -1 ) was purchased from Alamanda ® Polymers (Huntsville, Alabama, USA) and pentamidine isethionate (PTM) (molar mass, Mmol = 592.679 g•mol -1 ) from Sigma-Aldrich ® (St Quentin-Fallavier, France). Milli-Q water was obtained using a milli-Q Academic System (Merck Millipore ® , St Quentin-Fallavier, France). Sodium silicotungstate used for staining in TEM was supplied from Agar Scientific (Parsonage Lane, Stansted, UK). Dulbecco's Modified Eagle Medium (DMEM) without glucose, glutamine, phenol red and sodium pyruvate was bought from Gibco ® (Thermo Fisher Scientific ® , Waltham, Massachusetts, USA) and used to evaluate the stability in physiological medium. Human lung carcinoma cells (A549) and human breast adenocarcinoma cells (MDA-MB-231) were from ATCC and grown in complete Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific ® ), 100 U/mL penicillin (Thermo Fisher Scientific ® ), 100 mg•mL -1 streptomycin (Thermo Fisher Scientific ® ) and 1% L-glutamine (Life Technologies ® ). 3-(4,5-dimethylthiazol-2-yl)-2,5-solution of concentrations ranging from 0.83 to 3.3 mg•mL -1 were added. The mole ratio between HA (negatively charged) and PArg plus PTM (both positively charged) was ranging between 1.13 and 4.51. HA-PArg NPs and PTM-loaded NPs were characterized in terms of pH, size, polydispersity index, zeta potential and encapsulation efficiency. The main characteristics of all the compounds used to obtain PECs and NPs are listed in Table 1. was determined using the cumulants method that provides the z-average diameter and the polydispersity index (PdI). The zeta potential (ζ) was determined from the electrophoretic mobility (uE) (Equation 1). For all measurements, samples were diluted in milli-Q water and placed in a Ushaped fold capillary cell (DTS1070) made of polystyrene and containing two electrodes of goldplated beryllium/copper (Malvern). ζ potential was calculated according to the Henry Equation 28under the Smoluchowski approximation of the Henry factor f(κa):

= ( 1
)
where η is the viscosity of water (η = 0.887 mPa•s at 25°C), κ is the inverse Debye length, a is the radius of particles, and f(κa) = 3/2.

Turbidity of PECs was monitored by absorbance measurements at 600 nm using an UV spectrometer (UV-1280 from Shimadzu, Marne-la-Vallée, France) equipped with a cuvette with an optical path of 10 mm. The turbidity (τ) was calculated from the absorbance (Abs) reading as τ = ln (10) Abs = 2.3 Abs.

Ion-exchange chromatography

Isethionate ions released due to association of positively charged PTM to negatively charged HA were quantified using ion exchange chromatography (IC) (930 Compact IC Flex, Metrohm, Switzerland) equipped with a chemical suppressor and conductivity detection. PTM-loaded NPs were centrifuged (7,000 g, 30 min, 25 °C) using Amicon ® filter (Amicon Ultra-0.5, 30,000 NMWL, Millipore, Darmstadt, Germany), supernatants were recovered and injected into the IC. The analyses were conducted using Metrosep a Supp 5 250/4.0 column with an adequate pre-column at a temperature of 30°C. For the detection of anions, the mobile phase was 8 mmol•L -1 solution of Na2CO3 (Fischer Scientific, Illkirch, France) prepared in ultrapure water (resistivity > 18 МΩ•cm), filtered at 0.45 µm and degassed in an ultrasonic bath prior to use. The flow rate was 0.7 mL•min -1 .

Such conditions ensured linearity of calibration curve in a range from 0.005 to 5 mmol•L -1 of isethionate concentration.

Morphology of blank and loaded nanoparticles using transmission and cryogenic electron microscopy

Transmission electron microscopy (TEM) was performed with a Philips CM120 microscope at "Centre Technologique des Microstructures" (CTμ) at the University of Lyon 1 (Villeurbanne, France). A small drop of suspension (5 µL) was deposited on a carbon/formvar microscope grid (Delta Microscopies, Saint-Ybars, France), stained with a 1% w/w sodium silicotungstate aqueous solution, and slowly dried in open air. The dry samples were observed by TEM under 120 kV acceleration voltage.

Regarding cryogenic-transmission electron microscopy (Cryo-TEM), diluted samples of HA-PArg NPs and PTM-loaded NPs were dropped onto 300 mesh holey carbon films (Quantifoil R2/1) and quench-frozen in liquid ethane using a cryo-plunge workstation (made at Laboratoire de Physique des Solides-LPS Orsay, France). The specimens were then mounted on a precooled Gatan 626 specimen holder, transferred in the microscope (Phillips CM120) and observed at an accelerating voltage of 120 kV.

Association efficiency and loading capacity of pentamidine into nanoparticles

The association efficiency (AE) of PTM into loaded NPs was determined using an indirect method by measurements of the concentration of free PTM in the aqueous phase. PTM-loaded NPs were centrifuged (7,000 g, 30 min, 25°C) using Amicon ® filter (Amicon Ultra-0.5, 30.000 NMWL, Merck Millipore ® , Burlington, Massachusetts, USA), supernatants were recovered and analyzed for free PTM using UV absorbance at 270 nm. Calibration was performed using PTM solutions at different concentrations from 2.5 µg•mL -1 to 30 µg•mL -1 . From the AE values, the loading capacity (LC) of PTM-loaded NPs was calculated using the following Equations 2 and 3 respectively:

% = ! " × 100 (2) &' % = ( !! ) !! * ( !! ) " * ! × 100 (3)
All measurements were performed in triplicate using PTM aqueous solution as control.

Stability studies of blank and pentamidine-loaded nanoparticles

Colloidal stability of HA-PArg NPs and PTM-loaded NPs was evaluated over 4 weeks under storage at 4 °C. Size, polydispersity index and ζ potential were analyzed every week. Leakage of the drug was also evaluated at the end of the storage period. The stability in DMEM without glucose, glutamine, phenol red and sodium pyruvate was tested to check the effect of protein factors on NPs aggregation.

In vitro release study of pentamidine-loaded nanoparticles

In vitro release behavior of PTM-loaded NPs in simulated physiological conditions (PBS at 37 °C)

was performed using a bicompartmental diffusion device (Franz cells) mounted with a semisynthetic cellulose membrane (6-8 kDa MWCO from Spectra/Por, Spectrum Laboratories, The Netherlands). To ensure sink condition, 2 mL of PTM-loaded NPs prepared using 2.3 and 3.3 mg•mL -1 of HA and 0.50 mg•mL -1 of PTM were placed in the upper part of the cell (donor chamber). The lower part of the cell (receptor chamber) was filled with 10 mL of release media (PBS, pH 7.4 at 37 °C) with an horizontal shaking. At different time points (15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h,48 h, 72 h), 1 mL of each sample was collected and analyzed for PTM using UV absorption (at 270 nm). The amount of drug released over time was calculated from the difference between the initial total amount of the drug present in PTM-loaded NPs and the amount of PTM present in the receptor chamber. The experiments were performed for both NPs in triplicate.

Freeze-drying studies of blank and loaded pentamidine nanoparticles

Once the development of NPs has been completed, HA-PArg NPs and PTM-loaded NPs were freeze-dried using CRYONEXT pilot freeze dryer (Cryotec, Saint-Gély-du-Fesc, France). During the formulation step, the bulking agent mannitol was added to the formulations at different concentration (5%, 10% w/v). The freeze-drying program consisted of an initial freezing at -20°C in a freezer for 12 h. After that, the freeze-dryer was pre-cooled at -20°C and samples were introduced therein. Then, the temperature was decreased to -50°C at a rate of 1°C•min -1 and this temperature was kept for 12 h. The sublimation step was carried out at a temperature between -35°C and 5 °C and a pressure between 0.100 and 0.3000 mbar according to the recipe. Finally, a secondary drying step was carried out at 35 °C and 0.010 mbar. After freeze-drying, HA-PArg NPs and PTM-loaded NPs were resuspended in 1 mL of milli-Q water and left under magnetic stirring for 30 min. Size, polydispersity and association efficiency of nanoparticles were measured before and after resuspension. Also, morphological observations using TEM were performed.

In vitro cell viability studies

Human lung carcinoma cells (A549) and human breast adenocarcinoma cells (MDA-MB-231) were used to evaluate the cell viability. Cells (3000 cells/well) were seeded in 96 well plates in 100 µL media and left to adhere for 24 h. Then, the medium was replaced with a fresh one containing different concentrations (0.01-100 µM) of free PTM, HA-PArg NPs and PTM-loaded NPs. After 72 h of exposure, metabolically active cells were quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay according to the supplier's instructions. Briefly, 20 µL of MTT reagent (5 mg•mL -1 ) was added in each well and the plate was incubated at 37 °C for 2 h.

After that the supernatant was replaced by 100 µL of isopropanol/HCl/H2O (90/1/9 v/v/v) and the optical density was measured at 540 nm for purple intensity and at 690 nm for the subtraction of background using a multiwell-scanning spectrophotometer (Multiskan Ascent, Labsystems SA, Cergy-pontoise, France). Cell viability from the absorbance values was calculated using the following Equation 4:

'+,
IC50 values were determined using the CompuSyn software.

Results and discussion

Solubility of pentamidine at different pH

PTM isethionate is a synthetic amidine derivative, highly soluble in water (> 30 mg•mL -1 ) having a pKa of 12.5. Its solubility was above 30 mg•mL -1 in the pH range from 3 to 10 due to the ionization of amidine groups, while at pH values higher than 12 the solubility was drastically reduced (6.5 mg•mL -1 ) . Solubility also depended on ionic strength as the solubility of PTM in PBS pH 7.4 was around 6.9 mg•mL -1 . This decrease of PTM solubility could be due to an increase of the concentration of inert ions in the medium that causes a salting-out effect [START_REF] Long | Activity coefficients of nonelectrolyte solutes in aqueous salt solutions[END_REF] .

Preparation and physicochemical characterization of pentamidine-hyaluronic acid polyelectrolyte

Polyelectrolyte complexes (PECs) are defined as complexes formed through electrostatic interactions between oppositely charged structures. The formation of PTM-HA PECs was based on the electrostatic interaction between the positively charged drug PTM containing two guanidine groups, and the negatively charged polysaccharide HA which contains one carboxylic group for each repeat unit made of two sugar residues, neutral N-acetyl-D-glucosamine and negatively charged sodium salt of D-glucuronic acid as defined in Table 1.

The stoichiometry of the complex was defined as the PTM/HA mole ratio of PTM molecules to HA repeat units. Different PTM-HA PECs having a mole ratio PTM/HA between 0.2 and 2.4 and the mass ratio from 0.24 to 3.6 (PEC A -PEC G) were obtained (Table 2). HA solution was added to an equal volume of PTM at different concentration under magnetic stirring. Since the concentration of the low molar mass electrolyte strongly affects the formation of the complexes, only the amount of PTM was varied [START_REF] Dautzenberg | Polyelectrolyte complex formation in highly aggregating systems. 1. Effect of salt: Polyelectrolyte complex formation in the presence of NaCl[END_REF] .

All the formulations were prepared at pH values around 6-7 in order to obtain an optimal charge density that generates attractive interactions between the PTM and HA. The concentration regime of HA solutions was devised by taking measurements of overlapping concentrations (C * ) for aqueous solution of HA of various molar masses reported by Dodero et al. [START_REF] Dodero | Characterization of hyaluronic acid by dynamic light scattering and rheological techniques[END_REF] and extrapolating them down to the present low molar mass of HA (20 kDa). This gave C* = 15 mg•mL -1 . The largest present HA concentration of 2.5 mg•mL -1 was much lower than C*,

showing that HA concentrations were in the dilute regime.

DLS measurements were performed in order to study possible interactions between HA and PTM.

Figure 1A shows the experimental time correlation function as a function of time (at a scattering angle of 173°). For all PEC solutions, the correlation function showed a trimodal profile. This distribution was due to the existence of fast and slow relaxation modes in molecular motions.

The correlation function of the scattered amplitude G [START_REF] Marras | Polyelectrolyte Complexation of Oligonucleotides by Charged Hydrophobic -Neutral Hydrophilic Block Polymers[END_REF] (t) was modeled as the sum of three exponential decays as in Equation 5(Figure 2A):

; < 0 = < + 4 = > + + 4 = @ + + 4 = A (5)
where Ai and τi (i = 1, 2, 3) are the relative scattered intensities and relaxation times for the three relaxation modes respectively (A1 + A2 + A3 = 1). The first term in the right hand side of Equation 5is the fastest relaxation corresponding to the fastest motions. It was associated with the lateral diffusion of isolated HA-PTM complexes characterized by a mutual diffusion coefficient D (τ1 -1 = Dq 2 ). The diameter of such species given by the Stokes-Einstein relationship (B = C D E F ) was 12-14 nm (Figure 2B), in agreement with the expected size of a random coil of 3.9.10 4 g.mol -1 HA.

The second term is a slower mode that was ascribed to associations (aggregates) of HA-PTM macromolecules of mean size in the range 300-500 nm (Figure 1B). This interpretation was suggested by observations of such aggregates in dilute solutions of HA reported by Maleki et al. [START_REF] Maleki | Effect of pH on the behavior of hyaluronic acid in dilute and semidilute aqueous solutions[END_REF] , and in more complex systems containing HA [START_REF] Nyström | Dynamic and viscoelastic properties during the thermal gelation process of a nonionic cellulose ether dissolved in water in the presence of ionic surfactants[END_REF][START_REF] Maleki | Dynamical and structural behavior of hydroxyethylcellulose hydrogels obtained by chemical gelation[END_REF] . The nature of the third mode was difficult to figure out. The diameter of particles that would correspond to a translational diffusion was unrealistically large (∼50 µm) because they were not observed by optical microscopy. The origin of this third slowest relaxation might stand from slow internal motions inside HA-PTM aggregates that are not translational diffusion. Stronger binding of PTM cations than Na + coming from the divalent and hydrophobic natures of PTM was more directly detected by measurements of electrophoretic mobility (Figure 3). As an overall outcome, the conformation of the polymeric chain, turbidity and electrophoretic mobility measurements, confirmed the complexation of PTM by HA. Moreover, the electrostatic binding of PTM to HA was weak because the PTM cation was only divalent. Since the association of polyelectrolytes of opposite charges is usually quite strong, a cationic polyelectrolyte (polyarginine, PArg) was added in order to help at precipitation of nanoparticles and improve the encapsulation of PTM. To enhance the formation of NPs, the cationic poly(aminoacid) PArg was used as a cross-linking agent causing the formation of insoluble PECs and thereby allowing for the formation of nanoparticles. HA-PArg NPs were prepared by polyelectrolyte complexation in a similar manner as described by Oyarzun-Ampuero et al. [START_REF] Oyarzun-Ampuero | A new drug nanocarrier consisting of polyarginine and hyaluronic acid[END_REF] Five hundred microliters of a solution containing HA at different concentrations (from 1.25 to 5 mg•mL -1 ) were added to an aqueous solution of PArg (0.27 mg•mL -1 ) under magnetic stirring at room temperature for 30 min. The formation of NPs was ensured by the electrostatic interaction between the positively charged groups of PArg and the negatively charged carboxylate groups of HA. All the different formulations of HA-PArg NPs (Blank A -Blank I) were studied for their size, polydispersity index (PdI) and ζ potential (Table 3).

Preparation and physicochemical characterization of the blank hyaluronic acidpolyarginine nanoparticles

The average size of the resulting blank NPs ranged between 112 and 244 nm with a low polydispersity index (PdI < 0.

Development and physicochemical characterization of pentamidine-loaded hyaluronic acid-polyarginine nanoparticles

PTM-loaded NPs were prepared following the protocol described for blank NPs. Different concentrations of HA (0.83 mg•mL -1 to 3.3 mg•mL -1 ) were added to an aqueous solution containing PArg (0.18 mg•mL -1 ) and PTM of concentration ranging from 0.5 to 0.17 mg•mL -1 (Figure 4). The physicochemical characterizations of NPs obtained using a PTM solution of 0.5 mg•mL -1 concentration (Table 4) showed an average size between 155 and 203 nm with a low PdI (< 0.1) and a negative ζ potential ranging from -24 to -18 mV. Interestingly, the incorporation of the drug into NPs caused a reduction of NPs size compared to blank ones (Table 3). The balance between negatively (HA) and positively (PTM and PArg) charged species was reduced in the case of PTMloaded NPs as compared to the HA-PArg NPs. Therefore, the attractive force that modulates electrostatic interactions within the complexes determined a "condensation phenomenon" responsible of the reduction of the complex particle size [START_REF] Micale | A hyaluronic acid-pentamidine bioconjugate as a macrophage mediated drug targeting delivery system for the treatment of leishmaniasis[END_REF][START_REF] Paul | Physicochemical characteristics of pentamidine-loaded polymethacrylate nanoparticles: Implication in the intracellular drug release in Leishmania major infected mice[END_REF] . The ζ potential decreased from -24 to -26 mV for Loaded A to Loaded F, and then increased to -18 mV for Loaded G. The effect of NPs size reduction has been observed also for other particles suggesting the strong interaction between the drug and the polymeric chains [START_REF] Thomas | Paclitaxel loaded hyaluronic acid nanoparticles for targeted cancer therapy: In vitro and in vivo analysis[END_REF][START_REF] Lollo | Drug delivery system[END_REF] .

Since the formation of NPs, the association of PTM to the polymers causes its encapsulation inside the particles. Association efficiency AE is equivalent to the widely used encapsulation efficiency EE.

The amounts of associated PTM varied from AE= 26% (Loaded A) to as high as AE= 82% (Loaded G) upon increasing the HA/PArg mole ratio (Table 4). The highest encapsulation efficiencies were obtained when the amounts of PTM were low, so that high AE (82% for Loaded G) was correlated with low loading capacity (12% for Loaded G). Taking together all these results, PTM was efficiently associated in HA-PArg NPs at a mole ratio above 2.25 and with a loading capacity ranging from 20 to 10%. This data confirms that the addition of PArg to the system had the advantageous effect of promoting the association of PTM to the systems.

The fraction of associated PTM increased as a function of the concentration of HA (from Loaded A to Loaded G). The highest PTM binding efficiency was obtained for the formulation Loaded F containing 2.3 mg•mL -1 of HA and Loaded G containing 3.3 mg•mL -1 of HA. These results confirmed the ability of NPs to associate a high quantity of PTM by electrostatic interaction. Then, Loaded F and Loaded G were selected for the in vitro studies. Previous papers reported the different nanosystems for PTM encapsulation. Encapsulation of PTM inside liposome yielded an association efficiency below 50% due to the hydrophilic character of PTM [START_REF] Mérian | Development of a liposome formulation for improved biodistribution and tumor accumulation of pentamidine for oncology applications[END_REF] . PTM has also been encapsulated into other types polymeric NPs such as PLGA NPs. As described for the liposome formulation, the loading efficiency of PTM was very low (around 2.9%) [START_REF] Arias | Nanobody conjugated PLGA nanoparticles for active targeting of African Trypanosomiasis[END_REF] .

Micale et al. developed PTM-HA bioconjugates in which drug loading ranged from 20 to 30%.

However, the use of chemical reactions and organic solvents compromised the direct exploitation of the conjugate for drug delivery applications [START_REF] Micale | A hyaluronic acid-pentamidine bioconjugate as a macrophage mediated drug targeting delivery system for the treatment of leishmaniasis[END_REF] .

Quantification of isethionate ions using ion-exchange chromatography

According to electrostatic binding of counterions to polyelectrolytes in solution, the ion concentration in the counterion cloud that surrounds the charged molecules is significantly higher than that in the solution, especially at low ionic strength. Formation of an electrostatic complex of PTM with HA weakens electrostatic binding of isethionate counterions to PTM, so, isethionate counterions are released in bulk solution. The balance is a competitive binding of HA and isethionate to PTM which is shifted towards HA because of its large negative charge (it is a polyelectrolyte) compared to the monovalent isethionate anion. In case of full ion exchange of HA for isethionate, the overall charge of the particles varies as the PTM/HA mole ratio. In the absence of ion exchange (adsorption of the neutral PTM-isethionate ion pair), loading PTM does not change electrostatic phenomena with respect to blank NPs. The balance of isethionate binding to NPs controls the NPs charge, thus the onset of NPs formation. When two opposite charged macro-ions form a complex, as in the case of HA with PTM and PArg, the electrical double layer is perturbed and counterions are released to the bulk solution [START_REF] Van Der Guch | Polyelectrolyte complexes: Bulk phases and colloidal systems[END_REF] . To assess the influence of HA concentration on the formation of PTM-loaded NPs (Loaded B, C, D, E and F), the release of isethionate, the counterion of PTM, was quantified using ion exchange chromatography. The results show that the full amount of isethionate was released upon formation of all NPs irrespective of the amount of HA.

This shows that electrostatic interactions between HA and PTM are predominant. It should be kept in mind that association by strong interactions remains an equilibrium that could be shifted according to changes of external condition (pH, ionic strength).

Morphological analysis of blank and pentamidine-loaded nanoparticles

The morphological analysis of HA-PArg NPs (Blank H and I) and PTM-loaded NPs (Loaded F and G) was carried out using TEM and Cryogenic transmission electron microscopy (CryoTEM). As shown in Figure 5, HA-PArg NPs and PTM-loaded NPs formed monodispersed population of regular rounded-shaped particles as observed by DLS measurements. The addition of the drug did not modify the morphology of the NPs.

Besides, to prepare TEM samples, NPs were stained and dried before the analysis. This preparation step might modify the structure of the NPs. Observation NPs in their native state was achieved with Cryo-TEM. Both measurements showed similar results and confirmed the DLS analysis (Figure 6).

In vitro release study of pentamidine-loaded nanoparticles

The release study was performed for HA-PArg NPs (loaded F and loaded G) upon incubation with PBS at 37°C using vertical diffusion Franz cells. For both formulations a biphasic release profile characterized by an initial burst release in the first 8 h, 6% of release after 15 min increasing up to 50% after 8 h, was observed. Then, a plateau was reached and no further release was detected until 72 h (Figure 7). This behavior has been described also for others hydrophilic polymer-based NPs [START_REF] Chakraborty | Effects of drug solubility on the release kinetics of water soluble and insoluble drugs from HPMC based matrix formulations[END_REF] .

According to the authors, the encapsulation of a hydrophilic drug allows a faster penetration of water in the system causing the swelling of polymeric matrix and the formation of pores. The initial burst release is probably due to a fast erosion of the surface that cause the disaggregation of the polymeric matrix with a fast drug release. When the swelling of the matrix is compensated by erosion process a constant release towards water as receptor medium was observed. Moreover, the release behavior of PTM from NPs was similar to the control PTM solution. This shows that the rate-limiting step of the experiment was the diffusion of PTM through the dialysis membrane.

Stability studies of blank and pentamidine-loaded nanoparticles

The stability of colloidal suspensions of Blank H and I (Figure 8

Freeze-drying studies of blank and pentamidine-loaded nanoparticles

A dry form of blank and loaded NPs selected was prepared by freeze-drying in order to study the long-term stability. The optimal freeze-drying conditions that convert the aqueous suspension into dry powder and allow for reconstitution in physiological medium were set up. Blank H, Blank I, Loaded F and Loaded G were freeze-dried using different amount of mannitol (5%, 10% w/v) as bulking and isotonic agent. The aim was to obtain isotonic values close to physiological condition (280-300 mOsm•L -1 ). Tables 5 and6 show the physiochemical characteristics (size, PdI and osmolarity) of Blank H and Loaded F formulations prepared with different mannitol concentrations, before and after redispersion in water. Also, stability after redispersion was evaluated over 15 days at 4 °C. Blank H and Loaded F, were successfully dispersed in water irrespective of the amount of mannitol used. However, 5% mannitol was needed to obtain isotonic formulation. Following reconstitution in water, the size of HA-PArg NPs and PTM-loaded NPs increased. Moreover, the size continued to increase during the storage period for the both formulations (Table 56). However, the population remained monodispersed (PdI > 0.1) and no leakage of the drug during the time was observed. Figure 9 confirms that both Blank H and Loaded F recovered the initial morphology characteristics upon freeze-drying and reconstitution in water. Both formulations formed monodispersed population showing a regular round shape and no aggregation was detected.

In vitro cell viability studies of pentamidine-loaded nanoparticles

The antiproliferative activity of free and encapsulated PTM into Loaded F was assessed using the MTT assay. Lung (A549) and human breast cancer (MDA-MB-231) cell lines were selected based on their sensitivity to PTM [START_REF] Jung | Pentamidine reduces expression of hypoxia-inducible factor-1α in DU145 and MDA-MB-231 cancer cells[END_REF][START_REF] Lee | The novel combination of chlorpromazine and pentamidine exerts synergistic antiproliferative effects through dual mitotic action[END_REF] . The viability of A549 and MDA-MB-231 was monitored after incubation with different concentrations of free PTM and PTM-loaded NPs for 72 h. Blank H were used as controls to evaluate the biocompatibility of the nanocarrier. As shown in Table 7, the use of Loaded F was associated with a better activity than free PTM both on lung (IC50 = 0.21 ± 0.08 µM vs 1.2 ± 0.8 µM) and breast cancer cells (IC50 = 2.2 ± 1.8 µM vs 4.6 ± 3.7 µM). Blank I showed very low toxicity (IC50 > 40 µM) highlighting the biocompatibility of the nanocarrier. These data confirmed that PTM activity in vitro was improved when the drug was incorporated inside nanoparticles.

As reported in the literature both cell lines express CD44 receptors 41,42, . Hence, we suggest that the present NPs can target cancer cells in vivo through interaction with this receptor.

In line with our study, Battistini et al. [START_REF] Battistini | Equilibrium and release properties of hyaluronic acid-drug complexes[END_REF] observed that in A549 cells, cytotoxicity of HA-doxorubicin complexes was 3-fold higher than that of the reference free drug. These results indicated an increased cellular uptake of doxorubicin when complexed with HA due to the presence of CD44 membrane receptors [START_REF] Battistini | Hyaluronan as drug carrier. The in vitro efficacy and selectivity of Hyaluronan-Doxorubicin complexes to affect the viability of overexpressing CD44 receptor cells[END_REF] .Targeting CD44 using HA moieties has also been demonstrated in vivo in CD44-positive human breast tumor xenografts mice. HA based micelles loaded with paclitaxel exhibit a remarkably high accumulation and retention in the CD44 receptor-overexpressing tumor following intravenous. injection in comparison to the free drug [START_REF] Zhong | Hyaluronic acidshelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44overexpressing human breast tumor xenografts in vivo[END_REF] .

Conclusions

The present study provides insight into a new HA based nano-complex, namely nanoparticles, for encapsulation of positively charged hydrophilic drug. In a first set of experiments, HA-PTM complexes were studied. Such complexes were highly polydispersed species that remained soluble in water. To stabilize the complex and to maximize the amount of the drug associated to the system, HA-PArg NPs were developed using polyelectrolyte complexation technique. NPs were kinetically stabilized by the excess charge, which prevented their aggregation and ensured high encapsulation efficiency of PTM. Also, high encapsulation efficiency was associated to the release of the isethionate counterion in the solution quantified by IC. Following in vitro studies, PTM-loaded NPs were more effective in reducing cell viability as compared with free drug suggesting enhanced efficacy and cell internalization via CD44 receptor. Moreover, their optimum pharmaceutical properties, namely easy production using mild conditions, stability and possibility to obtain readyto-use dry powders, highlight the potential of HA-PArg nanoparticles as novel drug delivery system for nanomedicine applications.

Figure 1 .

 1 Figure 1. A) Normalized curve of correlation function (g 1 (t)) versus time of PECs C. The curve is fitted with the aid of the Equation5(see text). B) Diameter (nm) of aggregated (□) and isolated (■)

Figure 2 .

 2 Figure 2. Turbidity (λ = 600 nm) of PTM/HA PECs as a function of PTM/HA mole ratio. PEC A

  Electrophoretic mobility (uE) was negative over the whole range of PTM/HA stoichiometry, progressively shifting toward no mobility corresponding to electrical neutrality as increasing the concentration of PTM. Electrophoretic mobility experiments clearly indicated an increase of complexation at high PTM/HA mole ratio.

Figure 3 .

 3 Figure 3. Electrophoretic mobility (uE) of PTM-HA PECs in function of mole ratio PTM/HA (from 0.24 to 2.4). Values are given as mean ± sem (n = 3).

  2). Values of ζ potential ranged from +33 to -22 mV. When the charge ratio HA/PArg was lower than 1.24, NPs had a size around 120 nm with a positive ζ value (+33 mV), indicating that the charge brought about by PArg was larger than that of HA. At mole ratio higher than 1.24, NPs size increased from 166 to 244 nm, and an inversion of ζ potential to -31 mV occurred. The increase in the hydrodynamic size was correlated to the amount of HA used to obtain the NPs. Besides, inversion of surface potential indicated a conformational change which exposes carboxylic groups of HA in excess with respect to cationic groups of PArg towards the external surface of NPs. Globally, the HA/PArg interaction allows the preparation of NPs of 100-250 nm size with a narrow size distribution and reversal of the electrical charge according to the charge ratio of the two polymers.

Figure 4 .

 4 Figure 4. Preparation of PTM-loaded nanoparticles by polyelectrolyte association.

Figure 5 .

 5 Figure 5. TEM images of Blank H (A); Blank I (B); Loaded F (C) and Loaded G (D).

Figure 6 .

 6 Figure 6. Cryo-TEM images of Loaded F with 2.3 mg•mL -1 of HA (A) and Loaded G with 3.3 mg•mL -1 of HA (B).

Figure 7 .

 7 Figure 7. Release profile of PTM from loaded F and loaded G in PBS medium. The diffusion cells were thermoregulated with water at 37°C circulating in a double jacket. (Mean ± Sem.; n = 3).

  -A) and Loaded F and G (Figure8-B) NPs at 4 °C was evaluated over 1 month of storage. Size, polydispersity index and leakage of PTM were evaluated every week. As reported in Figure8-A and B, size and PdI of Blank H and I increased over time for both formulations. However, Blank (H-I) and Loaded NPs (F-G) were more 0.5 mg•mL-1 PTM) Loaded G (0.5 mg•mL-1 PTM) stable cause they were more negative and more stabilized by electrostatic repulsions. No leakage of the drug was observed for both formulations, demonstrating the ability of NPs to associate PTM in an efficient manner. In addition, Blank H and PTM-Loaded F did not aggregate in DMEM (data not shown).

Figure 8 .

 8 Figure 8. Stability studies, size and PdI, upon storage condition for 4 weeks at 4°C in aqueous solution of Blank H (3.5 mg•mL -1 of HA), Blank I (5 mg•mL -1 of HA) (A) and Loaded F

Table 1 .

 1 Chemical characteristics of the compounds used to obtain polyelectrolyte complexes and nanoparticles. The molar masses given for polymers are that of their repeat unit.

	Compound	Molecular Formula				Molecular structure	pKa	Mw (g•mol -1 )
	Pentamidine	C23H36N4O10S2				O					O		12.13	592.7
	isethionate		N H 2	NH	+ 21 1 O H		O	S	O OH		NH 2 NH
	Sodium Hyaluronate	(C14H20NO11Na)n	H	O	HOOC O H	O	H	O O HOH 2 C		O	OH	2.87	3.9•10 4
							OH				O		NH CH 3	n
	Poly(L-arginine	(C6H14N4O2)n		H 3	C	NH	O		n NH H	12.42	5.8•10 3
	hydrochloride)						H N H	N	NH 2	1	1+	HCl

2.5. Physicochemical characterization of polyelectrolyte complexes, blank and pentamidine-loaded nanoparticles

	water. Analyses were carried out at 25 °C with a scattering angle of 173°. Particle size distribution
	Size distribution and surface potential of the prepared particles (PECs and NPs) were analyzed
	using a Malvern Zetasizer ® (model Nano ZS, Malvern Panalytical, Malvern, UK). Particle size
	distributions were determined by Dynamic Light Scattering (DLS) of samples diluted with milli-Q

Table 2 .

 2 Formulation code for PTM-HA PECs. Mole ratio, mass ratio and pH values are given.

	Complexes	PTM/HA Mole ratio	PTM/HA Mass ratio	pH
	PEC A	0.16	0.24	7.17
	PEC B	0.34	0.5	7.07
	PEC C	0.67	1.0	6.78
	PEC D	0.94	1.48	6.76
	PEC E	1.48	2.2	6.79
	PEC F	2.01	3.0	6.65
	PEC G	2.42	3.6	7.17

Table 3 .

 3 Physicochemical characteristics of HA-PArg NPs. PdI: Polydispersity index. Values are given as mean ± sem (n ≥ 3).

		Mass ratio [HA/PArg]	Mole ratio [HA/PArg]	Size (nm)	PdI	Zeta potential (mV)
	Blank A	0.50: 0.27	0.82	112 ± 6	< 0.2	+ 33 ± 5

Table 4 .

 4 Physicochemical

		Mass ratio [HA: PArg: PTM]	Mole ratio [HA/PArg +PTM]	Size (nm)	PdI	Zeta potential (mV)	Loading capacity (%)	AE (%)
	Loaded A	1.25:0.27:0.75	1.13	155 ±3	< 0.1	-24 ±3	33	26 ±3
	Loaded B	1.5:0.27:0.75	1.35	159 ±4	< 0.1	-25 ±2	30	31 ±4
	Loaded C	2:0.27:0.75	1.80	191 ±20	< 0.1	-27 ±2	25	46 ±5
	Loaded D	2.5:0.27:0.75	2.25	157 ±6	< 0.1	-25 ±5	21	61 ±5
	Loaded E	3:0.27:0.75	2.70	172 ±14	< 0.1	-29 ±4	19	65 ±1
	Loaded F	3.5:0.27:0.75	3.15	179 ±4	< 0.1	-26 ±1	17	76 ±1
	Loaded G	5:0.27:0.75	4.51	203 ± 9	< 0.1	-18 ±1	12	82 ±1

properties of PTM-loaded NPs obtained using a PTM solution at 0.5 mg•mL -1 . PdI: polydispersity index; AE (%): association efficiency. Values are given as mean ± sem (n ≥ 3).

Table 5 .

 5 Physicochemical characterization of Blank H before and after freeze-drying and reconstitution. PdI: polydispersity index. Results are expressed as mean values ± sem; n = 3.

		5% mannitol			10% mannitol
	Days	Size (nm)	PdI	Osmol.	Size (nm)	PdI	Osmol.
				(mOsm•L -1 )			(mOsm•L -1 )

Table 7 .

 7 IC50 values of studied compounds on A549 and MDA-MB-231 cells. Results are expressed as mean values ± sem; n = 3.

	Cell line	Formulations	IC50 (µM)
		PTM	1.2 ± 0.8

Acknowledgements

Authors are thankful to Geraldine Agusti, Sébastien Urbaniak from University of Lyon 1, CNRS, LAGEPP UMR 5007, for their kind help with TEM images and lyophilization studies. We would like to acknowledge the contribution of Pierre-Yves Dugas (University of Lyon 1, C2P2 UMR 5265) for cryo-TEM observations at the "Centre Technologique des Microstructures" (CTμ -University of Lyon 1). Emeline Perrial and Zineb Bousfiha from CRCL, are gratefully acknowledged for cell culture.

Funding Sources

This work has been carried out within the research program RESOLVE, financially supported by EuroNanoMed-III (8 th call). Flavia Carton had a fellowship from the Ministry of Education of Italy.

The research leading to these results has received funding from Italian Ministry for University and Research (MIUR)-University of Torino, "Fondi Ricerca Locale (ex-60%)".

Notes

The authors declare no competing financial interests.