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
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S a is ical analysis o solid lipid nanopa icles p oduced by high-p essu e homogeniza ion: a
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p ac ical p edic ion app oach
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Ma ilde Du án-Loba o1*, Alicia Enguix-González2, Me cedes Fe nández-A é alo1, Lucía Ma ín-
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Bande as1
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1Dp o. Fa macia y Tecnología Fa macéu ica. Facul ad de Fa macia. Uni e sidad de Se illa. C/P o .
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Ga cía González nº2. 41012 Se illa (España).
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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
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s/n. 41012 Se illa (España).
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*Co esponding au ho add ess: Dp o. Fa macia y Tecnología Fa macéu ica. Facul ad de Fa macia.
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Uni e sidad de Se illa. c/P o . Ga cía González nº2. 41012 Se illa (España); Tel: +34 954556618; Fax:
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+34 954556085; Email: mdu [email protected]
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Manusc ip
Click he e o download Manusc ip : Manusc ip .docx
Click he e o iew linked Re e ences
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Abs ac
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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
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and high loading o lipophilic compounds. Among he LNP p oduc ion echniques, he easy scale up, lack
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o o ganic sol en s and sho p oduc ion imes o he high-p essu e homogeniza ion echnique (HPH)
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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
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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)
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unde -30 mV and smoo h su ace we e p oduced. Manageable equa ions based on commonly used
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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
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s a is ically explain he in luence o oil phase and su ac an concen a ion on inal nanopa icles size.
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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
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he numbe o mainly de e mined polydispe sion passes applied. α-Tocophe ol was used as a model d ug
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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
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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
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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
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ex ensi ely used in o mula ion.
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Keywo ds
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Solid lipid nanopa icles, high-p essu e homogeniza ion, s a is ical analysis, eg ession model, pa icle
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size p edic ion, ma hema ical model, d ug elease.
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In oduc ion
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Solid lipid nanopa icles (LNP) consis o ca ie sys ems made om lipids, in which d ug compounds
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can be inco po a ed. Thei mean pa icle size is in he submic on ange, anging om abou 40 o 1000
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(nm). Pa icle ma ix is made o a solid lipid o a blend o solid lipids, aiming o accu a e encapsula ion
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and deli e y o compounds (Pa deike e al. 2009). These ca ie sys ems adop ed some o he bes
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ea u es o o he colloidal ca ie s such as polyme ic nanopa icles and liposomes and can be made o
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physiological lipids (biocompa ible and biodeg adable), wha suppo s i s sa e y (Mülle e al. 2000b).
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Thei small pa icle size allows hem o be used o all ou es o adminis a ion (Sou o and Mülle 2006;
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Bondì e al. 2010; Mülle e al. 1997). In addi ion, hey ha e shown high encapsula ion a es o lipophilic
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compounds (Das and Chaudhu y 2011).
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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
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a e high-p essu e homogeniza ion echnique (Lied ke e al. 2000; Mehne and Mäde 2001),
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mic oemulsion echnique (Gasco 1997; P iano e al. 2007), emulsi ica ion-sol en e apo a ion me hod
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(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.
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2003), sol en injec ion (o sol en displacemen ) me hod (Schube and Mülle -Goymann 2003), phase
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in e sion me hod (Heu aul e al. 2002), mul iple emulsion echnique (Ga cía-Fuen es e al. 2002),
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ul asonica ion echnique (Puglia e al. 2008), memb ane con ac o echnique (Cha cosse e al. 2005; El-
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Ha a i e al. 2006), supe c i ical luid echnique (supe c i ical luid ex ac ion o emulsions (SFEE)
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(Cha opadhyay e al. 2007) and gas-assis ed mel ing a omiza ion (GAMA) (Salmaso e al. 2009)) and
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sp ay d ying echnique (Seb i and Amighi 2006).
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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
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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
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nanopa icles o he ma ke . This lack is due o basic echnological p oblems (e.g. basic scale-up p oblem,
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oxicologically p oblema ic esidues om he p oduc ion p ocess) and egula o y aspec s such as
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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.
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2000a). Conside ing his, he lack o o ganic sol en s, sho p oduc ion imes and easy scale-up p o ided
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by he high-p essu e homogeniza ion echnique (HPH) make his me hod highly sui able (Lied ke e al.
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2000; Mehne and Mäde 2001; Muchow e al. 2008). Ei he he ho o cold high-p essu e
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homogeniza ion echnique can be applied o ob ain lipid nanopa icles, enabling o adap he p oduc ion
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acco ding o he physicochemical p ope ies o compounds and he beha io expec ed om he pa icles
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(zu Mühlen e al. 1998; zu Mühlen and Mehne 1998). Fu he mo e, high-p essu e homogenize s a e
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widely used in many indus ies including he pha maceu ical indus y, e.g. o he p oduc ion o
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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
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his p oduc ion echnique, which can be conside ed as being indus ially he mos easible one (Mülle e
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al. 2000a).
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LNP p oduc ion me hods including HPH p esen howe e ce ain hu dles ha a ec he p oduc quali y,
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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,
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supe cooled mel s, lipid and pa icle shape modi ica ions and he co-exis ence o se e al colloidal species
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(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
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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
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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,
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wha e e p oduc ion echnique is used, iden i ying and op imizing he pa ame e s in luencing he inal
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p oduc is o pa amoun impo ance, since hese de e mine he d ug deli e y sys em p ope ies.
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Nowadays, he use o expe imen al designs has become a common me hod o simul aneously analyze he
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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),
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especially ega ding size (Vi o ino e al. 2011). In he case o HPH, many ho ough s udies ocused on
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emulsions and he homogeniza ion p ocess i sel ha e been ca ied ou (Moh 1987a; Moh 1987b; Flou y
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e al. 2000; Qian and McClemen s 2010; Mainda ka e al. 2012) bu ewe a ended o LNP (Se e ino e
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al. 2012).
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In his s udy, a s a is ical analysis is applied o he p oduc ion o LNP by HPH echnique om a
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comple ely p ac ical app oach. A wide ange o p essu e alues, excipien concen a ions and numbe o
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passes is analyzed. Commonly employed pa ame e s a e used o desc ibe he p ocess, leading o
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manageable equa ions p edic ing pa icle size. The in luence o pa icle size on he inal p ope ies o he
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deli e y sys em is u he illus a ed using ocophe ol as a model d ug.
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Expe imen al
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Ma e ials
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Glyce ol monos ea a e (Monos ea in, mel ing poin 63 – 68 ºC, Aco a ma, Spain) was used as lipid base.
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So bi an monos ea a e (Span® 60), polyso ba e 80 (Tween® 80) and α-Tocophe ol we e supplied by
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Sigma-Ald ich (Spain). Dihyd ogen sodium phospha e and phospho ic acid we e p o ided by Pan eac
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(Spain). Ace oni ile (HPLC g adien ) was supplied by VWR (Spain). Dis illed wa e was used o all he
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o mula ions.
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LNP p epa a ion
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LNP we e p epa ed by HPH as desc ibed elsewhe e (Zu Mühlen e al. 1998). Oil-in-wa e emulsions
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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
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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
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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-
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Tu ax (IKA®-WERKE, Ge many) a 75ºC. The sys ems ob ained we e passed h ough a homogenize
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(Panda 2K, Gea Ni o Soa i, I aly) o di e en numbe o passes (1-8) a a ious homogeniza ion
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p essu es (250-1500 ba ). On accoun o echnical ecommenda ion (Gea Ni o Soa i) and in o de o
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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
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o al p essu e o alues lowe and highe han 600 ba o inal p essu e espec i ely. Samples
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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
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compound was solubilized in he oil phase o ini ial emulsions a a concen a ion o 10 % (w/w) e e ed
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o he lipid ma ix.
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LNP cha ac e iza ion
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Mean pa icle size and pa icle size dis ibu ion we e measu ed by a lase sca e ing echnique based on
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Mie heo y (LA-950V2 Ho iba, Japan) a 25 ± 0.5 ºC. Measu emen s we e ca ied ou unde con inuous
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magne ic agi a ion. Measu e ange was ixed be ween 0.01 and 3000 µm. Samples we e measu ed
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di ec ly o a e dilu ion wi h dis illed wa e when necessa y.
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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
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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
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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
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dis illed wa e when necessa y.
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LNP mo phology and su ace we e cha ac e ized by image analysis ob ained by scanning elec on
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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
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o aluminum s ubs and d ied o e nigh . Samples we e coa ed wi h gold using a spu e coa e
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(EDWARDS Scancoa Six) and examined in a Jeol 6460LV. Besides, pa icles we e also obse ed by
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ansmission elec on mic oscopy (TEM). In his case, a d op o LNP dispe sion was sp ead on a ca bon-
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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
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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
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d ied a oom empe a u e and la e obse ed in a Philips CM-10 (Philips, Ge many).
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S a is ical analysis
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All measu emen s we e pe o med by iplica e on eshly samples p epa ed by iplica e as well. An
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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
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analysis was conside ed signi ican i he p alues we e lowe han 0.05. Fu he s a is ical analysis was
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pe o med using PASW S a is ics 18 (SPSS Inc., 2010).
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En apmen e iciency and loading capaci y
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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
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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
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calcula ed acco ding o he ollowing equa ions:
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The amoun o d ug con ained in he samples was measu ed by e e se phase high pe o mance liquid
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ch oma og aphy (HPLC) wi h spec opho ome ic de ec ion acco ding o a p e iously published me hod
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(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
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dissol ed in 100 µL DCM. A e 10 min o sonica ion, 900 µL o mobile phase we e added and samples
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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
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sys em. A 1-cm ca idge p ecolumn wi h 5-µm C18 Adso bosphe e packing was used. Mobile phase
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consis ed o 0.01 M dihyd ogen sodium phospha e/0.01 M phospho ic acid wi h ace oni ile (88:12, / )
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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 .
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D ug elease om LNP
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In o de o s udy he d ug elease om he pa icles, LNP samples we e suspended in phospha e bu e
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(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
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mechanically (100 pm) du ing he elease expe imen s (Uni onic OR, Selec a, Spain). Aliquo s (500 µL)
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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
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(Millex GV) (10 µL) we e injec ed in o he HPLC equipmen o quan i y he amoun o α- ocophe ol.
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Resul s and discussion
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LNP cha ac e iza ion
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The in luence o lipid concen a ion, su ac an concen a ion, applied p essu e and numbe o passes on
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mean pa icle size and size dis ibu ion was s udied. Mean pa icle size anged om 65 nm o 11.623 µm.
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The applied p essu e showed o be a high in luencing pa ame e on pa icle size and size dis ibu ion
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(Table 1), leading o a dec ease in LNP size as he p essu e was inc eased. The numbe o passes applied
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induced a s ong dec ease in pa icle size and size dis ibu ion as well, which changed om bimodal o
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monomodal as he numbe o cycles was inc eased (Fig. 1). Tempe a u e was moni o ed o each pass a
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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
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o passes applied and o mula ion composi ion. An inc ease in su ac an concen a ion led o a dec ease
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in LNP size, while inc easing bo h su ac an and lipid concen a ion in a cons an ela ionship did no
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induce signi ican changes on pa icle size (Fig. 2). Since he in luence o hese pa ame e s was
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s a is ically analyzed and ma hema ically desc ibed, hese esul s will be u he commen ed in he sec ion
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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
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shown).
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Inse Table 1 a ound he e
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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
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lase sca e ing
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Inse Fig. 1 a ound he e
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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
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0.5 % (w/ ) o Span® 60 a e : - one homogeniza ion cycle; - wo homogeniza ion cycles; - h ee homogeniza ion
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cycles; - ou homogeniza ion cycles; - i e homogeniza ion cycles; - six homogeniza ion cycles
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Inse Fig. 2 a ound he e
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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
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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
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% (w/ ) o Span® 60
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ZP o all samples was unde – 30 mV, hus indica ing he suspensions we e s able a p io i.
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SEM imaging showed sphe ical pa icles wi h a smoo h su ace (Fig. 3). Howe e , only la ge-sized
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pa icles could be obse ed by SEM, since he ol age needed o cap u e he smalles pa icles was
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ex emely high and made hem mel . Thus, TEM was applied o con i m he size o he smalles pa icles.
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T ansmission images enabled o obse e pa icles smalle han 500 nm (Fig. 4), con i ming he e o e he
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expe imen al esul s. No mo phological di e ences we e ound be ween emp y and ocophe ol-loaded
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LNP.
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Inse Fig. 3 a ound he e
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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
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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 %
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(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
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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 %
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(w/ ) o emulsi ie a 800 ba and 6 cycles
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Inse Fig. 4 a ound he e
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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
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% (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
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emulsi ie a 1500 ba and 8 cycles
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S a is ical analysis
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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
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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
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ob ained da a, we e eliable. Maximal p essu e alue was ixed a 1500 ba on accoun o echnical
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ecommenda ion (Gea Ni o Soa i). Lipid concen a ions we e kep unde 15 % (w/ ), since highe alues
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yielded low- luid emulsions ha could block homogenize channels. Finally, maximal su ac an
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concen a ion was ixed a 1.5 % (w/ ) o p e en he o ma ion o bubbles inside he equipmen .
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Following, and since he e we e no majo di e ences be ween in e media e alues, six alues om each
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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;
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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
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design simila o La in squa e was applied o ensu e ep esen a i eness in he combina ion o alues om
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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
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o 458 alid measu es o pa icle size.
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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.
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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
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condi ions employed in his wo k. Fu he mo e, main aining he wo king empe a u e unde he base lipid
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mel ing poin could con ibu e o hese di e ences. HPH o suspensions showed simila beha io s o
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hose p e iously exposed, ega ding p essu e and numbe o cycles in luence and he e olu ion o
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bimodal dis ibu ions (Kluge e al. 2012).
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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
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a io, he homogeniza ion p essu e de e mined he ul ima e pa icle size, while he numbe o passes
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applied de e mined he polydispe si y o ha size dis ibu ion.
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En apmen e iciency and loading capaci y
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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
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Table 4. Almos comple e loading o ocophe ol was achie ed o all pa icle sizes s udied, wi h EE
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alues o e 99 % and 10 % o LC. This can be a ibu ed o he ac ha ocophe ol is a highly lipophilic
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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
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in luence ocophe ol loading capaci y o he ange o d ug concen a ions assayed.
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Inse Table 4 a ound he e
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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
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homogeniza ion p essu e (ba )) and co esponding alues o encapsula ion e iciency (EE) (%) and loading capaci y
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(LC) (%).
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D ug elease om LNP
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In o de o s udy how pa icle size in luences d ug elease om he nanopa icles, samples o ocophe ol-
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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
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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
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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
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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
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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
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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
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Figu e
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Figu e
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Figu e
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Figu e
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Figu e
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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
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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