in-vivo evaluation of doxorubicin-loaded chitosan-alginate nanoparticles using a melanoma mouse model

Accepted Manuscript Optimization and in-vitro/in-vivo evaluation of doxorubicin-loaded chitosanalginate nanoparticles using a melanoma mouse model Krassimira Yoncheva, Maria Merino, Aslihan Shenol, Nikolay T. Daskalov, Petko St. Petkov, Georgi N. Vayssilov, Maria J. Garrido PII: DOI: Reference:

S0378-5173(18)30899-8 https://doi.org/10.1016/j.ijpharm.2018.11.070 IJP 17966

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

15 August 2018 6 November 2018 26 November 2018

Please cite this article as: K. Yoncheva, M. Merino, A. Shenol, N.T. Daskalov, P.S. Petkov, G.N. Vayssilov, M.J. Garrido, Optimization and in-vitro/in-vivo evaluation of doxorubicin-loaded chitosan-alginate nanoparticles using a melanoma mouse model, International Journal of Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm. 2018.11.070

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Optimization and in-vitro/in-vivo evaluation of doxorubicin-loaded chitosan-alginate nanoparticles using a melanoma mouse model

Krassimira Yonchevaa*, Maria Merinob, Aslihan Shenola, Nikolay T. Daskalovc, Petko St. Petkovc, Georgi N. Vayssilovc, Maria J. Garridob*

a

Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy,

Medical University of Sofia, 1000 Sofia, Bulgaria b Department c Faculty

of Pharmaceutical Technology, University of Navarra, 31008 Pamplona, Spain

of Chemistry and Pharmacy, University of Sofia, 1126 Sofia, Bulgaria

* Corresponding authors Krassimira Yoncheva, PhD Department of Pharmaceutical Technology Medical University of Sofia, Faculty of Pharmacy 2 Dunav Str. 1000 Sofia Bulgaria Tel: +35929236544 Fax: +35929879874 E-mail: [email protected] Maria Garrido, PhD Department of Pharmaceutical Technology University of Navarra, Faculty of Pharmacy 31008 Pamplona Spain Tel: +34948425600 (ext.806529) Fax: +34948296500 E-mail: [email protected] 1

Abstract The present study evaluates the potential of encapsulated doxorubicin to reduce both the viability of melanoma cells and the tumor growth in a mouse melanoma model. The prepared doxorubicin loaded chitosan/alginate nanoparticles possessed mean diameter around 300 nm and negative zeta-potential. Classical molecular dynamic simulations revealed that the high encapsulation efficiency (above 90 %) was mainly due to electrostatic interaction between doxorubicin and sodium alginate, although dipole-dipole and hydrophobic interactions might also contribute. The in vitro dissolution tests showed slower doxorubicin release in slightly alkaline medium (pH=7.4) and faster release in acid one (pH=5.5), indicating that higher concentration of doxorubicin might reach the acidic tumor tissue. The free and the encapsulated doxorubicin decreased the viability of melanoma cell lines (B16-F10 and B16OVA) in a similar degree. However, the cytotoxic effect of the encapsulated doxorubicin still occurred in the more resistant B16-F10 cells even after removing the extracellular drug. The experiments on a syngeneic melanoma mouse model revealed that free and encapsulated doxorubicin elicited the control of the tumor growth (dose of 3 mg/kg). Thus, the encapsulation of doxorubicin into chitosan/alginate nanoparticles could be considered advantageous because of the better intracellular accumulation and longer cytotoxic effect on the investigated melanoma cells.

Keywords: Nanoparticles, Chitosan, Sodium alginate, Doxorubicin, Antitumor activity, Melanoma

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1. Introduction Doxorubicin is an anthracycline antibiotic derived from the actinobacteria Streptomyces peucetius var. caesius. The drug possesses a broad-spectrum of anticancer activity, including breast, ovarian, lung, bladder cancer etc. Some studies have reported that doxorubicin was less effective in patients with metastatic malignant melanoma (Smylie et al., 2007; Vorobiof et al., 2003). In vitro studies in melanoma cells revealed that the crucial role for the low cytotoxic activity of doxorubicin is due to a high cell resistance (Panneerselvam et al., 1987). Generally, the tumor cell resistance is associated with transport proteins that have doxorubicin as a substrate (Cox and Weinman, 2016; Shen at al., 2008). The activity of these proteins is directly related with the reduction of drug accumulation in tumor cells and limitation of its cytotoxic potential. One of them is an efflux transporter P-glycoprotein, although others also were found to contribute to doxorubicin efflux (Pajic et al., 2009). Frank et al. (2005) have reported that a highly expressed ABCB5 transporter acted as a drug-efflux pump for doxorubicin in chemoresistant G3361 malignant melanoma cells. Another study has considered that a member of the ABC family, a mitochondrial transporter ABCB8, participated in mediating of doxorubicin resistance in human melanoma cells (Elliott and AlHajj, 2009). The incorporation of doxorubicin in nanoparticulate systems might overcome the cell resistance. Different approaches have been explored for avoidance of tumor resistance, e.g. simultaneous delivery of doxorubicin and P-gp inhibitor, co-loading of doxorubicin and P-gp siRNA to silence P-gp expression etc. (Frank et al., 2014; Meng et al., 2010; Tang et al., 2016). One of the approaches capable to overcome the tumor cell resistance is to select appropriate carrier, which might control drug delivery and accumulation in the resistant cells (Li et al., 2016). The small size of nanoparticles together with the appropriate properties of the carrier might govern nanoparticle circulation, location into tumor tissue through the

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enhanced permeability and retention (EPR) effect and consequent intracellular delivery and accumulation (Maeda et al., 2001). The loading of doxorubicin in mesoporous silica nanoparticles induced higher accumulation of doxorubicin vs. the free drug in a drug resistant and P-gp over-expressing cancer cells (Shen et al., 2011). The authors have concluded that the higher accumulation and lower IC50 of encapsulated doxorubicin were due to a downregulation of P-gp expression by the mesoporous nanoparticles themselves. Huang et al. (2011) loaded doxorubicin in endosomal pH-sensitive mesoporous silica nanoparticles (MSNHydrazone-Dox) that were transported by endocytosis in human uterine sarcoma MESSA/Dox-resistant tumor cells. This pH-governed release and endocytosis allowed bypassing the efflux pump. The comparison between liposomal and free doxorubicin in resistant colon cancer cells expressing P-gp showed that the liposomal doxorubicin was less transported out of the cells (Riganti et al., 2011). The scientific group reported that the inhibition of P-gp was associated with the fact that the liposomal shell altered the composition of rafts in the cells and decreased the lipid raft-associated amount of Pgp. The achievement of suitable sustained release of doxorubicin also might improve its efficacy (Chavanpatil et al., 2007a; Jin et al., 2017). For example, the encapsulation of doxorubicin in combined surfactant/alginate nanoparticles resulted in sustained release of doxorubicin which further correlated with increased intracellular level and sustained cytotoxicity in breast cancer cells (Chavanpatil et al., 2007a). Chitosan/alginate nanoparticles are attractive platform for doxorubicin loading since both polysaccharides are biocompatible, biodegradable and non-toxic (Baldrick, 2010; Varum et al., 1997). Further, the preparation of such nanoparticles represents the advantage of simple and mild procedure in water. The latter is due to the formation of polyelectrolyte complex between positively charged amino-groups of chitosan and negatively charged carboxyl groups of sodium alginate. Both have different pH-dependent solubility, i.e. sodium alginate is less

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soluble at acidic pH and chitosan is less soluble in slightly alkaline pH, which might sustain the drug release rate. The sustained release could be considered an important prerequisite for the achievement of sustained cytotoxic effect. Chavanpatil et al. (2007b) have reported that nanoparticles based on sodium alginate and surfactant enhanced and sustained cellular delivery of doxorubicin. In addition, the sustained release of doxorubicin could solve the problems with its dose-dependent toxicity. The aim of the present study is to encapsulate doxorubicin in chitosan/alginate nanoparticles by electrostatic binding of the drug and to explore whether this strategy could enhance the therapeutic response in melanoma cells. The study describes the optimization of doxorubicin loading in chitosan/alginate nanoparticles and its in-vitro/in-vivo evaluation using a syngeneic melanoma mouse model. Melanoma cells and model were selected aiming to examine the capacity of the encapsulated doxorubicin in the treatment of this highly aggressive cancer. The first part of the work represents detailed information about the interaction and loading of doxorubicin in the chitosan/alginate nanoparticles explored by molecular dynamic simulations. Further studies evaluate the cytotoxic effect of the encapsulated doxorubicin in-vitro in two melanoma cell lines. Finally, the potential of the encapsulated doxorubicin to inhibit tumor growth was investigated in a syngeneic melanoma mouse model.

2. Materials and Methods 2.1. Materials Doxorubicin hydrochloride and chitosan (Mv 110 000 - 150 000) were provided by Sigma Aldrich. Sodium alginate (very low viscosity) was supplied by Alfa Aesar GmBH & CoKG (Karlsruhe, Germany). Mouse syngeneic melanoma cell lines (B16-F10 and B16OVA) were supplied by Dr. P. Berraondo (Center for Applied Medical Research (CIMA),

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University of Navarra, Spain). B16-OVA cells are derived from B16F10 syngeneic mouse melanoma cell line, transfected with chicken ovalbumin (OVA) protein, which is expressing as surrogate tumor antigen (Ag).

2.2. Preparation of doxorubicin-loaded polysaccharide nanoparticles The nanoparticles were prepared by electrostatic gelation of both biopolymers by adaptation of previously established procedure (Li et al., 2008; Rajaonarivony et al., 1993). Here, doxorubicin was dissolved in 0.250 ml of calcium chloride solution (3.35 mg/ml) and slowly added to aqueous solution of sodium alginate (3 mg/ml, pH=5.3). Both solutions were incubated under agitation (300 rpm) for 60 min. After that, calcium chloride (0.750 ml) was added to the mixture under ultrasound for 1 min (0.04 wats). The resulted mixture was incubated for new 30 min under stirring (300 rpm). After that, the solution of chitosan (0.75 mg/ml, pH=4.5) was added drop by drop under ultrasound for 4 min (0.04 wats). The resulted nanoparticulate dispersion was stirred for 4 h (300 rpm) and centrifuged for 15 min at 20 000 rpm. The resulted pellets were rinsed with purified water, centrifuged as discussed above and dispersed in purified water for further evaluation.

2.3. Characterization of the nanoparticles The nanoparticle morphology was studied by scanning electron microscopy (SEM) (Lyra/Tescan). The dispersions were deposited on SEM stubs by evaporation at room temperature. Nanoparticle diameter, polydispersity and zeta-potential were determined by photon correlation spectroscopy and electrophoretic laser doppler velocimetry using a Zetasizer Nano Series (Malvern Instruments, UK). The aqueous dispersions of the nanoparticles were measured at 25 oC with a scattering angle of 90o.

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Encapsulation of doxorubicin was evaluated by the difference between its initial concentration and the concentration found in the supernatant after centrifugation of freshly prepared dispersion. The amount of the non-encapsulated doxorubicin was measured by spectrophotometry at 480 nm (BioTek PowerWave HT UV/Vis, Belgium). The concentration of doxorubicin was calculated according to a standard curve prepared in the range of 0.1-1 mg/ml (r=0.9991). The loading degree (LD) and encapsulation efficiency (EE) were calculated using the following equations: LD (%) = [(Total amount of drug – Non-encapsulated drug) / Weight of nanoparticles] x 100 EE (%) = [(Total amount of drug – Non-encapsulated drug) / Total amount of drug] x 100

2.4. Computational details for the classical molecular dynamics simulations of the complexation of doxorubicin with sodium alginate Classical molecular dynamics simulations of the complexation of doxorubicin with sodium alginate were run with Gromacs 5.1.2 (Abraham et al., 2015) employing Gromos 53a6 force field, used earlier for simulation of alginates (Tan et al., 2014). The force field parameters were modified with additional Lennard Jones atom types used by the ATB force field (Oostenbrink et al., 2004). Topologies were automatically generated using Automated Force Field Topology Builder (ATB). Simulations were performed with the pressure fixed at 1 atm and temperature at 300 K. Chloride anions were used to neutralize the positive charge of doxorubicin, while sodium cations were used as counterions of the carboxyl groups of the alginate oligomers. For alginate molecules we used fragments containing eight monosaccharide residues. For the long range electrostatic and van der Waals interactions, the particle mesh Ewald (PME) algorithm was used (Essmann et al., 1995). All cut-offs ware set

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to 1 nm. The temperature was controlled through velocity rescaling (Bussi et al. 2007) and Parrinello-Rahman barostat was used to control the pressure (Parrinello et al., 1981). All compounds were solvated in explicit SPC/E (extended simple point charge model) water and periodic boundary conditions were applied (Berendsen et al., 1987). This SPC/E model includes both Coulombic and long-range Lennard-Jones interactions between water molecules. The time step was 1 fs and integration of the equations of motion was done with the leapfrog algorithm as the geometry of each 50th step was recorded. The systems were simulated using the following protocol - energy minimization, equilibration (0.1 ns), and production runs of 10 ns, which were extended for some systems to 100 ns. The visual analysis of the molecular structures and images were made with VMD software (VMD version 1.9.3, 2016. VMD is developed with NIH support by the Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at UrbanaChampaign).

2.5. In vitro release In vitro release studies were performed by dialysis method applying phosphate buffers (pH=5.5 and pH=7.4, Eur. Ph.) as acceptor phases (50 ml). The nanoparticulate dispersion was placed in a membrane (10 000 MWCO), which was immersed in the acceptor phase under gentle stirring and temperature of 37 oC. Samples of 1 ml were withdrawn from the acceptor phase at predetermined intervals and the concentration of the released doxorubicin was defined by spectrophotometry as described above.

2.6. In vitro cytotoxicity studies Two mouse syngeneic melanoma cell lines, B16-F10 and B16-OVA, were used to evaluate the antiproliferative effect of encapsulated doxorubicin. Cells were maintained in a

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mixture of DMEM cell culture medium, 10 % FBS and 1 % penicillin-streptomycin. Cell lines were grown as adherent monolayers in 75 cm2 flasks at 37 ºC in a 5 % CO2 humidified atmosphere. The cell proliferation and morphology were controlled daily until to reach 70-75 % of confluence. The presence of Mycoplasma was tested for each new culture cell. Cells detached with 1ml of trypsin-EDTA were centrifuged for 5 min at 20 oC and counted by microscopy using the vital stain Trypan Blue. Cell density of 1x104 cells/well was seeded in 96-wells microtiter plates and after 24h, different concentrations of free or encapsulated doxorubicin (ranging 1-100 M) were added for 72 h. At selected time points culture medium was removed and Alamar Blue solution was added in a ratio of 1:10 (AB:DMEM, v/v) into wells, according to the manufacture´s specifications. A negative control was also prepared without cells to determine the background signal of nanoparticles. Each plate was incubated in darkness at 37 oC for 2 h and the absorbance was measured at 570 and 600 nm, respectively, using the Power Wave XS micro-plate spectrophotometer (Biotek, Belgium). Viable cell number was calculated according to the equation: Number of cells= (A570 – A600) - (Abackground at 570 – Abackground at 600) where, (A570 – A600) represents the absorbance for treated cells and (Abackground at 570 – Abackground at 600)

for the corresponding control.

2.7. In-vivo study Female C57B6 mice weighing 20-25 g (Harlan, Barcelona, Spain) were housed in plastic cages under standard and sterile conditions (25 °C, 50 % relative humidity, 12 hours dark/light), with water and food at libitum. All experiments were performed according to European animal care regulations and the protocol was approved by Ethical Committee for Animal Research of University of Navarra (ref. 046/14).

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A subcutaneous tumor was induced by the inoculation of 5x105 B16-OVA cells in 100 μl of PBS, in the right flank of the mice. When tumors reached a diammeter of approx. 3-4 mm, mice were randomly divided into different groups: control, free doxorubicin (Dox), empty nanoparticles (NP) and doxorubicin loaded nanoparticles (NP-Dox). Mice were intravenously (i.v.) injected with a dose 3 mg/kg of doxorubicin every 7 days during two cycles. Tumor measurements were performed using an electronic caliper and then, tumor volume was calculated by the following formula: Tumor volume (mm3) = (L* (W)2)/2 where, L represents the length diameter and W is the width.

2.8. Statistical analysis Data were expressed as mean ± Standard deviation (SD), whereas for in-vivo experiment, data corresponded to the average ± SEM. The statistical analysis was performed using a parametric test, ANOVA to compare all treatments followed by the Student’s unpaired t-test. The significance level was set at p<0.05.

3. Results and Discussion The encapsulation of doxorubicin in chitosan/alginate nanoparticles by binding to sodium alginate could sustain the drug release in extracellular fluids and provide efficient cytotoxicity in resistant melanoma cells. Therefore, the main tasks include optimization of the loading of doxorubicin in chitosan/alginate nanoparticles, evaluation of its binding to sodium alginate and examination of the capacity of encapsulated doxorubicin to overcome the resistance of melanoma cells.

3.1. Preparation and characterization of doxorubicin loaded chitosan/alginate nanoparticles The first part of the present study explored the optimal conditions for encapsulation of doxorubicin in chitosan/alginate nanoparticles. Our previous study showed that the variation of the ratio between sodium alginate and chitosan (10:1 or 1:10) resulted in nanoparticles with 10

different diameter and charge (Aluani et al., 2017). In the present study, the encapsulation was performed by pre-gelation method at a ratio between sodium alginate and chitosan 10:1 (wt/wt). This ratio was applied taking into account that the amino-groups of doxorubicin would interact with the carboxyl groups of sodium alginate. In particular, the drug and the alginate are oppositely charged into the aqueous solution of sodium alginate (pH=5.3), which ensures their electrostatic interaction. For this reason, the first stage of the preparation procedure included incubation of doxorubicin and sodium alginate accompanied by pregelation with CaCI2, while the second step consisted of chitosan addition. Five ratios between doxorubicin and sodium alginate were investigated aiming to consider their influence on drug loading. The results revealed that the increase of the initial amount of doxorubicin augmented the loading degree (Fig. 1). As seen, the highest loading was achieved at 1:5 and 1:4 ratios of doxorubicin to sodium alginate. Further ratios were not performed since the loading degree at 1:5 and 1:4 was equal but the encapsulation efficiency slightly decreased at a ratio of 1:4. In general, the high encapsulation efficiency at all investigated ratios suggested a high capacity for doxorubicin loading. As stated above, the high encapsulation efficiency of doxorubicin might be due to electrostatic interactions between the drug and sodium alginate. Thus, the understanding of the binding of doxorubicin to sodium alginate was our next task, knowing that it might influence the release kinetics and the processes at cellular level. Scanning electron microscopy was performed aiming to evaluate the nanoparticle morphology. The microscopy revealed a spherical shape of the nanoparticles and diameter ranging 300-500 nm, which was closed to the size determined by DLS (Fig. 2 and 3). As seen in Fig. 3, the size and polydispersity of the nanoparticles prepared at the different ratios between doxorubicin and sodium alginate were similar. In all cases, the mean diameter was around 300 nm and the polydispersity index approximated 0.2. For comparison, the empty nanoparticles were characterized by a mean diameter of 352 nm and polydispersity index of

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0.227. Azevedo et al. (2014) reported similar decrease of the size and polydispersity after loading of chitosan/alginate nanoparticles with vitamin B2. The authors explained this phenomenon with the interactions between positively charged vitamin B2 and negatively charged sodium alginate during gelation procedure. Regarding zeta-potential, the developed encapsulation procedure in the present study allowed preparation of negatively charged nanoparticles. The zeta-potential of non-loaded nanoparticles was -32 mV, whereas the zetapotential of the drug loaded nanoparticles ranged from -22.5 to -25.0 mV. The difference between loaded and empty nanoparticles was considered as indication for the interaction with doxorubicin. Nevertheless, the retention of the negative value of zeta-potential indicated an absence of free amino-groups of doxorubicin on nanoparticle surface that correlated with the observed high loading and encapsulation efficiency. Thus, the further studies, including in vitro release tests, cytotoxicity and in vivo study in a syngeneic melanoma mouse model were performed with the nanoparticles prepared at a ratio 1:5 (doxorubicin : sodium alginate) because of their optimal loading, encapsulation efficiency and physicochemical properties.

3.2. Classical molecular dynamics simulations of the complexation of doxorubicin with sodium alginate In order to clarify the interactions between the drug and sodium alginate we performed series of classical molecular dynamic simulations using various ratios between the two components in water environment. Initially, the simulations of six molecules doxorubicin in the simulation box resulted in aggregation of five of the drug molecules in a single complex in the first nanosecond of the simulation (Fig. 4). During the simulation of 6.0 ns the molecules change their positions but remain aggregated (Fig. 4, B and C). The aggregation is accomplished by hydrophobic interactions and pi-stacking as in the internal part of the

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aggregate the antraquinone moieties of four drug molecules are aligned in parallel. The more polar oxane fragments surround the internal part and are exposed to the water media. Similar result was obtained after simulation with 12 doxorubicin molecules in the box, where after 10 ns of MD simulation small aggregates of two to four drug molecules were formed. As described above, for the alginates we used fragment containing eight monosaccharide residues. Analyzing various structures obtained during the simulations of the modeled systems we observed two types of complexes. In the initial stages of the simulation an external types of complexes are formed in which one or more doxorubicin molecules are attached to different parts of the alginate (Fig. 5, A and B). After longer simulations times, which depend on the size of the system, an aggregate type of complexes is formed. In these complexes doxorubicin molecules form an aggregate, which is partially covered by the alginate molecules (Fig. 5, C and D). For the systems with four doxorubicin and three alginates the formation of aggregate complex was accomplished after about 6 ns, while for the larger system with 12 doxorubicin and three alginates the complex is formed after about 40 ns. The internal structure of the aggregates of doxorubicin molecules is similar to that in absence of alginates, described above – most of them are aligned in parallel to another doxorubicin molecule to maximize the pi-stacking between them. In order to clarify the contribution of the electrostatic interactions between doxorubicin and alginate molecules we extracted from the simulation data the interatomic distances between the positively charged nitrogen center of doxorubicin and the negatively charged carboxyl oxygen centers of alginates for some of the modeled systems (Fig. 6). In the both systems, shown in Fig. 6, there is a fraction of N-O distances in the range 2.9-3.9 Å, suggesting close location of those charged centers and we may conclude that the electrostatic interaction contributes to the binding of doxorubicin to the alginate. For the model with four doxorubicin molecules the area of this peak (dashed line in the left panel of Fig. 6) suggests

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that between two and three of the drug molecules interact electrostatically with the alginate, while for the system with twelve doxorubicin molecule, in average five doxorubicin molecules interact electrostatically. Taking into account that both the drug molecule and the alginate are relatively large, a single electrostatic interaction is likely not sufficient to keep the complex stable. Thus, one may assume substantial contribution to the binding also from dipole-dipole interactions and hydrophobic interactions between doxorubicin and alginate. The latter is in agreement with previous studies that have reported possible hydrophobic interactions of doxorubicin (Munnier at al., 2007; Tsoneva et al., 2015).

3.3. In vitro release of encapsulated doxorubicin In vitro drug release tests were performed in buffer media having pH-values 5.5 and 7.4, representing the acidic extracellular pH-value of tumor tissue and slightly alkaline pH of several body fluids, respectively. The release profiles in both media were biphasic, in particular initial burst effect and gradual sustained release during the longer second phase (Fig. 7). The burst effect was less pronounced in the slightly alkaline medium, where for 6 hours 35 % doxorubicin was released vs. 52 % in the acid medium. A similar tendency for slower doxorubicin release at pH=7.4 was observed during the second phase of the release process. These results were explained by the stronger interaction between doxorubicin and sodium alginate at pH=7.4 due to ionization either of amino-groups of doxorubicin or carboxyl groups of sodium alginate. Similarly, the release rate of doxorubicin from poly(acrylic acid) - poly (N-isopropylacrylamide) capsules was lower in a medium with pH=7.4 than in pH=1.2, due to electrostatic interaction between the drug and poly(acrylic acid) at the higher pH (Nan et al., 2014). In this way, the lower burst release at pH=7.4 might contribute to a higher amount of the drug capable to reach the slightly acidic tumor tissue. In

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our opinion, the lower burst was achieved due to the appearance of electrostatic, dipole-dipole and hydrophobic interactions between doxorubicin and alginates as molecular dynamic simulations revealed. Nevertheless, from clinical point of view, the slower doxorubicin release at physiological pH (pH=7.4) is considered advantageous since it could reduce the drug loss until it reaches the target tumor tissue.

3.4. In vitro cytotoxicity studies The cytotoxic studies were performed on two mouse syngeneic melanoma cell lines B16-F10 and B16-OVA. The empty nanoparticles did not show cytotoxic effect in both cell lines after 72 h exposure (not shown). At the same treatment time, encapsulated and free doxorubicin demonstrated well pronounced cytotoxic effect at concentrations similar to previously reported concentrations on other cell lines (Fig. 8) (Arif et al., 2013; Zhang et al., 2015). Our results showed slightly stronger cytotoxic effect of free doxorubicin compared to the encapsulated drug (Table 1). This observation could be explained by the sustained doxorubicin release from the nanoparticles (Fig. 7), which was in accordance with previous studies with polymeric nanoparticles (Moreno et al., 2010; Tian et al., 2017). Comparing the results from both cell lines, the cytotoxic effect of doxorubicin was higher for B16-OVAcells. This suggests a lower resistance to this drug in comparison with B16-F10 cells, a very aggressive metastatic cell line (Seliger et al., 2001; Weigelin et al., 2015). In fact, metastatic melanoma is one of the most resistant cancers to single agent chemotherapy and combination chemotherapy (Rodriguez et al., 2013). Since the resistance is a challenge for the therapy, our in vitro experiments continued with B16-F10 cells. In particular, the further study aimed to evaluate the impact elicited by the encapsulated doxorubicin on cell viability after a removal of extracellular drug. Fig. 9 represents the results obtained after 72 h treatment of the cells followed by their resuspension

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in drug-free medium for 24 h. The evaluation after the resuspension of the cells in drug-free medium showed that the cytotoxic effect of free doxorubicin was not changed after resuspension of the cells, demonstrating that the activity stopped with the removal of the extracellular drug. In comparison, the cytotoxic effect on the cells treated with the encapsulated doxorubicin increased. In particular, the cell viability decreased from 40 % to 18 % independent on the removal of the extracellular drug. These data suggested better transport and higher intracellular concentration of encapsulated doxorubicin compared to the nonencapsulated drug. Consequently, the encapsulation of doxorubicin into chitosan/alginate nanoparticles promoted more efficient intracellular transport and achievement of prolonged activity. This result is important taking into account that doxorubicin acts by intercalating into DNA and its intracellular accumulation is essential to anti-tumor efficacy (Tewey et al., 1984).

3.5. In vivo study Doxorubicin is indicated in breast cancer but in the present study the treatment of melanoma was studied aiming to extend the knowledge of its potential. It is known that B16 cells produced large and aggressive tumors in C57B6 mice and it is impossible to eliminate them completely (Zhang et al., 2015). In the present study, the effect of free and encapsulated doxorubicin on tumor size was evaluated in B16-OVA tumor-bearing C57B6 mice. Both B16OVA and B16-F10 are aggressive cell line with a rapid progression but we selected B16OVA due to the higher doxorubicin sensitivity. The animals received two cycles of treatment (every 7 days) and the tumor size was measured during 21 days (Fig. 10). As shown, during 15 days there was no significant difference between the groups treated with free or encapsulated doxorubicin. The tumor size of the control group was similar to the treated groups. However, after 15 days, the tumor size of control group and the group treated with the

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empty nanoparticles increased, whereas it was maintained reduced in the groups treated with the free and encapsulated doxorubicin. This similar effect for both treatments may be due to the effect of protein absorption on nanoparticles, which can not be observed in the in-vitro experiments, but it is often described in animals. Indeed, this effect is higher for large particle (approx. 300 nm) inducing the nanoparticle uptake by macrophages and thereby, a rapid blood clearance (Alexis et al., 2008). But, although with this possible limitation, the encapsulated doxorubicin elicited the control of the growth, being more evidenced after the second dose (days 17 and 21) achieving a statistical difference in comparison with the empty nanoparticles, which behave similar to the control group. This suggests that dose regimen need to be also explored in depth because it plays a relevant role, as well as the particle size. Nevertheless even with the current regimen and using a drug resistant tumor model, doxorubicin-loaded chitosan/alginate nanoparticles provided tumor shrinkage.

4. Conclusions The present study demonstrated that the encapsulation of doxorubicin in chitosan/alginate nanoparticles was due to different interactions (electrostatic, dipole-dipole or eventually hydrophobic) between sodium alginate and doxorubicin, that further ensured sustained release of the drug. Both, the properties of the developed nanoparticles and the sustained release, provided better accumulation and longer cytotoxic effect of encapsulated doxorubicin in melanoma cell lines compared to the free drug. The free and encapsulated doxorubicin elicited the control of the tumor growth in a syngeneic melanoma mouse model and maintained this effect over time. Thus, doxorubicin loaded chitosan/alginate nanoparticles could be considered an appropriate doxorubicin delivery platform and further optimization of in vivo dose schedule might improve their potential against melanoma.

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Figure legends

Fig. 1. Encapsulation efficiency and loading degree of doxorubicin hydrochloride in chitosan/alginate nanoparticles prepared at different ratios between doxorubicin and sodium alginate (1:20, 1:15, 1:10, 1:5 and 1:4, wt/wt).

Fig. 2. Scanning electron microscopy of doxorubicin loaded chitosan/alginate nanoparticles prepared at a ratio between doxorubicin and sodium alginate of 1:5 (wt/wt).

Fig. 3. Mean diameter and polydispersity index of chitosan/alginate nanoparticles prepared at different ratios between doxorubicin and sodium alginate (1:20, 1:15, 1:10, 1:5 and 1:4, wt/wt).

Fig. 4. Snapshots of the simulations of six doxorubicin molecules (shown in different color) in water: initial structure (A), formation of initial complex after 1.1 ns simulation (B) and formation of stable complex after 5.8 ns simulation (C).

Fig. 5. Snapshots of the simulations of doxorubicin (in yellow, denoted as Dox) and alginate molecules (in red, denoted as ALG) in water: external complexes for 3 doxorubicin molecules and 2 alginates (A) and atomistic model showing interaction of 2 doxorubicin molecules with

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1 alginate (B), aggregate complex with 4 doxorubicin molecules and 3 alginates (C), and aggregate complex with 12 doxorubicin molecules and 3 alginates (D).

Fig. 6. Interatomic distance between the positively charged N center of doxorubicin and the negatively charged carboxyl oxygen centers of alginate for the models with four doxorubicin and four alginates (left panel) and for 12 doxorubicin and 3 alginates (right panel). Solid line – relative number of distances, dashed line – integrated number of contacts.

Fig. 7. In vitro doxorubicin release from the developed chitosan/alginate nanoparticles in phosphate buffers with pH-values of 5.5 and 7.4. Data represent the means ± S.D.

Fig. 8. Cytotoxicity of free (Dox) and encapsulated doxorubicin (NP-Dox) after 72 h treatment of B16-F10 and B16-OVA cells. Data represent the means ± S.D.

Fig. 9. Cytotoxicity of free (Dox) and encapsulated doxorubicin (NP-Dox) (0.5 μM) after 72 h treatment of B16-F10 cells followed by their resuspension in drug-free medium for 24 h. Data represent the means ± S.D.

Fig. 10. Time profiles of tumor growth evaluated in B16-OVA melanoma-bearing mice after two doses of 3 mg/kg of free (Dox) and encapsulated doxorubicin (NP-Dox). Lines represent the mean ± SEM (n=6/group). Arrows represent the dose administration. *, P<0.05 for treated vs. control.

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Table 1. IC50 values calculated from the linear regression of the dose response curves after 72 h exposure of both cell lines to free and encapsulated doxorubicin.

IC50 (μM)

IC50 (μM)

B16-OVA cells

B16-F10 cells

Free doxorubicin

0.24

0.35

Encapsulated doxorubicin

0.68

0.73

120

EE

50

Loading degree

100

40

EE (%)

80

30

60 20

40

10

20 0

Loading degree (%)

Sample

0 1 : 20

1 : 15

1 : 10

1:5

1:4

Initial ratio between doxorubicin and sodium alginate (w/w)

Figure 1 26

Figure 2

1

400 PDI

0,8

300

0,6 200 0,4 100

PDI

Mean diameter (nm)

Mean diameter

0,2

0

0 1 : 20

1 : 15

1 : 10

1:5

1:4

Initial ratio between doxorubicin and sodium alginate (w/w)

Figure 3

27

Fig. 6 28

Released DOX (%)

100 80 60 40

pH=7.4 20

pH=5.5

0 0

50

100

150

200

250

Time (h)

Fig. 7

29

Fig. 8

30

Cell viability (%) ,

120

Dox

NP-Dox

100 80 60 40 20 0 72 h treatment with the drug

Control

24 h after drug removal

Fig. 9

1000 Control NP

800

Dox

Tumor (mm3)

Dox-NP

600

400

* *

200

0 0

7

14

21

Time (days)

Fig. 10 31

32