Comparative study of chitosan- and PEG-coated lipid and PLGA nanoparticles as oral delivery systems for cannabinoids

Title: Comparative Study of Chitosanand PEG-Coated Lipid and PLGA Nanoparticles as Oral Delivery Systems for Cannabinoids Authors: Matilde Durán-Lobato, Lucía Martín-Banderas, Lídia M. D. Gonçalves, Mercedes Fernández-Arévalo, António J. Almeida Note: This is the preprint version of the manuscript submitted to Journal of Nanoparticle Research. This version has not been peer-reviewed or edited and may differ significantly from the final published version. For the peer-reviewed version, please refer to: Durán-Lobato, M., Martín-Banderas, L., Gonçalves, L. M. D., Fernández-Arévalo, M., & Almeida, A. J. (2015). Comparative study of chitosanand PEG-coated lipid and PLGA nanoparticles as oral delivery systems for cannabinoids. Journal of Nanoparticle Research, 17(61). https://doi.org/10.1007/s11051-015-2875-y 1 Comparative study of chitosanand PEG-coated lipid and polymeric nanoparticles 1 as oral delivery systems for cannabinoids 2 3 4 Matilde Durán-Lobato1,2, Lucía Martín-Banderas1*, Lídia M.D. Gonçalves2, Mercedes 5 Fernández-Arévalo1, Antonio J. Almeida2. 6 7 1Facultad de Farmacia, Universidad de Sevilla, Profesor García González, 2, 41012 8 Sevilla, Spain. 9 10 2Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade 11 de Farmácia da Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 12 Lisboa, Portugal. 13 14 15 * Corresponding author address: Dpto. Farmacia y Tecnología Farmacéutica, Facultad 16 de Farmacia, Universidad de Sevilla. C/ Profesor García González, 2, 41012, Sevilla, 17 España; Tel; +34 954556754; Fax: 954556085; E-mail: [email protected]. 18 19 20 Manuscript Click here to download Manuscript: renamed_cd002.docx Click here to view linked References 2 Abstract 21 The cannabinoid derivative 1-naphthalenyl[4-(pentyloxy)-1-naphthalenyl]methanone 22 (CB13) has an important therapeutic potential as analgesic in chronic pain states that 23 respond poorly to conventional drugs. However, the incidence of its mild-to-moderate 24 and dose-dependent adverse effects (AEs), as well as its pharmacokinetic profile 25 actually holds back its use in humans. Thus, the use of a suitable carrier system for oral 26 delivery of CB13 becomes an attractive strategy to develop a valuable therapy. 27 Polymeric PLGA and lipid nanoparticles are widely studied delivery vehicles that 28 improve the bioavailability of lipophilic compounds and present special interest in oral 29 delivery. Their surface can be modified to improve the adhesion of particles to the oral 30 mucosa and increase their circulation time in blood with additives such as chitosan (CS) 31 and polyethylene glycol (PEG), which can be feasibly incorporated onto these particles 32 in a post-production step. In this work, CSand PEG-modified polymeric PLGA and 33 lipid nanoparticles were comparatively evaluated under the same experimental 34 conditions as oral carriers for CB13, a Class II (BCS) model drug with high 35 therapeutical potential. Physicochemical characteristics of modified and non-modified 36 NPs, blood compatibility, cytotoxicity and uptake in Caco-2 and THP-1 cell lines were 37 studied as a comparison of their potential as oral delivery systems of CB13. 38 Keywords 39 cannabinoids, oral administration, neuropathic pain, lipid nanoparticles, PLGA 40 nanoparticles 41 42 3 Introduction 43 44 Cannabinoids present an important therapeutic potential in a wide range of syndromes 45 and diseases (Ben Amar 2006; Pertwee 2001), especially as analgesics in chronic pain 46 states that respond poorly to conventional drugs such as morphine (Iversen and 47 Chapman 2002). However, cannabinoids also present undesirable physicochemical 48 properties such as poor stability and solubility as well as adverse side effects (AEs), 49 which actually hold back their use in humans (Attal et al. 2004; Hall and Solowij 1998). 50 The cannabinoid derivative 1-naphthalenyl[4-(pentyloxy)-1-naphthalenyl]methanone 51 (cannabinoid receptor agonist 13, CRA-13, CB13) is a novel cannabinoid receptor 52 agonist that stands out owing to its less penetration into the brain than other 53 cannabinoids and consequently less pronounced AEs (Dziadulewicz et al. 2007). In 54 these terms, the incidence of mild-to-moderate and dose-dependent AEs as well as the 55 pharmacokinetics profile of this compound should be considered (Gardin et al. 2009). 56 CB13 belongs to Class II of compounds (low solubility and high permeability) of the 57 Biopharmaceutics Classification System (BCS), which determines a highly variable 58 absorption (Gardin et al. 2009) and hence uneven plasmatic concentrations and with 59 consequent incidence of AEs. 60 Thus, the use of a suitable carrier system for oral delivery of CB13 becomes an 61 attractive strategy to develop a valuable therapy. Specifically, polymeric poly(DL62 lactide-co-glycolide) (PLGA) and lipid nanoparticles (LNPs) are some of the most 63 widely studied therapeutic delivery vehicles to improve the bioavailability of lipophilic 64 compounds and present special interest in oral delivery (Zhang et al. 2013). PLGA 65 nanoparticles (NPs) provide biocompatibility, controlled drug release and degradation 66 into completely safe products (PLGA monomers). In addition, this polymer is 67 4 commercially available in different grades and its glass transition temperature (Tg) is 68 above physiological temperature of 37 °C, lending the required mechanical strength for 69 formulation development (Martin-Banderas et al. 2012). On the other hand, LNPs can 70 be made of physiological lipids (biocompatible and biodegradable) with a wide range of 71 methodologies (Almeida and Souto 2007; Parhi and Suresh 2010). Their use for oral 72 drug delivery has been strongly developed (Fricker et al. 2010; Harde et al. 2011) and 73 they have shown high rates of encapsulation for lipophilic compounds and improvement 74 of gastrointestinal (GI) absorption and oral bioavailabity of several drugs (Das and 75 Chaudhury 2011). 76 These NPs present some drawbacks as oral delivery carriers as well, e.g. their slightly 77 negative surface charge that tends to prevent the interaction with the intestinal mucosa 78 (Martin-Banderas et al. 2012; Semete et al. 2012; Zhang et al. 2012). Nonetheless, 79 surface properties of NPs can be generally modified either by coating their surface with 80 hydrophilic stabilizers, bioadhesive polymers or surfactants or by incorporating in the 81 formulation biodegradable copolymers containing hydrophilic moieties (Martin82 Banderas et al. 2013). These modifications mainly change the zeta potential (ZP) and 83 hydrophobicity of the NPs, and therefore determine their colloidal stability, 84 mucoadhesion properties, oral absorption and the adsorption of proteins on the surface 85 (des Rieux et al. 2006). 86 Chitosan (CS) is a biodegradable and biocompatible polymer that has been widely 87 studied as potential oral transmucosal absorption enhancer (Issa et al. 2005; Zhang et al. 88 2012) and can be feasibly incorporated onto negatively charged surface of nanoparticles 89 in a post-production step with no influence over the preparation procedure (Durán90 Lobato et al. 2014; Sarmento et al. 2011). Polyethylene glycol (PEG) is a hydrophilic 91 and also biocompatible polymer that has been shown to facilitate the transport through 92 5 the Peyer’s p atches GALT (Garinot et al. 2007), prevent the enzymatic degradation of 93 NPs in GI fluids (Tobıo et al. 2000; Vila et al. 2002; Vila et al. 2004), minimize 94 opsonization (Singh and Lillard 2009) and increase the systemic circulation time in vivo 95 of particles (Knop et al. 2010; Semete et al. 2012), leading to a significant enhancement 96 of bioavailability of the encapsulated drug in blood stream and lymphatics (Tobio et al. 97 ; Tobı o et al. 2000; Vila et al. 2002 . The addition of PEG molecules to NPs 98 surface can be achieved via a number of different routes (Martin-Banderas et al. 2013). 99 In this work, polymeric PLGA NPs and LNPs were produced and evaluated under the 100 same experimental conditions and in a comparative manner as carriers for oral delivery 101 of CB13. CS and PEG have been employed as surface-modifying additives to improve 102 CB13-loaded PLGA NPs and LNPs intestinal uptake and prevent biological clearance 103 mechanisms. Physicochemical characteristics of modified and non-modified NPs either 104 with CS or PEG -mean particle size, zeta potential and drug loading-, cytotoxicity and 105 uptake in Caco-2 cell line and uptake in THP1 cell line were studied. The present 106 investigation aims to show and accurately compare the potential of CS and PEG107 modified PLGA NPs and LNPs to improve the oral delivery of CB13, a BCS Class II 108 and cannabinoid model drug with high therapeutic potential. 109 Materials and methods 110 Materials 111 CB13 was provided by Tocris Cookson Ltd. (Bristol, UK). Poly(DL-lactide-co112 glycolide) (PLGA 50:50) Resomer® RG 502 was obtained from Boehringer-Ingelheim 113 (Ingelheim, Germany). Chitosan (CS) low MW, sodium deoxycholate (SD), Span® 60, 114 Tween20, Tween 80, stearylamine (SA), cetyl trimethylammonium bromide (CTAB), 115 and Pluronic® F-68 and, Nile Red and rhodamine were provided by Sigma-Aldrich. 116 6 Precirol® ATO 5 (glyceryl palmitostearate, melting point: 53-56 ºC) was kindly given 117 by Gattefossé (Saint-Priest, Cedex, France). Soya lecithin (Lipoid S100) was purchased 118 from Lipoid (Ludwigshafen, Germany). Threalose was obtained from VWR 119 International Eurolab S.L. (Barcelona, Spain). HPLC-grade acetonitrile, acetic acid, and 120 ethyl acetate were purchased from Panreac (Spain). Glycerol was obtained from 121 Acofarma Distribución S.A. (Barcelona, Spain). 122 For cell line experiments, human colon adenocarcinoma cells (Caco-2) cells were 123 obtained from the European Collection of Cell Cultures (ECACC) (Salisbury, UK). 124 Differentiate macrophage THP1 cells (human monocytic cell line) were obtained from 125 ATCC TIB-202™ (Barcelona, Spain). Minimum Essential Medium Eagle (MEM with 126 Earle´s salts without L-glutamine), RPMl 1640 medium, sodium pyruvate, MEM non127 essential amino acids, L-glutamine and fetal bovine serum were obtained from PAA 128 Laboratories (Pasching, Austria). Gentamicin was purchased from Gibco® Life 129 Technologies Corporation (NY, USA). Trypsin/EDTA, MTT (Thiazolyl Blue 130 Tetrazolium Bromide or Methylthiazolyldiphenyl-tetrazolium bromide), SDS (Sodium 131 Dodecyl Sulfate), PFA (paraformaldehyde) and Hoechst 33258 were purchased from 132 Sigma-Aldrich (St Louis, MO). The ProLong® Gold antifade reagent containing the 133 blue-fluorescent nuclear counterstain DAPI was obtained from Invitrogen. 134 Methods 135 LNPs preparation 136 LNPs were produced using the emulsification-solvent evaporation method previously 137 described (Lopes et al. 2012). Briefly, Precirol® and lecithin were dissolved in 138 dichloromethane and then added to the aqueous phase containing Tween®20 and 139 sodium deoxycholate. The dispersion step was performed during 2.5 min period of 140 7 sonication (Branson Sonifier 250, Danbury, USA). Afterwards, this dispersion was 141 homogenized for 3.5 min at 125000 rpm (Silverson High Speed Mixer L5M, Silverson 142 Machines, UK). The nanoparticle dispersion was then kept under stirring for 4 h at 143 room temperature until complete evaporation of the dichloromethane. When 144 incorporating CB 13, the drug was added to the organic phase at 10% drug/lipid 145 respectively. In the case of fluorochrome-loaded particles, 20 μL of a mg/mL 146 rhodamine solution were added to the organic phase instead. 147 To produce cationic LNPs, stearylamine (Pedersen et al. 2006) and/or cetyl 148 trimethylammonium bromide (CTAB) (Tabatt et al. 2004) were included in the 149 formulation in absence of lecithin. When incorporated, 10 mg of stearylamine were 150 added to the organic phase prior to homogenization. CTAB replaced sodium 151 deoxycholate in the aqueous phase and was added at a concentration of 0.1 %w/v. 152 Lecithin-free formulations were purified using size exclusion chromatography in PD10 153 columns (GE Healthcare, Germany) and re-suspended in a 6 % w/v threalose solution 154 used as cryoprotectant. Formulations containing lecithin were purified using an 155 ultrafiltration-centrifugation method using centrifugal filters (Amicon Ultra-4, 156 Millipore, Germany) with a 100 kDa molecular weight cut-off (4º, 4000g, 10 min in 157 triplicate Beckman L8-60M ultracentrifuge (Beckman Instruments, Inc., USA) (Lopes 158 et al. 2012). Following, the particles were frozen in liquid nitrogen and lyophilized at 159 −80.0 ± 0.5 °C and 0.057 mbar (Telstar Cryodos, Spain). All the formulations were 160 prepared in triplicate (n = 3). 161 PLGA NPs preparation 162 PLGA NPs were prepared by the nanoprecipitation method (NPP) with some 163 modification (Durán-Lobato et al. 2014). Briefly, a weighed amount of PLGA was co164 8 dissolved with Span® 60 in acetone to reach a concentration of 1.5 % w/v. 5 mL of 165 such solution were subsequently added dropwise at 5 mL/min using a syringe pump 166 (Harvard Apparatus, USA) into 15 mL of a Pluronic® F68 aqueous solution (0.5 % 167 w/v) under magnetic stirring. The acetone was then evaporated at r.t. for 4 h. 168 Following, the particles suspension was centrifuged over a glycerol bed (100 µL) at 169 10000 rpm for 15 min at 4ºC to collect the NPs. After washing twice, the NPs were re170 suspended in a 5 % w/v threalose solution used as cryoprotectant and freeze-dried 171 (frozen in liquid nitrogen and lyophilized at − 0.0 ± 0.5 °C and 0.057 mbar; Telstar 172 Cryodos, Spain) to obtain a fine powder. All the formulations were prepared in triplicate 173 (n = 3). 174 In the case loaded PLGA NPs, CB13 was co-dissolved with the polymer in acetone at 175 13 % w/w drug/polymer. In the case of rhodamine-loaded NPs, 20 μ L of a mg/mL of 176 fluorochrome solution were added to the organic phase instead. 177 NPs surface modification 178 To promote the internalization of the particles by Caco-2 cells (accepted in the scientific 179 literature as gastrointestinal barrier model) both kinds of particles were coated with CS. 180 For this purpose, and due to the hydrophilic nature of CS, this additive was added in an 181 additional step after NPs formation (Durán-Lobato et al. 2014; Sarmento et al. 2011). 182 The NPs were incubated into a 0.25 % w/v CS solution in acetic acid 1% v/v for 30 min 183 and then collected by centrifugation. 184 In order to prevent biological clearance mechanisms as well as favor GI cells uptake, 185 plain polymeric and lipid particles were also coated with PEG. For this purpose, the 186 NPs were in turn incubated in a 4.5 % w/v PEG6000 solution for 4h under stirring. An 187 additional approach was also followed with LNPs formulations, consisting of 188 15 Therefore, stearylamine and/or CTAB were included in the formulation to reduce the 325 intensity of the electrostatic interaction and allow for similar size values before and after 326 the coating process (Fig. 1B). However, in the case of particles containing stearylamine, 327 the addition of chitosan led to a marked increase in particle size and size distribution, 328 and thus this formulation was excluded from further studies. Fig. 1A also displays the 329 size distribution of anionic LNPs after the coating process with PEG in a post330 production step, which did not lead to significant differences in size values (p < 0.05). 331 Thus, cationic excipients stearylamine and CTAB were not employed in this case. In 332 addition, LNPs formulated with PEG incorporated in the aqueous phase of the emulsion 333 resulted in large sizes and wide size distributions (Fig. 1A), and therefore were 334 excluded from further studies. 335 Regarding PLGA NPs, plain particles showed a mean particle size in the range of 310336 360 nm, which was increased after CS and PEG coating steps (Fig. 1C). The increased 337 size after CS and PEG adsorption on particle surface has been previously reported 338 (Garcia-Fuentes et al. 2005; Nafee et al. 2009; Parveen and Sahoo 2011) and attributed 339 to the deposition of multilayer surface-coating components. Nonetheless, surface 340 modification of nanoparticles with hydrophilic components is expected to improve their 341 cellular uptake, as well as avoid the opsonization process, regardless of the reported size 342 increase (Gref et al. 2000; Parveen and Sahoo 2011). 343 Insert Fig. 1 around here 344 Fig. 1 Size distribution of A plain and coated anionic LNPs (PEG-LNPsA prepared with PEG 345 added in a post-production step; PEG-LNPsB prepared with PEG added in the organic phase of 346 the emulsion); B plain and coated cationic LNPs; C plain and coated PLGA NPs. 347 16 In terms of ZP, a similar behaviour was observed in both polymeric and lipid 348 nanoparticles, as depicted in Fig. 2. The modification with CS strongly turned ZP 349 values to positive values (-35.6 mV to +60.7 mV for PLGA NPs and -34.2 ± 1.3 mV to 350 +47.33 ± 0.56 mV for cationic LNPs), which evidences the deposition of CS onto the 351 particles surface and is in accordance with the published literature (Durán-Lobato et al. 352 2014; Sarmento et al. 2011; Vila et al. 2002). 353 The surface modification with PEG induced nonetheless a shift in ZP in both types of 354 particles to values near neutrality (Fig. 2) also in accordance with previous published 355 data (Garcia-Fuentes et al. 2005; Garinot et al. 2007; Vila et al. 2002). The presence of 356 PEG chains displaces the diffuse ionic layer to a greater distance from the particle 357 surface, resulting in a decrease in absolute ZP values (Garcia-Fuentes et al. 2005; Patel 358 et al. 2012). 359 Insert Fig. 2 around here 360 Fig. 2 Zeta potential values of formulations. LNPs plain anionic lipid nanoparticles; CTAB361 LNPs CTAB-containing plain cationic lipid nanoparticles; CS-CTAB-LNPs chitosan-coated 362 CTAB-containing LNPs; SA-CTAB-LNPs plain stearylamineand CTABcontaining lipid 363 nanoparticles; CS-SA-CTAB-LNPs chitosan-coated stearylamineand CTAB-containing LNPs; 364 SA-LNPs plain stearylamine-containing LNPs; CS-SA-LNPs chitosan-coated stearylamine365 containing LNPs; PEG-LNPs PEG-coated anionic LNPs; PLGA NPs; CS-PLGA NPs chitosan366 coated PLGA NPs; PEG-PLGA NPs PEG-coated PLGA NPs (error bars SD, n = 3). 367 Both PLGA and lipid particles were spherical and non-aggregated. After the coating 368 process, PLGA NPs showed a well-defined core-shell structure. As an example, Fig. 3 369 shows the morphology of CB13 loaded-PLGA NPs obtained by TEM imaging. This 370 particular structure was not observed for LNPs probably due to a smaller amount of CS 371 on the particle surface (data not shown). 372 17 Insert Fig. 3 around here 373 Fig. 3 TEM image of CS-coated PLGA NPs core-shelf structure. 374 Drug loading and entrapment efficiency 375 Values of EE (%) and LC (%) ranged between 89 – 100 % and 7 – 10 %, respectively 376 for LNPs, and 70 – 80 % and 8 – 10 %, respectively for PLGA nanoparticles. The 377 corresponding data is shown in Table 1. The high values of encapsulation achieved are 378 probably due to the lipophilic nature of the drug, which presents low affinity to water 379 phases and thus tends to migrate to the organic phase. There were no statistically 380 significant differences between plain and the corresponding surface-modified 381 formulations, probably due to coating methodology: the additives were added in a post382 production step, therefore not influencing the encapsulation process (Durán-Lobato et al. 383 2014). 384 Insert Table 1 around here 385 Table 1 EE and LC of formulations. LNPs plain anionic lipid nanoparticles; CTAB-LNPs 386 CTAB-containing plain cationic lipid nanoparticles; SA-CTAB-LNPs plain stearylamineand 387 CTABcontaining lipid nanoparticles; CS-SA-CTAB-LNPs chitosan-coated stearylamineand 388 CTAB-containing LNPs; SA-LNPs plain stearylamine-containing LNPs; CS-SA-LNPs 389 chitosan-coated stearylamine-containing LNPs; PEG-LNPs PEG-coated anionic LNPs; PLGA 390 NPs; CS-PLGA NPs chitosan-coated PLGA NPs; PEG-PLGA NPs PEG-coated PLGA NPs (SD, 391 n = 3). 392 In vitro drug release profiles 393 CB13 presents extremely low water solubility. Hence, to maintain sink conditions, a 394 0.1 % w/v Tween 80 solution was used as dissolution medium. The in vitro release of 395 CB13 from plain, CS-coated and PEG-coated LNPs and PLGA NPs is illustrated in Fig. 396 18 4. Where the release of CB13 from PLGA NPs is shown to be significantly lower than 397 from LNPs, following a prolonged release pattern with no burst effect. LNPs released 398 more than 50 % of CB13 in the first 2 h and 90 % in the first 8 h. In comparison, PLGA 399 NPs (plain, CS and PEG-coated) released less than 10 % of CB13 at the end of the first 400 2 h and less than 50 % in the first 8 h. 401 With regard to coated NPs, in the case of polymeric particles the presence of a surface 402 coating led to a decrease of CB13 release rate compared to the corresponding plain 403 particles (Fig. 4). Surface-modified PLGA particles did not attain complete release of 404 the drug within the analyzed period (maximum value of released drug below 60 %), 405 which can be attributed to the additional layer of additive on the surface acting as an 406 additional barrier to drug diffusion (Durán-Lobato et al. 2014; Parveen and Sahoo 2011). 407 Nonetheless, in the case of LNPs, the surface coating did not lead to significant 408 differences in drug release profiles with respect to the corresponding plain particles. The 409 similarity between plain and coated LNPs drug release can be related to the similar 410 particle size values obtained before and after the coating process, suggesting the 411 absence of the multiple layers obtained with coated PLGA NPs that hindered the release 412 process. 413 Insert Fig. 4 around here 414 Fig. 4 Release from plain and CSand PEG-coated PLGA NPs and LNPs. LNPs plain anionic 415 lipid nanoparticles; CS-CTAB-LNPs chitosan-coated CTAB-containing LNPs; PEG-LNPs 416 PEG-coated anionic LNPs; PLGA NPs; CS-PLGA NPs chitosan-coated PLGA NPs; PEG417 PLGA NPs PEG-coated PLGA NPs (error bars SD, n = 3). 418 FT-IR 419 19 FT-IR analysis was carried out to further confirm the presence of the additives in the 420 final NPs suspensions. FT-IR spectra of plain and PEGand CS-coated PLGA NPs and 421 LNPs are depicted in Fig. 5 and were compared to the signal of the corresponding 422 excipients (data not shown) and published literature. The spectra of plain (A), PEG423 coated (B) and CS-coated (C) PLGA formulations show the characteristic alkane C-H 424 bond absorption peaks at 2978 and 2960 cm-1 and -COOstretching peak at 1745 cm-1 425 of PLGA (Shen et al. 2011). In turn, in the FT-IR spectra of plain (D), PEG-coated (E) 426 and CS-coated (F) LNPs formulations, absorption peaks at 2917 and 2843 cm-1 427 attributed to Precirol®, the main component of particle matrix (Reitz et al. 2008) were 428 detected. The presence of PEG in PEG-coated PLGA (B) and LNP (E) formulations 429 was confirmed by the absorption bands at 2883 cm-1 (B) due to stretching C-H vibration, 430 and at 1146 cm-1 and 1102 cm-1 (E) attributed to C-C stretching and the characteristic C431 O-C stretching vibration of the repeated -OCH2CH2units of the PEG backbone 432 respectively (Kassim et al. 2006; Petrova et al. 2008). Finally, CS-coated PLGA (C) and 433 LNP (F) formulations presented absorption bands at 1647 (C) and 1653 (F) attributed to 434 amide I (C=O) and at 1312 cm-1 (F) due to the bending vibration of C-N, confirming the 435 presence of CS in the formulations (Wang et al. 2007). 436 Insert Fig. 5 around here 437 Fig. 5 FT-IR spectra of plain and surface-modified PLGA NPs and LNPs. A) PLGA NPs; B) 438 PEG-PLGA NPs; C) CS-PLGA NPs; D) LNPs; E) PEG-LNPs; F) CS-CTAB-LNPs. 439 Blood compatibility 440 Data obtained from blood compatibility studies is presented in Table 2 and show a 441 broad in vivo safety margin for the different types of nanoparticles assayed. All the 442 formulations showed values below 2.5 % of lysis, indicating these carriers can be 443 20 considered hemocompatible for drug delivery applications. The formulations are 444 expected to exhibit a negligible effect on hemolysis and not to influence sP-selectin 445 release levels from platelet activation quantification, complement system activation or 446 plasma clotting times. Similar results have been reported for other nanoparticulate 447 systems containing the materials employed in the formulations assayed in this work 448 (Date et al. 2007; Martin-Banderas et al. 2012). 449 Insert Table 2 around here 450 Table 2 Blood compatibility of CB13-loaded NPs formulations in terms of hemolysis (%), 451 platelet activation (sP-selectin release, ng/mL), complement activation (C3a release: C3a 452 desArg, ng/mL), and plasma recalcification time (T1/2max, min). LNPs; CS-CTAB-LNPs 453 chitosan-coated CTAB containing LNPs; PEG-LNPs PEG-coated LNPs; PLGA NPs; CS-PLGA 454 NPs chitosan-coated PLGA NPs; PEG-PLGA NPs PEG-coated PLGA NPs (n = 3). 455 MTT 456 A MTT assay (Durán-Lobato et al. 2014) was carried out to determine cell viability in 457 the presence of plain and coated PLGA NPs and LNPs. The study was conducted with 458 Caco-2 cells since they are considered a gastrointestinal permeability model in the 459 literature (Alhamoruni et al. 2010). It should be noted that, although cannabinoids have 460 been proven to inhibit cell growth and induce apoptosis in tumour cells (Gustafsson et 461 al. 2009), their influence on cell viability and proliferation depends on the specific cell 462 culture assayed and the dose administrated (Hart et al. 2004; Ligresti et al. 2003; Lopez463 Rodriguez et al. 2005; Sarne et al. 2011). 464 Cells were incubated with CB13 concentrations ranging from 0.003 to 30 μM in the 465 form of free drug and loaded into plain and surface-modified PLGA and lipid 466 nanoparticles. Blank NPs were tested as well at the amount equivalent to those of 467 21 CB13-loaded particles assayed (0.004 – 4 μg NPs). DMSO used as positive control 468 demonstrated to be toxic at the concentration assayed and therefore validated the assay. 469 Fig. 6 illustrates the results obtained from the study, showing cell viability values were 470 maintained around 100 % for all the formulations assayed, thus indicating the lack of 471 toxicity of the particles tested at the concentrations employed. 472 Insert Fig. 6 around here 473 Fig. 6 Caco-2 cell viability (%) obtained through the MTT assay after incubation with NPs 474 LNPs plain anionic lipid nanoparticles; CTAB-LNPs CTAB-containing plain cationic lipid 475 nanoparticles; CS-CTAB-LNPs chitosan-coated CTAB-containing LNPs; SA-CTAB-LNPs 476 plain stearylamineand CTABcontaining lipid nanoparticles; CS-SA-CTAB-LNPs chitosan477 coated stearylamineand CTAB-containing LNPs; SA-LNPs plain stearylamine-containing 478 LNPs; CS-SA-LNPs chitosan-coated stearylamine-containing LNPs; PEG-LNPs PEG-coated 479 anionic LNPs; PLGA NPs; CS-PLGA NPs chitosan-coated PLGA NPs; PEG-PLGA NPs PEG480 coated PLGA NPs (error bars SD, n = 3). 481 Cell uptake 482 Caco-2 cells were also used to simulate the gastrointestinal (GI) drug barrier for oral 483 delivery (Zhang and Feng 2006) in uptake studies. NPs coated with hydrophilic 484 polymers or moieties such as CS and PEG have been reported in the literature to display 485 higher intestinal transport compared with that of unmodified nanoparticles (des Rieux et 486 al. 2006; Durán-Lobato et al. 2014; Martin-Banderas et al. 2013). 487 Fig. 7 shows similar levels of CS-coated LNPs and PLGA NPs (Fig. 7c, 7f, 7g), also 488 higher than the corresponding plain and PEG-coated formulations (Fig. 7). The results 489 are in accordance with previous literature and account for a higher interaction between 490 particles and cells due to the positive charges of chitosan molecules on the NPs surface 491 (Durán-Lobato et al. 2014; Garcia-Fuentes et al. 2005). Moreover, PEG-coated 492 22 formulations showed the lowest uptake (Fig. 7b, 7e, 7g), which could be attributed to 493 the the steric impediment exerted by the PEG coating that is known to prevent the 494 interaction with cells, being the basis of its “stealth” properties (Gref et al. 2000; 495 Martin-Banderas et al. 2013; Owens and Peppas 2006). It should be noted that these 496 results seem to be in disagreement with the higher transport of PEG-coated PLA NPs 497 compared to plain PLA NPs previously reported in the literature (Vila et al. 2004). 498 However, although a mucoadhesion/chain penetration mechanism has been proposed as 499 the basis of a higher transport of PEG-coated NPs (Tobı o et al. 2000 , the same authors 500 have stated the still unclear question of whether PEG-coating of NPs increases 501 bioavailability due to an improved GI transport or to a simply greater stability in GI 502 fluids and blood stream (Vila et al. 2004). 503 When comparing both types of particles, plain and CS-coated PLGA and lipid NPs 504 showed similar cell uptake values respectively. However, PEG-coated LNPs showed 505 slightly higher uptake values than the corresponding PLGA formulations (Fig. 7b, 7e, 506 7g). It should be noted that particle size is known to be an influencing factor in NPs 507 transport through mucosa membranes (Froehlich 2012; Vila et al. 2005; Vila et al. 508 2004) and the larger size of coated PLGA NPs could be influencing the lower cell 509 internalization at the time point assayed in this study. 510 Based on the results, it could be concluded that CS-coating of nanoparticles potentially 511 provides the higher cell internalization at the GI level, a previous and necessary step for 512 reaching blood circulation. Additional considerations such as NPs stability prior to 513 absorption and bioavailability should be taken into account when comparing the general 514 performance of both types of coatings. 515 Insert Fig. 7 around here 516 23 Fig. 7 Caco-2 uptake CLSM images and particle counting algorithms. a) LNPs plain anionic 517 lipid nanoparticles; b) PEG-LNPs PEG-coated anionic LNPs; c) CS-CTAB-LNPs chitosan518 coated CTAB-containing LNPs; d) PLGA NPs; e) PEG-PLGA NPs PEG-coated PLGA NPs; f) 519 CS-PLGA NPs chitosan-coated PLGA NPs; g) number of particles per cell (error bars SD, n = 520 3). 521 THP1 cell uptake 522 Uptake studies in THP1 cells showed a common trend of uptake PEG-coated << CS523 coated < plain NPs for polymeric and lipid formulations. A marked decrease of PEG524 coated NPs uptake compared to the corresponding plain and chitosan-coated 525 formulations was observed (Fig. 8), as expected and supported by previous literature 526 (Bocca et al. 1998), due to the hydrophilic properties that the additive provides to the 527 surface of particles (Owens and Peppas 2006; Vonarbourg et al. 2006). Chitosan-coated 528 formulations showed also a decrease in THP1 uptake with respect to plain formulations, 529 though not as marked as in the case of PEG-coated formulations (Fig. 8). A lower 530 phagocytic uptake has been attributed to the hydrophilic properties of chitosan as well 531 as its positive charges (Sarmento et al. 2011), since the hydrophobic surface of particles 532 as well as negative surface charges resembling bacteria’s surface favor clearance 533 mechanisms within the body (Froehlich 2012). In accordance, plain formulations 534 presented the highest THP1 uptake values (Fig. 8). 535 Insert Fig. 8 around here 536 Fig. 8 Quantitative analysis by flow cytometry of uptake in THP1 cells after 120 min of 537 incubation with rhodamine label particles. Plots (a) LNPs; (b) PLGA; (c) CS-LNPs; (d) CS538 PLGA NPs; (e) PEG-LNPs; (f) PEG-PLGA NPs. Percentage plot corresponds to total events 539 included in R2 region. Y-axis corresponds to number of counts. 540 24 However, a slightly increase in uptake could be observed in the case of CSand PEG541 coated PLGA NPs with regard to CSand PEG-coated LNPs respectively, which could 542 be attributed to the larger size of PLGA formulations obtained after the deposition of 543 multiple layers of additive in the coating process. Particle size has been reported to be a 544 key factor influencing the extent of opsonization and phagocytic uptake (Froehlich 545 2012), specifically in the case of PEG-coated PLGA NPs for the size range studied in 546 this work (Yang et al. 2012). 547 According to the results, PEG-coated NPs provide the highest protection against 548 opsonization and phagocytic uptake and thus a higher chance for prolonged circulation 549 in vivo. However, and as reported by previous works (Garcia-Fuentes et al. 2005), PEG 550 coating of LNPs did not lead to in vivo improvements, while CS-coating of the same 551 formulation did. It should be noted that in vitro results do not always correlate with in 552 vivo data, since there are many influencing factors for the latter that are not present in in 553 vitro experiments. However, in vitro data allow obtaining a deeper understanding of the 554 performance of the carriers that is a key factor towards the optimization of functional 555 carrier systems. Further studies on the interaction of these particles with biological 556 surfaces and the influence of their components and coating additives should be carried 557 out covering a broader range of in vitro conditions, to ultimately be able to fully 558 understand their behaviour in vivo. 559 Conclusions 560 PLGA NPs with size of 320-420 nm in diameter, narrow size distribution and negative 561 zeta potential, and LNPs with 12060 nm in diameter with Pdi ≈ 0.3 and negative zeta 562 potential were obtained. Modification of NPs surface with CS and PEG strongly turned 563 ZP to positive and near neutrality values respectively. Electron microscopy imaging 564 31 830 831 832 Fig. 1 833 834 32 835 Fig. 2 836 837 -40 -20 0 20 40 60 80 Zeta Potential (mV) 33 838 839 840 841 842 843 844 Fig. 3 845 846 34 847 848 Fig. 4 849 850 0 20 40 60 80 100 012345678910 11 12 13 14 15 Drug released (%) Time (h) LNPs CS-CTAB-LNPs PEG-LNPs A 0 20 40 60 80 100 010 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Drug released (%) Time (h) PLGA NPs CS-PLGA NPs PEG-PLGA NPs B 35 851 Fig. 5 852 853 36 854 Fig. 6 855 856 0 20 40 60 80 100 0.001 0.01 0.1 110 100 Viability (%) CB13 concentration (μM) LNPs CTAB-LNPs CS-CTAB-LNPs SA-CTAB-LNPs CS-SA-CTAB-LNPs SA-LNPs CS-SA-LNPs PEG-LNPs PLGA NPs CS-PLGA NPs PEG-PLGA NPs DMSO 37 857 Fig. 7 858 859 A B C D E F G 38 860 Fig. 8 861 862 39 EE ± SD (%) LC ± SD (%) LNPs 89.0 ± 9.0 3.6 ± 0.4 CTAB-LNPs 85.4 ± 6.9 7.7 ± 0.6 CS-CTAB-LNPs 86.5 ± 7.4 7.5 ± 0.7 SA-CTAB-LNPs 90.9 ± 7.5 7.0 ± 0.6 CS-SA-CTAB-LNPs 91.3 ± 7.5 5.9 ± 0.8 SA-LNPs 90.9 ± 7.5 7.0 ± 0.6 CS-SA-LNPs 89.0 ± 6.5 6.8 ± 0.7 PEG-LNPs 90 ± 9.0 3.8 ± 0.7 PLGA NPs 73,4 ± 8.0 3,7 ± 0.5 CS-PLGA NPs 75,3 ± 7.2 3,5 ± 0.7 PEG-PLGA NPs 79,4 ± 9.0 3,9 ± 0.6 863 Table 1 864 865 40 Sample Haemolysis (%) sP-selectin release (ng/mL) C3a desArg (ng/mL) T1/2max (min) LNPs 2.2 ± 0.6 98 ± 10 289 ± 9 11.5 ± 1.0 CS-CTAB-LNPs 2.0 ± 0.4 101 ± 8 292 ± 7 10.1 ± 0.9 PEG-LNPs 2.1 ± 0.5 103 ± 7 288 ± 9 10.9 ± 1.3 PLGA NPs 1.9 ± 0.7 104 ± 5 295 ± 5 11.5 ± 0.9 CS-PLGA NPs 2.2 ± 0.6 99 ± 6 289 ± 8 11.9 ± 1.0 PEG-PLGA NPs 2.3 ± 0.5 102 ± 5 296 ± 8 12.0 ± 0.9 Control (PBS solution) 0 99 ± 12 289 ± 10 11.3 ± 1.2 866 Table 2 867 Figure 4b Click here to download high resolution image Figure 5 Click here to download high resolution image Figure 6 Click here to download high resolution image Figure 7 Click here to download high resolution image Figure 8 Click here to download high resolution image 1 1 EE ± SD (%) LC ± SD (%) LNPs 89.0 ± 9.0 3.6 ± 0.4 CTAB-LNPs 85.4 ± 6.9 7.7 ± 0.6 CS-CTAB-LNPs 86.5 ± 7.4 7.5 ± 0.7 SA-CTAB-LNPs 90.9 ± 7.5 7.0 ± 0.6 CS-SA-CTAB-LNPs 91.3 ± 7.5 5.9 ± 0.8 SA-LNPs 90.9 ± 7.5 7.0 ± 0.6 CS-SA-LNPs 89.0 ± 6.5 6.8 ± 0.7 PEG-LNPs 90 ± 9.0 3.8 ± 0.7 PLGA NPs 73,4 ± 8.0 3,7 ± 0.5 CS-PLGA NPs 75,3 ± 7.2 3,5 ± 0.7 PEG-PLGA NPs 79,4 ± 9.0 3,9 ± 0.6 2 table Click here to download table: Table 1.doc 1 1 Sample Haemolysis (%) sP-selectin release (ng/mL) C3a desArg (ng/mL) T1/2max (min) LNPs 2.2 ± 0.6 98 ± 10 289 ± 9 11.5 ± 1.0 CS-CTAB-LNPs 2.0 ± 0.4 101 ± 8 292 ± 7 10.1 ± 0.9 PEG-LNPs 2.1 ± 0.5 103 ± 7 288 ± 9 10.9 ± 1.3 PLGA NPs 1.9 ± 0.7 104 ± 5 295 ± 5 11.5 ± 0.9 CS-PLGA NPs 2.2 ± 0.6 99 ± 6 289 ± 8 11.9 ± 1.0 PEG-PLGA NPs 2.3 ± 0.5 102 ± 5 296 ± 8 12.0 ± 0.9 Control (PBS solution) 0 99 ± 12 289 ± 10 11.3 ± 1.2 2 table Click here to download table: Table 2.doc