Statistical analysis of solid lipid nanoparticles produced by high-pressure homogenization: a practical prediction approach

Ti le: S a is ical Analysis o Solid Lipid Nanopa icles P oduced by High-P essu e Homogeniza ion: A P ac ical P edic ion App oach Au ho s: Ma ilde Du án-Loba o, Alicia Enguix-González, Me cedes Fe nández-A é alo, Lucía Ma ín-Bande as No e: This is he accep ed manusc ip o he a icle published in Jou nal o Nanopa icle Resea ch. This e sion has been pee - e iewed bu has no unde gone inal edi ing, o ma ing, o ypese ing by Sp inge . Fo he inal published e sion, please e e o: Du án-Loba o, M., Enguix-González, A., Fe nández-A é alo, M., & Ma ín-Bande as, L. (2013). S a is ical Analysis o Solid Lipid Nanopa icles P oduced by High-P essu e Homogeniza ion: A P ac ical P edic ion App oach. Jou nal o Nanopa icle Resea ch, 15(2), A icle 1443. h ps://doi.o g/10.1007/s11051-013-1443-6 1 S a is ical analysis o solid lipid nanopa icles p oduced by high-p essu e homogeniza ion: a 1 p ac ical p edic ion app oach 2 Ma ilde Du án-Loba o1*, Alicia Enguix-González2, Me cedes Fe nández-A é alo1, Lucía Ma ín- 3 Bande as1 4 1Dp o. Fa macia y Tecnología Fa macéu ica. Facul ad de Fa macia. Uni e sidad de Se illa. C/P o . 5 Ga cía González nº2. 41012 Se illa (España). 6 2Dp o. Es adís ica e In es igación Ope a i a. Facul ad de Ma emá icas. Uni e sidad de Se illa. C/Ta ia 7 s/n. 41012 Se illa (España). 8 9 *Co esponding au ho add ess: Dp o. Fa macia y Tecnología Fa macéu ica. Facul ad de Fa macia. 10 Uni e sidad de Se illa. c/P o . Ga cía González nº2. 41012 Se illa (España); Tel: +34 954556618; Fax: 11 +34 954556085; Email: mdu [email protected] 12 13 14 15 16 17 18 19 20 21 22 23 24 Manusc ip Click he e o download Manusc ip : Manusc ip .docx Click he e o iew linked Re e ences 2 Abs ac 25 Lipid Nanopa icles (LNP) a e a p omising ca ie o all adminis a ion ou es due o i s sa e y, small size 26 and high loading o lipophilic compounds. Among he LNP p oduc ion echniques, he easy scale up, lack 27 o o ganic sol en s and sho p oduc ion imes o he high-p essu e homogeniza ion echnique (HPH) 28 make his me hod s and ou . In his s udy, a s a is ical analysis was applied o he p oduc ion o LNP by 29 HPH. Sphe ical LNP wi h mean size anging om 65 nm o 11.623 m, nega i e ze a po en ial (ZP) 30 unde -30 mV and smoo h su ace we e p oduced. Manageable equa ions based on commonly used 31 pa ame e s in he pha maceu ical ield we e ob ained. The lipid o emulsi ie a io (RL/S) was p o ed o 32 s a is ically explain he in luence o oil phase and su ac an concen a ion on inal nanopa icles size. 33 Besides, he homogeniza ion p essu e was ound o ul ima ely de e mine LNP size o a gi en RL/S, while 34 he numbe o mainly de e mined polydispe sion passes applied. α-Tocophe ol was used as a model d ug 35 o illus a e elease p ope ies o LNP as a unc ion o pa icle size, wha was op imized by he eg ession 36 models. This s udy is in ended as a i s s ep o op imize p oduc ion condi ions p io o LNP p oduc ion a 37 bo h labo a o y and indus ial scale om an eminen ly p ac ical app oach, based on pa ame e s 38 ex ensi ely used in o mula ion. 39 Keywo ds 40 Solid lipid nanopa icles, high-p essu e homogeniza ion, s a is ical analysis, eg ession model, pa icle 41 size p edic ion, ma hema ical model, d ug elease. 42 In oduc ion 43 Solid lipid nanopa icles (LNP) consis o ca ie sys ems made om lipids, in which d ug compounds 44 can be inco po a ed. Thei mean pa icle size is in he submic on ange, anging om abou 40 o 1000 45 (nm). Pa icle ma ix is made o a solid lipid o a blend o solid lipids, aiming o accu a e encapsula ion 46 and deli e y o compounds (Pa deike e al. 2009). These ca ie sys ems adop ed some o he bes 47 ea u es o o he colloidal ca ie s such as polyme ic nanopa icles and liposomes and can be made o 48 physiological lipids (biocompa ible and biodeg adable), wha suppo s i s sa e y (Mülle e al. 2000b). 49 Thei small pa icle size allows hem o be used o all ou es o adminis a ion (Sou o and Mülle 2006; 50 Bondì e al. 2010; Mülle e al. 1997). In addi ion, hey ha e shown high encapsula ion a es o lipophilic 51 compounds (Das and Chaudhu y 2011). 52 3 Se e al di e en me hods o he p oduc ion o LNP ha e been desc ibed in he li e a u e. These me hods 53 a e high-p essu e homogeniza ion echnique (Lied ke e al. 2000; Mehne and Mäde 2001), 54 mic oemulsion echnique (Gasco 1997; P iano e al. 2007), emulsi ica ion-sol en e apo a ion me hod 55 (Sjös öm and Be gens ahl 1992), emulsi ica ion-sol en di usion me hod (Hu e al. 2002; T o a e al. 56 2003), sol en injec ion (o sol en displacemen ) me hod (Schube and Mülle -Goymann 2003), phase 57 in e sion me hod (Heu aul e al. 2002), mul iple emulsion echnique (Ga cía-Fuen es e al. 2002), 58 ul asonica ion echnique (Puglia e al. 2008), memb ane con ac o echnique (Cha cosse e al. 2005; El- 59 Ha a i e al. 2006), supe c i ical luid echnique (supe c i ical luid ex ac ion o emulsions (SFEE) 60 (Cha opadhyay e al. 2007) and gas-assis ed mel ing a omiza ion (GAMA) (Salmaso e al. 2009)) and 61 sp ay d ying echnique (Seb i and Amighi 2006). 62 Howe e , he lack o a la ge-scale p oduc ion me hod yielding a p oduc o a quali y ha is accep able by 63 he egula o y au ho i ies (e.g., Food and D ug Adminis a ion) gene ally hinde s he in oduc ion o solid 64 nanopa icles o he ma ke . This lack is due o basic echnological p oblems (e.g. basic scale-up p oblem, 65 oxicologically p oblema ic esidues om he p oduc ion p ocess) and egula o y aspec s such as 66 sui abili y o he p oduc ion uni and p oduc ion p ocess o be quali ied and alida ed (Mülle e al. 67 2000a). Conside ing his, he lack o o ganic sol en s, sho p oduc ion imes and easy scale-up p o ided 68 by he high-p essu e homogeniza ion echnique (HPH) make his me hod highly sui able (Lied ke e al. 69 2000; Mehne and Mäde 2001; Muchow e al. 2008). Ei he he ho o cold high-p essu e 70 homogeniza ion echnique can be applied o ob ain lipid nanopa icles, enabling o adap he p oduc ion 71 acco ding o he physicochemical p ope ies o compounds and he beha io expec ed om he pa icles 72 (zu Mühlen e al. 1998; zu Mühlen and Mehne 1998). Fu he mo e, high-p essu e homogenize s a e 73 widely used in many indus ies including he pha maceu ical indus y, e.g. o he p oduc ion o 74 emulsions o pa en e al nu i ion. Hence, no egula o y p oblems exis o he p oduc ion o LNP using 75 his p oduc ion echnique, which can be conside ed as being indus ially he mos easible one (Mülle e 76 al. 2000a). 77 LNP p oduc ion me hods including HPH p esen howe e ce ain hu dles ha a ec he p oduc quali y, 78 i.e. d ug deg ada ion induced du ing he manu ac u ing p ocess, lipid c ys alliza ion, gela ion phenomena, 79 supe cooled mel s, lipid and pa icle shape modi ica ions and he co-exis ence o se e al colloidal species 80 (Mish a e al. 2012; Sinha e al. 2010). Ne e heless, hese limi a ions can be o e come by moni o ing he 81 4 p oduc ion condi ions ( empe a u e ange, shea s ess, ligh ) and imp o ing he selec ion o he d ug 82 ca ie , he o mula ion and he d ug loading echnique (Mish a e al. 2012; Sinha e al. 2010). The e o e, 83 wha e e p oduc ion echnique is used, iden i ying and op imizing he pa ame e s in luencing he inal 84 p oduc is o pa amoun impo ance, since hese de e mine he d ug deli e y sys em p ope ies. 85 Nowadays, he use o expe imen al designs has become a common me hod o simul aneously analyze he 86 in luence o di e en a iables on he p oduc ion o pa icles (A aujo e al. 2010; Va shosaz e al. 2010), 87 especially ega ding size (Vi o ino e al. 2011). In he case o HPH, many ho ough s udies ocused on 88 emulsions and he homogeniza ion p ocess i sel ha e been ca ied ou (Moh 1987a; Moh 1987b; Flou y 89 e al. 2000; Qian and McClemen s 2010; Mainda ka e al. 2012) bu ewe a ended o LNP (Se e ino e 90 al. 2012). 91 In his s udy, a s a is ical analysis is applied o he p oduc ion o LNP by HPH echnique om a 92 comple ely p ac ical app oach. A wide ange o p essu e alues, excipien concen a ions and numbe o 93 passes is analyzed. Commonly employed pa ame e s a e used o desc ibe he p ocess, leading o 94 manageable equa ions p edic ing pa icle size. The in luence o pa icle size on he inal p ope ies o he 95 deli e y sys em is u he illus a ed using ocophe ol as a model d ug. 96 Expe imen al 97 Ma e ials 98 Glyce ol monos ea a e (Monos ea in, mel ing poin 63 – 68 ºC, Aco a ma, Spain) was used as lipid base. 99 So bi an monos ea a e (Span® 60), polyso ba e 80 (Tween® 80) and α-Tocophe ol we e supplied by 100 Sigma-Ald ich (Spain). Dihyd ogen sodium phospha e and phospho ic acid we e p o ided by Pan eac 101 (Spain). Ace oni ile (HPLC g adien ) was supplied by VWR (Spain). Dis illed wa e was used o all he 102 o mula ions. 103 LNP p epa a ion 104 LNP we e p epa ed by HPH as desc ibed elsewhe e (Zu Mühlen e al. 1998). Oil-in-wa e emulsions 105 we e p epa ed by dispe sing 0.5; 2.5; 5.0; 7.5; 10; 15 % w/ o lipid in dis illed wa e wi h emulsi ie a a 106 concen a ion o 0.12; 0.25; 0.5; 1.0; 1.25; 1.5 % (w/ ) o each lipid concen a ion assayed, yielding a 107 o al numbe o 62 o mula ions. A dispe sing s ep p io o homogeniza ion was pe o med wi h an Ul a- 108 5 Tu ax (IKA®-WERKE, Ge many) a 75ºC. The sys ems ob ained we e passed h ough a homogenize 109 (Panda 2K, Gea Ni o Soa i, I aly) o di e en numbe o passes (1-8) a a ious homogeniza ion 110 p essu es (250-1500 ba ). On accoun o echnical ecommenda ion (Gea Ni o Soa i) and in o de o 111 p e en d op ecoalescence, p essu e a he second al e was ixed a a ela ion 1:5 and 1:10 e e ed o 112 o al p essu e o alues lowe and highe han 600 ba o inal p essu e espec i ely. Samples 113 empe a u e a ia ions due o p essu e we e moni o ed. In he case o α- ocophe ol-loaded LNP, he 114 compound was solubilized in he oil phase o ini ial emulsions a a concen a ion o 10 % (w/w) e e ed 115 o he lipid ma ix. 116 LNP cha ac e iza ion 117 Mean pa icle size and pa icle size dis ibu ion we e measu ed by a lase sca e ing echnique based on 118 Mie heo y (LA-950V2 Ho iba, Japan) a 25 ± 0.5 ºC. Measu emen s we e ca ied ou unde con inuous 119 magne ic agi a ion. Measu e ange was ixed be ween 0.01 and 3000 µm. Samples we e measu ed 120 di ec ly o a e dilu ion wi h dis illed wa e when necessa y. 121 LNP su ace cha ge was de e mined by ze a po en ial (ZP) measu emen s. Pa icles ZP was de e mined 122 by lase Dopple (Ze amas e 300, Mal e n Ins umen s L d, Mal e n, UK) a 25 ± 0.5 ºC. ZP 123 measu emen s we e ca ied ou in iplica e. Samples we e measu ed di ec ly o a e dilu ion wi h 124 dis illed wa e when necessa y. 125 LNP mo phology and su ace we e cha ac e ized by image analysis ob ained by scanning elec on 126 mic oscopy (SEM). Fo his pu pose, a d op o LNP dispe sion was sp ead on ca bon ab p e iously s uck 127 o aluminum s ubs and d ied o e nigh . Samples we e coa ed wi h gold using a spu e coa e 128 (EDWARDS Scancoa Six) and examined in a Jeol 6460LV. Besides, pa icles we e also obse ed by 129 ansmission elec on mic oscopy (TEM). In his case, a d op o LNP dispe sion was sp ead on a ca bon- 130 coa ed 200-mesh coppe g id and d ied o e nigh . Then, a d op o 2 % (w/ ) u anyl ace a e in e hanol was 131 placed on o he g id o 10 min, ollowed by a d op o 2 % (w/ ) lead ci a e o 15 min. The g id was 132 d ied a oom empe a u e and la e obse ed in a Philips CM-10 (Philips, Ge many). 133 S a is ical analysis 134 6 All measu emen s we e pe o med by iplica e on eshly samples p epa ed by iplica e as well. An 135 ini ial s a is ical e alua ion o da a was ca ied ou by one-way analysis o a iance (ANOVA). S a is ical 136 analysis was conside ed signi ican i he p alues we e lowe han 0.05. Fu he s a is ical analysis was 137 pe o med using PASW S a is ics 18 (SPSS Inc., 2010). 138 En apmen e iciency and loading capaci y 139 Conside ing he en apmen e iciency (EE) he amoun o d ug ha can be inco po a ed in o he pa icles 140 and he loading capaci y (LC) he amoun o d ug inco po a ed pe mg o lipid, bo h pa ame e s we e 141 calcula ed acco ding o he ollowing equa ions: 142 The amoun o d ug con ained in he samples was measu ed by e e se phase high pe o mance liquid 143 ch oma og aphy (HPLC) wi h spec opho ome ic de ec ion acco ding o a p e iously published me hod 144 (T ombino e al. 2009) wi h some modi ica ion. B ie ly, 4-5 mg o LNP we e accu a ely weighed and 145 dissol ed in 100 µL DCM. A e 10 min o sonica ion, 900 µL o mobile phase we e added and samples 146 we e sonica ed again o 5 minu es. Then, samples we e il e ed by 420 nm il e and injec ed in o HPLC 147 sys em. A 1-cm ca idge p ecolumn wi h 5-µm C18 Adso bosphe e packing was used. Mobile phase 148 consis ed o 0.01 M dihyd ogen sodium phospha e/0.01 M phospho ic acid wi h ace oni ile (88:12, / ) 149 pH 2.3, a a low a e o 0.5 mL/min. Wa eleng h was ixed a 280 nm on a Jasco UV-2075 de ec o . 150 D ug elease om LNP 151 In o de o s udy he d ug elease om he pa icles, LNP samples we e suspended in phospha e bu e 152 (pH 7.4) con aining Tween® 80 a a concen a ion o 0.1 % (w/ ), main ained a 37 ºC and s i ed 153 mechanically (100 pm) du ing he elease expe imen s (Uni onic OR, Selec a, Spain). Aliquo s (500 µL) 154 we e wi hd awn a ixed ime in e als and il e ed upon cen i uga ion a 8000 pm. Fil e ed samples 155 (Millex GV) (10 µL) we e injec ed in o he HPLC equipmen o quan i y he amoun o α- ocophe ol. 156 Resul s and discussion 157 7 LNP cha ac e iza ion 158 The in luence o lipid concen a ion, su ac an concen a ion, applied p essu e and numbe o passes on 159 mean pa icle size and size dis ibu ion was s udied. Mean pa icle size anged om 65 nm o 11.623 µm. 160 The applied p essu e showed o be a high in luencing pa ame e on pa icle size and size dis ibu ion 161 (Table 1), leading o a dec ease in LNP size as he p essu e was inc eased. The numbe o passes applied 162 induced a s ong dec ease in pa icle size and size dis ibu ion as well, which changed om bimodal o 163 monomodal as he numbe o cycles was inc eased (Fig. 1). Tempe a u e was moni o ed o each pass a 164 e e y alue o p essu e (Table 1), emaining cons an o each alue o p essu e ega dless o he numbe 165 o passes applied and o mula ion composi ion. An inc ease in su ac an concen a ion led o a dec ease 166 in LNP size, while inc easing bo h su ac an and lipid concen a ion in a cons an ela ionship did no 167 induce signi ican changes on pa icle size (Fig. 2). Since he in luence o hese pa ame e s was 168 s a is ically analyzed and ma hema ically desc ibed, hese esul s will be u he commen ed in he sec ion 169 o s a is ical analysis. Inco po a ion o ocophe ol did no in luence pa icle size dis ibu ion (da a no 170 shown). 171 172 Inse Table 1 a ound he e 173 Table 1 Values o applied p essu e and co esponding o mula ion empe a u e and mean pa icle size ob ained by 174 lase sca e ing 175 176 Inse Fig. 1 a ound he e 177 Fig. 1 LNP size dis ibu ions o o mula ions p oduced a 1500 ba o p essu e wi h 0.5 % (w/ ) o Monos ea in and 178 0.5 % (w/ ) o Span® 60 a e : - one homogeniza ion cycle; - wo homogeniza ion cycles; - h ee homogeniza ion 179 cycles; - ou homogeniza ion cycles; - i e homogeniza ion cycles; - six homogeniza ion cycles 180 181 Inse Fig. 2 a ound he e 182 Fig. 2 LNP size dis ibu ion o o mula ions p oduced wi h: - 0.50 % (w/ ) o Monos ea in and 0.25 % (w/ ) o 183 Span® 60; - 2.50 % (w/ ) o Monos ea in and 1.25 % (w/ ) o Span® 60; - 10.00 % (w/ ) o Monos ea in and 5.00 184 % (w/ ) o Span® 60 185 8 ZP o all samples was unde – 30 mV, hus indica ing he suspensions we e s able a p io i. 186 SEM imaging showed sphe ical pa icles wi h a smoo h su ace (Fig. 3). Howe e , only la ge-sized 187 pa icles could be obse ed by SEM, since he ol age needed o cap u e he smalles pa icles was 188 ex emely high and made hem mel . Thus, TEM was applied o con i m he size o he smalles pa icles. 189 T ansmission images enabled o obse e pa icles smalle han 500 nm (Fig. 4), con i ming he e o e he 190 expe imen al esul s. No mo phological di e ences we e ound be ween emp y and ocophe ol-loaded 191 LNP. 192 Inse Fig. 3 a ound he e 193 Fig. 3 SEM images o LNP o mula ions. F om le o igh , op o bo om: pa icles p oduced wi h 10.00 % (w/ ) o 194 lipid and 1.25 % (w/ ) o emulsi ie a 800 ba and 5 cycles; pa icles p oduced wi h 2.50 % (w/ ) o lipid and 0.25 % 195 (w/ ) o emulsi ie a 800 ba and 5 cycles; pa icles p oduced wi h 2.50 % (w/ ) o lipid and 0.12 % (w/ ) o 196 emulsi ie a 1000 ba and 5 cycles; pa icles p oduced wi h 0.25 % o α- ocophe ol, 2.50 % (w/ ) o lipid and 0.25 % 197 (w/ ) o emulsi ie a 800 ba and 6 cycles 198 Inse Fig. 4 a ound he e 199 Fig. 4 TEM images o LNP o mula ions. F om op o bo om: pa icles p oduced wi h 2.50 % (w/ ) o lipid and 1.25 200 % (w/ ) o emulsi ie a 1500 ba and 5 cycles; pa icles p oduced wi h 0.50 % (w/ ) o lipid and 0.5 % (w/ ) o 201 emulsi ie a 1500 ba and 8 cycles 202 S a is ical analysis 203 S a is ical analysis o da a was s uc u ed in sepa a e s ages. Fi s , unc ional alues o p oduc ion 204 condi ions we e es ed o de e mine he ope a ing ange whe e he equipmen pe o mance, and hence he 205 ob ained da a, we e eliable. Maximal p essu e alue was ixed a 1500 ba on accoun o echnical 206 ecommenda ion (Gea Ni o Soa i). Lipid concen a ions we e kep unde 15 % (w/ ), since highe alues 207 yielded low- luid emulsions ha could block homogenize channels. Finally, maximal su ac an 208 concen a ion was ixed a 1.5 % (w/ ) o p e en he o ma ion o bubbles inside he equipmen . 209 Following, and since he e we e no majo di e ences be ween in e media e alues, six alues om each 210 o he abo e a iables we e selec ed; 300; 500; 800; 1000; 1300; 1500 ba o p essu e; 0.5; 2.5; 5.0; 7.5; 211 10; 15 % (w/ ) lipid concen a ion; 0.12; 0.25; 0.5; 1.0; 1.25; 1.5 % (w/ ) emulsi ie concen a ion. A 212 design simila o La in squa e was applied o ensu e ep esen a i eness in he combina ion o alues om 213 p oduc ion condi ions, wi h 288 measu es om 36 ba ches and 8 passes, which p o ided a o al numbe 214 o 458 alid measu es o pa icle size. 215 15 P essu es o 500 ba and 1 o 3 passes a e ypically enough o ob ain small pa icle sizes (Se e ino e al. 385 2012; zu Mühlen e al. 1998), o which highe p essu es and numbe o cycles we e needed in his 386 s udy. This could be owed o he highe d ople sizes o coa se p e-emulsions ha a e p oduced unde he 387 condi ions employed in his wo k. Fu he mo e, main aining he wo king empe a u e unde he base lipid 388 mel ing poin could con ibu e o hese di e ences. HPH o suspensions showed simila beha io s o 389 hose p e iously exposed, ega ding p essu e and numbe o cycles in luence and he e olu ion o 390 bimodal dis ibu ions (Kluge e al. 2012). 391 In iew o all he a iables analyzed in his s udy, i could be deduced ha , o a gi en lipid o su ac an 392 a io, he homogeniza ion p essu e de e mined he ul ima e pa icle size, while he numbe o passes 393 applied de e mined he polydispe si y o ha size dis ibu ion. 394 En apmen e iciency and loading capaci y 395 D ug con en o ocophe ol-loaded LNP was analyzed in ega d o pa icle size. The esul s a e shown in 396 Table 4. Almos comple e loading o ocophe ol was achie ed o all pa icle sizes s udied, wi h EE 397 alues o e 99 % and 10 % o LC. This can be a ibu ed o he ac ha ocophe ol is a highly lipophilic 398 d ug (logP 9.959) and hus has a high a ini y owa ds lipid ma ix. In addi ion, pa icle size did no 399 in luence ocophe ol loading capaci y o he ange o d ug concen a ions assayed. 400 Inse Table 4 a ound he e 401 Table 4 Tocophe ol-loaded LNP size, p oduc ion condi ions (L lipid concen a ion, RL/S lipid o emulsi ie a io, P 402 homogeniza ion p essu e (ba )) and co esponding alues o encapsula ion e iciency (EE) (%) and loading capaci y 403 (LC) (%). 404 D ug elease om LNP 405 In o de o s udy how pa icle size in luences d ug elease om he nanopa icles, samples o ocophe ol- 406 loaded LNP wi h di e en pa icle size bu equal d ug con en we e compa ed. Release s udies esul s a e 407 illus a ed in Fig. 8. All o mula ions showed an ini ial bu s elease, p obably due o d ug and su ac an 408 adso bed in pa icles su ace and a d ug shell-en iched s uc u e o he nanopa icles (Schä e -Ko ing e 409 al. 2007). On one hand, he p esence o su ac an was epo ed o accele a e d ug elease (R. H. Mülle e 410 al. 1994). On he o he hand, a d ug shell-en iched s uc u e is ypically ob ained when he d ug mel ing 411 poin is unde he ma ix lipid mel ing poin , as in his case (2 ºC o α- ocophe ol; 63-68 ºC o 412 16 Monos ea in). In he p oduc ion p ocess, lipid p ecipi a ion akes place i s leading o a phase sepa a ion 413 du ing he cooling p ocess. The lipid occupies he e o e he pa icle co e while he compound is 414 accumula ed wi hin he shell, eaching he elease medium as e . Besides, i can be clea ly app ecia ed 415 ha smalle pa icle sizes induced a as e elease. This was expec ed since smalle sizes imply a highe 416 con ac su ace o he pa icle wi h he ex e nal medium, so d ug di usion is a o ed. Consequen ly, a 417 de e mined pa icle size should be aimed o a desi ed elease, wha can be easily done by means o he 418 equa ions p esen ed in his s udy. 419 Inse Figu e 8 a ound he e 420 Fig. 8 Tocophe ol elease om LNP wi h 10 % o ocophe ol LC as a unc ion o pa icle size 421 Conclusions 422 LNP a e a p omising d ug deli e y sys em o all adminis a ion ou es. HPH is a highly ad an ageous 423 LNP p oduc ion echnique a bo h labo a o y and indus ial scale. In his wo k, a s a is ical analysis was 424 ca ied ou aiming o unde s and and con ol he pa ame e s in luencing he p oduc ion o LNP by HPH, 425 and he e o e hei inal p ope ies. Manageable equa ions based on commonly used pa ame e s in he 426 pha maceu ical ield we e ob ained. The lipid o emulsi ie concen a ion a io (RL/S) was p o ed o 427 s a is ically explain he in luence o oil phase and su ac an concen a ion on inal nanopa icles size. 428 Besides, he homogeniza ion p essu e was ound o ul ima ely de e mine LNP size o a gi en RL/S, while 429 polydispe sion was mainly de e mined by he numbe o passes applied. Fu he s udies ega ding 430 di e en lipids and ypes o emulsi ie could ex end he condi ions co e ed by hese eg ession models. 431 This s udy is in ended as a i s s ep o op imize p oduc ion condi ions p io o LNP p oduc ion a bo h 432 labo a o y and indus ial scale om an eminen ly p ac ical app oach, based on ex ensi ely used 433 pa ame e s in o mula ion. 434 Acknowledgmen s 435 M.D.L. hanks Uni e si y o Se ille o a g an om IV Resea ch Plan o Uni e si y o Se ille. L.M.B. is 436 especially g a e ul o Jun a de Andalucía (Spain) o inancial suppo (P ojec N . P09-CTS5029). 437 Mic oscopy Se ices (Cen o de In es igación, Tecnología e Inno ación de la Uni e sidad de Se illa, 438 CITIUS) echnical suppo is also g a e ul. Au ho s also hank D . Ál a ez-Fuen es o echnical suppo . 439 17 440 Re e ences 441 A aujo J, Gonzalez-Mi a E, Egea MA, Ga cia ML, Sou o EB (2010) Op imiza ion and physicochemical 442 cha ac e iza ion o a iamcinolone ace onide-loaded NLC o ocula an iangiogenic applica ions. 443 In J Pha m 393:167–175 444 Biasu i M, Veni E, Ma chesini G, Innocen e N (2010) Rheological p ope ies o model dai y emulsions 445 as a ec ed by high p essu e homogeniza ion. Inno Food Sci & Eme g Tech 11:580–586 446 Bondì ML, C apa o EF, Giammona G, D ago F (2010) B ain- a ge ed solid lipid nanopa icles con aining 447 iluzole: p epa a ion, cha ac e iza ion and biodis ibu ion. Nanomed (London, England) 5:25–32 448 Cha cosse C, El-Ha a i A, Fessi H (2005) P epa a ion o solid lipid nanopa icles using a memb ane 449 con ac o . 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Langmui 24:12758–12765 551 Figu e Click he e o download Figu e: Fig1.docx Figu e Click he e o download Figu e: Fig2.docx Figu e Click he e o download Figu e: Fig3.docx Figu e Click he e o download Figu e: Fig4.docx Figu e Click he e o download Figu e: Fig5.docx Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 Va iable Va iable Va iable Va iable Va iable Va iable Va iable Va iable 0.758 -0.652 -0.803 -0.778 -0.873 0.912 0.927 0.925 -0.751 -0.644 -0.785 -0.769 0.852 -0.895 -0.915 -0.908 0.733 0.618 0.773 0.739 -0.847 0.892 0.904 0.907 -0.71 -0.612 -0.745 -0.733 -0.802 0.863 0.871 0.879 0.699 -0.609 -0.74 -0.729 -0.796 0.862 0.87 0.88 0.698 -0.567 0.71 -0.683 0.794 -0.84 -0.86 0.855 0.668 0.555 -0.69 0.668 -0.743 0.835 0.842 -0.85 -0.656 -0.53 0.646 -0.641 0.731 -0.78 -0.799 -0.786 -0.664 0.493 -0.646 0.598 0.731 -0.771 -0.789 -0.776 -0.607 0.492 0.645 0.597 0.683 -0.71 -0.726 -0.709 -0.566 0.447 0.597 0.545 -0.697 -0.663 -0.675 -0.656 -0.376 0.33 0.148 0.157 0.149 0.226 0.223 0.271 -0.002 0.011 -0.009 -0.003 0.004 -0.019 -0.024 -0.025 able Click he e o download able: Table3.docx L (% w/ ) RL/S P* (ba ) Cycle Mean size (nm) EE (%) LC (%) 0.50 1 1500 8 71 ± 8 91 ± 9 9 ± 1 0.50 2 1300 5 188 ± 10 96 ± 5 8 ± 2 0.50 2 1000 6 305 ± 12 94 ± 9 10 ± 2 2.50 20 1000 6 487 ± 15 98 ± 6 10 ± 1 2.50 20 1000 5 608 ± 14 97 ± 7 9 ± 2 2.50 10 800 6 886 ± 18 92 ± 10 10 ± 2 0.50 2 800 6 994 ± 22 94 ± 7 7 ± 2 able Click he e o download able: Table4.docx