Ti le: Compa a i e S udy o Chi osan- and PEG-Coa ed Lipid and PLGA Nanopa icles as O al
Deli e y Sys ems o Cannabinoids
Au ho s: Ma ilde Du án-Loba o, Lucía Ma ín-Bande as, Lídia M. D. Gonçal es, Me cedes
Fe nández-A é alo, An ónio J. Almeida
No e:
This is he p ep in e sion o he manusc ip submi ed o Jou nal o Nanopa icle Resea ch.
This e sion has no been pee - e iewed o edi ed and may di e signi ican ly om he inal
published e sion.
Fo he pee - e iewed e sion, please e e o:
Du án-Loba o, M., Ma ín-Bande as, L., Gonçal es, L. M. D., Fe nández-A é alo, M., &
Almeida, A. J. (2015). Compa a i e s udy o chi osan- and PEG-coa ed lipid and PLGA
nanopa icles as o al deli e y sys ems o cannabinoids. Jou nal o Nanopa icle Resea ch,
17(61). h ps://doi.o g/10.1007/s11051-015-2875-y
1
Compa a i e s udy o chi osan- and PEG-coa ed lipid and polyme ic nanopa icles
1
as o al deli e y sys ems o cannabinoids
2
3
4
Ma ilde Du án-Loba o1,2, Lucía Ma ín-Bande as1*, Lídia M.D. Gonçal es2, Me cedes
5
Fe nández-A é alo1, An onio J. Almeida2.
6
7
1Facul ad de Fa macia, Uni e sidad de Se illa, P o eso Ga cía González, 2, 41012
8
Se illa, Spain.
9
10
2Resea ch Ins i u e o Medicines and Pha maceu ical Sciences (iMed.UL), Faculdade
11
de Fa mácia da Uni e sidade de Lisboa, A enida P o esso Gama Pin o, 1649-003
12
Lisboa, Po ugal.
13
14
15
* Co esponding au ho add ess: Dp o. Fa macia y Tecnología Fa macéu ica, Facul ad
16
de Fa macia, Uni e sidad de Se illa. C/ P o eso Ga cía González, 2, 41012, Se illa,
17
España; Tel; +34 954556754; Fax: 954556085; E-mail: [email p o ec ed].
18
19
20
Manusc ip
Click he e o download Manusc ip : enamed_cd002.docx
Click he e o iew linked Re e ences
2
Abs ac
21
The cannabinoid de i a i e 1-naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone
22
(CB13) has an impo an he apeu ic po en ial as analgesic in ch onic pain s a es ha
23
espond poo ly o con en ional d ugs. Howe e , he incidence o i s mild- o-mode a e
24
and dose-dependen ad e se e ec s (AEs), as well as i s pha macokine ic p o ile
25
ac ually holds back i s use in humans. Thus, he use o a sui able ca ie sys em o o al
26
deli e y o CB13 becomes an a ac i e s a egy o de elop a aluable he apy.
27
Polyme ic PLGA and lipid nanopa icles a e widely s udied deli e y ehicles ha
28
imp o e he bioa ailabili y o lipophilic compounds and p esen special in e es in o al
29
deli e y. Thei su ace can be modi ied o imp o e he adhesion o pa icles o he o al
30
mucosa and inc ease hei ci cula ion ime in blood wi h addi i es such as chi osan (CS)
31
and polye hylene glycol (PEG), which can be easibly inco po a ed on o hese pa icles
32
in a pos -p oduc ion s ep. In his wo k, CS- and PEG-modi ied polyme ic PLGA and
33
lipid nanopa icles we e compa a i ely e alua ed unde he same expe imen al
34
condi ions as o al ca ie s o CB13, a Class II (BCS) model d ug wi h high
35
he apeu ical po en ial. Physicochemical cha ac e is ics o modi ied and non-modi ied
36
NPs, blood compa ibili y, cy o oxici y and up ake in Caco-2 and THP-1 cell lines we e
37
s udied as a compa ison o hei po en ial as o al deli e y sys ems o CB13.
38
Keywo ds
39
cannabinoids, o al adminis a ion, neu opa hic pain, lipid nanopa icles, PLGA
40
nanopa icles
41
42
3
In oduc ion
43
44
Cannabinoids p esen an impo an he apeu ic po en ial in a wide ange o synd omes
45
and diseases (Ben Ama 2006; Pe wee 2001), especially as analgesics in ch onic pain
46
s a es ha espond poo ly o con en ional d ugs such as mo phine (I e sen and
47
Chapman 2002). Howe e , cannabinoids also p esen undesi able physicochemical
48
p ope ies such as poo s abili y and solubili y as well as ad e se side e ec s (AEs),
49
which ac ually hold back hei use in humans (A al e al. 2004; Hall and Solowij 1998).
50
The cannabinoid de i a i e 1-naph halenyl[4-(pen yloxy)-1-naph halenyl]me hanone
51
(cannabinoid ecep o agonis 13, CRA-13, CB13) is a no el cannabinoid ecep o
52
agonis ha s ands ou owing o i s less pene a ion in o he b ain han o he
53
cannabinoids and consequen ly less p onounced AEs (Dziadulewicz e al. 2007). In
54
hese e ms, he incidence o mild- o-mode a e and dose-dependen AEs as well as he
55
pha macokine ics p o ile o his compound should be conside ed (Ga din e al. 2009).
56
CB13 belongs o Class II o compounds (low solubili y and high pe meabili y) o he
57
Biopha maceu ics Classi ica ion Sys em (BCS), which de e mines a highly a iable
58
abso p ion (Ga din e al. 2009) and hence une en plasma ic concen a ions and wi h
59
consequen incidence o AEs.
60
Thus, he use o a sui able ca ie sys em o o al deli e y o CB13 becomes an
61
a ac i e s a egy o de elop a aluable he apy. Speci ically, polyme ic poly(DL-
62
lac ide-co-glycolide) (PLGA) and lipid nanopa icles (LNPs) a e some o he mos
63
widely s udied he apeu ic deli e y ehicles o imp o e he bioa ailabili y o lipophilic
64
compounds and p esen special in e es in o al deli e y (Zhang e al. 2013). PLGA
65
nanopa icles (NPs) p o ide biocompa ibili y, con olled d ug elease and deg ada ion
66
in o comple ely sa e p oduc s (PLGA monome s). In addi ion, his polyme is
67
4
comme cially a ailable in di e en g ades and i s glass ansi ion empe a u e (Tg) is
68
abo e physiological empe a u e o 37 °C, lending he equi ed mechanical s eng h o
69
o mula ion de elopmen (Ma in-Bande as e al. 2012). On he o he hand, LNPs can
70
be made o physiological lipids (biocompa ible and biodeg adable) wi h a wide ange o
71
me hodologies (Almeida and Sou o 2007; Pa hi and Su esh 2010). Thei use o o al
72
d ug deli e y has been s ongly de eloped (F icke e al. 2010; Ha de e al. 2011) and
73
hey ha e shown high a es o encapsula ion o lipophilic compounds and imp o emen
74
o gas oin es inal (GI) abso p ion and o al bioa ailabi y o se e al d ugs (Das and
75
Chaudhu y 2011).
76
These NPs p esen some d awbacks as o al deli e y ca ie s as well, e.g. hei sligh ly
77
nega i e su ace cha ge ha ends o p e en he in e ac ion wi h he in es inal mucosa
78
(Ma in-Bande as e al. 2012; Seme e e al. 2012; Zhang e al. 2012). None heless,
79
su ace p ope ies o NPs can be gene ally modi ied ei he by coa ing hei su ace wi h
80
hyd ophilic s abilize s, bioadhesi e polyme s o su ac an s o by inco po a ing in he
81
o mula ion biodeg adable copolyme s con aining hyd ophilic moie ies (Ma in-
82
Bande as e al. 2013). These modi ica ions mainly change he ze a po en ial (ZP) and
83
hyd ophobici y o he NPs, and he e o e de e mine hei colloidal s abili y,
84
mucoadhesion p ope ies, o al abso p ion and he adso p ion o p o eins on he su ace
85
(des Rieux e al. 2006).
86
Chi osan (CS) is a biodeg adable and biocompa ible polyme ha has been widely
87
s udied as po en ial o al ansmucosal abso p ion enhance (Issa e al. 2005; Zhang e al.
88
2012) and can be easibly inco po a ed on o nega i ely cha ged su ace o nanopa icles
89
in a pos -p oduc ion s ep wi h no in luence o e he p epa a ion p ocedu e (Du án-
90
Loba o e al. 2014; Sa men o e al. 2011). Polye hylene glycol (PEG) is a hyd ophilic
91
and also biocompa ible polyme ha has been shown o acili a e he anspo h ough
92

5
he Peye ’s p a ches GALT (Ga ino e al. 2007), p e en he enzyma ic deg ada ion o
93
NPs in GI luids (Tobıo e al. 2000; Vila e al. 2002; Vila e al. 2004), minimize
94
opsoniza ion (Singh and Lilla d 2009) and inc ease he sys emic ci cula ion ime in i o
95
o pa icles (Knop e al. 2010; Seme e e al. 2012), leading o a signi ican enhancemen
96
o bioa ailabili y o he encapsula ed d ug in blood s eam and lympha ics (Tobio e al.
97
; Tobı o e al. 2000; Vila e al. 2002 . The addi ion o PEG molecules o NPs
98
su ace can be achie ed ia a numbe o di e en ou es (Ma in-Bande as e al. 2013).
99
In his wo k, polyme ic PLGA NPs and LNPs we e p oduced and e alua ed unde he
100
same expe imen al condi ions and in a compa a i e manne as ca ie s o o al deli e y
101
o CB13. CS and PEG ha e been employed as su ace-modi ying addi i es o imp o e
102
CB13-loaded PLGA NPs and LNPs in es inal up ake and p e en biological clea ance
103
mechanisms. Physicochemical cha ac e is ics o modi ied and non-modi ied NPs ei he
104
wi h CS o PEG -mean pa icle size, ze a po en ial and d ug loading-, cy o oxici y and
105
up ake in Caco-2 cell line and up ake in THP1 cell line we e s udied. The p esen
106
in es iga ion aims o show and accu a ely compa e he po en ial o CS and PEG-
107
modi ied PLGA NPs and LNPs o imp o e he o al deli e y o CB13, a BCS Class II
108
and cannabinoid model d ug wi h high he apeu ic po en ial.
109
Ma e ials and me hods
110
Ma e ials
111
CB13 was p o ided by Toc is Cookson L d. (B is ol, UK). Poly(DL-lac ide-co-
112
glycolide) (PLGA 50:50) Resome ® RG 502 was ob ained om Boeh inge -Ingelheim
113
(Ingelheim, Ge many). Chi osan (CS) low MW, sodium deoxychola e (SD), Span® 60,
114
Tween20, Tween 80, s ea ylamine (SA), ce yl ime hylammonium b omide (CTAB),
115
and Plu onic® F-68 and, Nile Red and hodamine we e p o ided by Sigma-Ald ich.
116
6
P eci ol® ATO 5 (glyce yl palmi os ea a e, mel ing poin : 53-56 ºC) was kindly gi en
117
by Ga e ossé (Sain -P ies , Cedex, F ance). Soya leci hin (Lipoid S100) was pu chased
118
om Lipoid (Ludwigsha en, Ge many). Th ealose was ob ained om VWR
119
In e na ional Eu olab S.L. (Ba celona, Spain). HPLC-g ade ace oni ile, ace ic acid, and
120
e hyl ace a e we e pu chased om Pan eac (Spain). Glyce ol was ob ained om
121
Aco a ma Dis ibución S.A. (Ba celona, Spain).
122
Fo cell line expe imen s, human colon adenoca cinoma cells (Caco-2) cells we e
123
ob ained om he Eu opean Collec ion o Cell Cul u es (ECACC) (Salisbu y, UK).
124
Di e en ia e mac ophage THP1 cells (human monocy ic cell line) we e ob ained om
125
ATCC TIB-202™ (Ba celona, Spain). Minimum Essen ial Medium Eagle (MEM wi h
126
Ea le´s sal s wi hou L-glu amine), RPMl 1640 medium, sodium py u a e, MEM non-
127
essen ial amino acids, L-glu amine and e al bo ine se um we e ob ained om PAA
128
Labo a o ies (Pasching, Aus ia). Gen amicin was pu chased om Gibco® Li e
129
Technologies Co po a ion (NY, USA). T ypsin/EDTA, MTT (Thiazolyl Blue
130
Te azolium B omide o Me hyl hiazolyldiphenyl- e azolium b omide), SDS (Sodium
131
Dodecyl Sul a e), PFA (pa a o maldehyde) and Hoechs 33258 we e pu chased om
132
Sigma-Ald ich (S Louis, MO). The P oLong® Gold an i ade eagen con aining he
133
blue- luo escen nuclea coun e s ain DAPI was ob ained om In i ogen.
134
Me hods
135
LNPs p epa a ion
136
LNPs we e p oduced using he emulsi ica ion-sol en e apo a ion me hod p e iously
137
desc ibed (Lopes e al. 2012). B ie ly, P eci ol® and leci hin we e dissol ed in
138
dichlo ome hane and hen added o he aqueous phase con aining Tween®20 and
139
sodium deoxychola e. The dispe sion s ep was pe o med du ing 2.5 min pe iod o
140
7
sonica ion (B anson Soni ie 250, Danbu y, USA). A e wa ds, his dispe sion was
141
homogenized o 3.5 min a 125000 pm (Sil e son High Speed Mixe L5M, Sil e son
142
Machines, UK). The nanopa icle dispe sion was hen kep unde s i ing o 4 h a
143
oom empe a u e un il comple e e apo a ion o he dichlo ome hane. When
144
inco po a ing CB 13, he d ug was added o he o ganic phase a 10% d ug/lipid
145
espec i ely. In he case o luo och ome-loaded pa icles, 20 μL o a mg/mL
146
hodamine solu ion we e added o he o ganic phase ins ead.
147
To p oduce ca ionic LNPs, s ea ylamine (Pede sen e al. 2006) and/o ce yl
148
ime hylammonium b omide (CTAB) (Taba e al. 2004) we e included in he
149
o mula ion in absence o leci hin. When inco po a ed, 10 mg o s ea ylamine we e
150
added o he o ganic phase p io o homogeniza ion. CTAB eplaced sodium
151
deoxychola e in he aqueous phase and was added a a concen a ion o 0.1 %w/ .
152
Leci hin- ee o mula ions we e pu i ied using size exclusion ch oma og aphy in PD10
153
columns (GE Heal hca e, Ge many) and e-suspended in a 6 % w/ h ealose solu ion
154
used as c yop o ec an . Fo mula ions con aining leci hin we e pu i ied using an
155
ul a il a ion-cen i uga ion me hod using cen i ugal il e s (Amicon Ul a-4,
156
Millipo e, Ge many) wi h a 100 kDa molecula weigh cu -o (4º, 4000g, 10 min in
157
iplica e Beckman L8-60M ul acen i uge (Beckman Ins umen s, Inc., USA) (Lopes
158
e al. 2012). Following, he pa icles we e ozen in liquid ni ogen and lyophilized a
159
−80.0 ± 0.5 °C and 0.057 mba (Tels a C yodos, Spain). All he o mula ions we e
160
p epa ed in iplica e (n = 3).
161
PLGA NPs p epa a ion
162
PLGA NPs we e p epa ed by he nanop ecipi a ion me hod (NPP) wi h some
163
modi ica ion (Du án-Loba o e al. 2014). B ie ly, a weighed amoun o PLGA was co-
164
8
dissol ed wi h Span® 60 in ace one o each a concen a ion o 1.5 % w/ . 5 mL o
165
such solu ion we e subsequen ly added d opwise a 5 mL/min using a sy inge pump
166
(Ha a d Appa a us, USA) in o 15 mL o a Plu onic® F68 aqueous solu ion (0.5 %
167
w/ ) unde magne ic s i ing. The ace one was hen e apo a ed a . . o 4 h.
168
Following, he pa icles suspension was cen i uged o e a glyce ol bed (100 µL) a
169
10000 pm o 15 min a 4ºC o collec he NPs. A e washing wice, he NPs we e e-
170
suspended in a 5 % w/ h ealose solu ion used as c yop o ec an and eeze-d ied
171
( ozen in liquid ni ogen and lyophilized a − 0.0 ± 0.5 °C and 0.057 mba ; Tels a
172
C yodos, Spain) o ob ain a ine powde . All he o mula ions we e p epa ed in iplica e
173
(n = 3).
174
In he case loaded PLGA NPs, CB13 was co-dissol ed wi h he polyme in ace one a
175
13 % w/w d ug/polyme . In he case o hodamine-loaded NPs, 20 μ L o a mg/mL o
176
luo och ome solu ion we e added o he o ganic phase ins ead.
177
NPs su ace modi ica ion
178
To p omo e he in e naliza ion o he pa icles by Caco-2 cells (accep ed in he scien i ic
179
li e a u e as gas oin es inal ba ie model) bo h kinds o pa icles we e coa ed wi h CS.
180
Fo his pu pose, and due o he hyd ophilic na u e o CS, his addi i e was added in an
181
addi ional s ep a e NPs o ma ion (Du án-Loba o e al. 2014; Sa men o e al. 2011).
182
The NPs we e incuba ed in o a 0.25 % w/ CS solu ion in ace ic acid 1% / o 30 min
183
and hen collec ed by cen i uga ion.
184
In o de o p e en biological clea ance mechanisms as well as a o GI cells up ake,
185
plain polyme ic and lipid pa icles we e also coa ed wi h PEG. Fo his pu pose, he
186
NPs we e in u n incuba ed in a 4.5 % w/ PEG6000 solu ion o 4h unde s i ing. An
187
addi ional app oach was also ollowed wi h LNPs o mula ions, consis ing o
188
15
The e o e, s ea ylamine and/o CTAB we e included in he o mula ion o educe he
325
in ensi y o he elec os a ic in e ac ion and allow o simila size alues be o e and a e
326
he coa ing p ocess (Fig. 1B). Howe e , in he case o pa icles con aining s ea ylamine,
327
he addi ion o chi osan led o a ma ked inc ease in pa icle size and size dis ibu ion,
328
and hus his o mula ion was excluded om u he s udies. Fig. 1A also displays he
329
size dis ibu ion o anionic LNPs a e he coa ing p ocess wi h PEG in a pos -
330
p oduc ion s ep, which did no lead o signi ican di e ences in size alues (p < 0.05).
331
Thus, ca ionic excipien s s ea ylamine and CTAB we e no employed in his case. In
332
addi ion, LNPs o mula ed wi h PEG inco po a ed in he aqueous phase o he emulsion
333
esul ed in la ge sizes and wide size dis ibu ions (Fig. 1A), and he e o e we e
334
excluded om u he s udies.
335
Rega ding PLGA NPs, plain pa icles showed a mean pa icle size in he ange o 310-
336
360 nm, which was inc eased a e CS and PEG coa ing s eps (Fig. 1C). The inc eased
337
size a e CS and PEG adso p ion on pa icle su ace has been p e iously epo ed
338
(Ga cia-Fuen es e al. 2005; Na ee e al. 2009; Pa een and Sahoo 2011) and a ibu ed
339
o he deposi ion o mul ilaye su ace-coa ing componen s. None heless, su ace
340
modi ica ion o nanopa icles wi h hyd ophilic componen s is expec ed o imp o e hei
341
cellula up ake, as well as a oid he opsoniza ion p ocess, ega dless o he epo ed size
342
inc ease (G e e al. 2000; Pa een and Sahoo 2011).
343
Inse Fig. 1 a ound he e
344
Fig. 1 Size dis ibu ion o A plain and coa ed anionic LNPs (PEG-LNPsA p epa ed wi h PEG
345
added in a pos -p oduc ion s ep; PEG-LNPsB p epa ed wi h PEG added in he o ganic phase o
346
he emulsion); B plain and coa ed ca ionic LNPs; C plain and coa ed PLGA NPs.
347

16
In e ms o ZP, a simila beha iou was obse ed in bo h polyme ic and lipid
348
nanopa icles, as depic ed in Fig. 2. The modi ica ion wi h CS s ongly u ned ZP
349
alues o posi i e alues (-35.6 mV o +60.7 mV o PLGA NPs and -34.2 ± 1.3 mV o
350
+47.33 ± 0.56 mV o ca ionic LNPs), which e idences he deposi ion o CS on o he
351
pa icles su ace and is in acco dance wi h he published li e a u e (Du án-Loba o e al.
352
2014; Sa men o e al. 2011; Vila e al. 2002).
353
The su ace modi ica ion wi h PEG induced none heless a shi in ZP in bo h ypes o
354
pa icles o alues nea neu ali y (Fig. 2) also in acco dance wi h p e ious published
355
da a (Ga cia-Fuen es e al. 2005; Ga ino e al. 2007; Vila e al. 2002). The p esence o
356
PEG chains displaces he di use ionic laye o a g ea e dis ance om he pa icle
357
su ace, esul ing in a dec ease in absolu e ZP alues (Ga cia-Fuen es e al. 2005; Pa el
358
e al. 2012).
359
Inse Fig. 2 a ound he e
360
Fig. 2 Ze a po en ial alues o o mula ions. LNPs plain anionic lipid nanopa icles; CTAB-
361
LNPs CTAB-con aining plain ca ionic lipid nanopa icles; CS-CTAB-LNPs chi osan-coa ed
362
CTAB-con aining LNPs; SA-CTAB-LNPs plain s ea ylamine- and CTAB- con aining lipid
363
nanopa icles; CS-SA-CTAB-LNPs chi osan-coa ed s ea ylamine- and CTAB-con aining LNPs;
364
SA-LNPs plain s ea ylamine-con aining LNPs; CS-SA-LNPs chi osan-coa ed s ea ylamine-
365
con aining LNPs; PEG-LNPs PEG-coa ed anionic LNPs; PLGA NPs; CS-PLGA NPs chi osan-
366
coa ed PLGA NPs; PEG-PLGA NPs PEG-coa ed PLGA NPs (e o ba s SD, n = 3).
367
Bo h PLGA and lipid pa icles we e sphe ical and non-agg ega ed. A e he coa ing
368
p ocess, PLGA NPs showed a well-de ined co e-shell s uc u e. As an example, Fig. 3
369
shows he mo phology o CB13 loaded-PLGA NPs ob ained by TEM imaging. This
370
pa icula s uc u e was no obse ed o LNPs p obably due o a smalle amoun o CS
371
on he pa icle su ace (da a no shown).
372
17
Inse Fig. 3 a ound he e
373
Fig. 3 TEM image o CS-coa ed PLGA NPs co e-shel s uc u e.
374
D ug loading and en apmen e iciency
375
Values o EE (%) and LC (%) anged be ween 89 – 100 % and 7 – 10 %, espec i ely
376
o LNPs, and 70 – 80 % and 8 – 10 %, espec i ely o PLGA nanopa icles. The
377
co esponding da a is shown in Table 1. The high alues o encapsula ion achie ed a e
378
p obably due o he lipophilic na u e o he d ug, which p esen s low a ini y o wa e
379
phases and hus ends o mig a e o he o ganic phase. The e we e no s a is ically
380
signi ican di e ences be ween plain and he co esponding su ace-modi ied
381
o mula ions, p obably due o coa ing me hodology: he addi i es we e added in a pos -
382
p oduc ion s ep, he e o e no in luencing he encapsula ion p ocess (Du án-Loba o e al.
383
2014).
384
Inse Table 1 a ound he e
385
Table 1 EE and LC o o mula ions. LNPs plain anionic lipid nanopa icles; CTAB-LNPs
386
CTAB-con aining plain ca ionic lipid nanopa icles; SA-CTAB-LNPs plain s ea ylamine- and
387
CTAB- con aining lipid nanopa icles; CS-SA-CTAB-LNPs chi osan-coa ed s ea ylamine- and
388
CTAB-con aining LNPs; SA-LNPs plain s ea ylamine-con aining LNPs; CS-SA-LNPs
389
chi osan-coa ed s ea ylamine-con aining LNPs; PEG-LNPs PEG-coa ed anionic LNPs; PLGA
390
NPs; CS-PLGA NPs chi osan-coa ed PLGA NPs; PEG-PLGA NPs PEG-coa ed PLGA NPs (SD,
391
n = 3).
392
In i o d ug elease p o iles
393
CB13 p esen s ex emely low wa e solubili y. Hence, o main ain sink condi ions, a
394
0.1 % w/ Tween 80 solu ion was used as dissolu ion medium. The in i o elease o
395
CB13 om plain, CS-coa ed and PEG-coa ed LNPs and PLGA NPs is illus a ed in Fig.
396
18
4. Whe e he elease o CB13 om PLGA NPs is shown o be signi ican ly lowe han
397
om LNPs, ollowing a p olonged elease pa e n wi h no bu s e ec . LNPs eleased
398
mo e han 50 % o CB13 in he i s 2 h and 90 % in he i s 8 h. In compa ison, PLGA
399
NPs (plain, CS and PEG-coa ed) eleased less han 10 % o CB13 a he end o he i s
400
2 h and less han 50 % in he i s 8 h.
401
Wi h ega d o coa ed NPs, in he case o polyme ic pa icles he p esence o a su ace
402
coa ing led o a dec ease o CB13 elease a e compa ed o he co esponding plain
403
pa icles (Fig. 4). Su ace-modi ied PLGA pa icles did no a ain comple e elease o
404
he d ug wi hin he analyzed pe iod (maximum alue o eleased d ug below 60 %),
405
which can be a ibu ed o he addi ional laye o addi i e on he su ace ac ing as an
406
addi ional ba ie o d ug di usion (Du án-Loba o e al. 2014; Pa een and Sahoo 2011).
407
None heless, in he case o LNPs, he su ace coa ing did no lead o signi ican
408
di e ences in d ug elease p o iles wi h espec o he co esponding plain pa icles. The
409
simila i y be ween plain and coa ed LNPs d ug elease can be ela ed o he simila
410
pa icle size alues ob ained be o e and a e he coa ing p ocess, sugges ing he
411
absence o he mul iple laye s ob ained wi h coa ed PLGA NPs ha hinde ed he elease
412
p ocess.
413
Inse Fig. 4 a ound he e
414
Fig. 4 Release om plain and CS- and PEG-coa ed PLGA NPs and LNPs. LNPs plain anionic
415
lipid nanopa icles; CS-CTAB-LNPs chi osan-coa ed CTAB-con aining LNPs; PEG-LNPs
416
PEG-coa ed anionic LNPs; PLGA NPs; CS-PLGA NPs chi osan-coa ed PLGA NPs; PEG-
417
PLGA NPs PEG-coa ed PLGA NPs (e o ba s SD, n = 3).
418
FT-IR
419
19
FT-IR analysis was ca ied ou o u he con i m he p esence o he addi i es in he
420
inal NPs suspensions. FT-IR spec a o plain and PEG- and CS-coa ed PLGA NPs and
421
LNPs a e depic ed in Fig. 5 and we e compa ed o he signal o he co esponding
422
excipien s (da a no shown) and published li e a u e. The spec a o plain (A), PEG-
423
coa ed (B) and CS-coa ed (C) PLGA o mula ions show he cha ac e is ic alkane C-H
424
bond abso p ion peaks a 2978 and 2960 cm-1 and -COO- s e ching peak a 1745 cm-1
425
o PLGA (Shen e al. 2011). In u n, in he FT-IR spec a o plain (D), PEG-coa ed (E)
426
and CS-coa ed (F) LNPs o mula ions, abso p ion peaks a 2917 and 2843 cm-1
427
a ibu ed o P eci ol®, he main componen o pa icle ma ix (Rei z e al. 2008) we e
428
de ec ed. The p esence o PEG in PEG-coa ed PLGA (B) and LNP (E) o mula ions
429
was con i med by he abso p ion bands a 2883 cm-1 (B) due o s e ching C-H ib a ion,
430
and a 1146 cm-1 and 1102 cm-1 (E) a ibu ed o C-C s e ching and he cha ac e is ic C-
431
O-C s e ching ib a ion o he epea ed -OCH2CH2- uni s o he PEG backbone
432
espec i ely (Kassim e al. 2006; Pe o a e al. 2008). Finally, CS-coa ed PLGA (C) and
433
LNP (F) o mula ions p esen ed abso p ion bands a 1647 (C) and 1653 (F) a ibu ed o
434
amide I (C=O) and a 1312 cm-1 (F) due o he bending ib a ion o C-N, con i ming he
435
p esence o CS in he o mula ions (Wang e al. 2007).
436
Inse Fig. 5 a ound he e
437
Fig. 5 FT-IR spec a o plain and su ace-modi ied 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 compa ibili y
440
Da a ob ained om blood compa ibili y s udies is p esen ed in Table 2 and show a
441
b oad in i o sa e y ma gin o he di e en ypes o nanopa icles assayed. All he
442
o mula ions showed alues below 2.5 % o lysis, indica ing hese ca ie s can be
443
20
conside ed hemocompa ible o d ug deli e y applica ions. The o mula ions a e
444
expec ed o exhibi a negligible e ec on hemolysis and no o in luence sP-selec in
445
elease le els om pla ele ac i a ion quan i ica ion, complemen sys em ac i a ion o
446
plasma clo ing imes. Simila esul s ha e been epo ed o o he nanopa icula e
447
sys ems con aining he ma e ials employed in he o mula ions assayed in his wo k
448
(Da e e al. 2007; Ma in-Bande as e al. 2012).
449
Inse Table 2 a ound he e
450
Table 2 Blood compa ibili y o CB13-loaded NPs o mula ions in e ms o hemolysis (%),
451
pla ele ac i a ion (sP-selec in elease, ng/mL), complemen ac i a ion (C3a elease: C3a
452
desA g, ng/mL), and plasma ecalci ica ion ime (T1/2max, min). LNPs; CS-CTAB-LNPs
453
chi osan-coa ed CTAB con aining LNPs; PEG-LNPs PEG-coa ed LNPs; PLGA NPs; CS-PLGA
454
NPs chi osan-coa ed PLGA NPs; PEG-PLGA NPs PEG-coa ed PLGA NPs (n = 3).
455
MTT
456
A MTT assay (Du án-Loba o e al. 2014) was ca ied ou o de e mine cell iabili y in
457
he p esence o plain and coa ed PLGA NPs and LNPs. The s udy was conduc ed wi h
458
Caco-2 cells since hey a e conside ed a gas oin es inal pe meabili y model in he
459
li e a u e (Alhamo uni e al. 2010). I should be no ed ha , al hough cannabinoids ha e
460
been p o en o inhibi cell g ow h and induce apop osis in umou cells (Gus a sson e
461
al. 2009), hei in luence on cell iabili y and p oli e a ion depends on he speci ic cell
462
cul u e assayed and he dose adminis a ed (Ha e al. 2004; Lig es i e al. 2003; Lopez-
463
Rod iguez e al. 2005; Sa ne e al. 2011).
464
Cells we e incuba ed wi h CB13 concen a ions anging om 0.003 o 30 μM in he
465
o m o ee d ug and loaded in o plain and su ace-modi ied PLGA and lipid
466
nanopa icles. Blank NPs we e es ed as well a he amoun equi alen o hose o
467

21
CB13-loaded pa icles assayed (0.004 – 4 μg NPs). DMSO used as posi i e con ol
468
demons a ed o be oxic a he concen a ion assayed and he e o e alida ed he assay.
469
Fig. 6 illus a es he esul s ob ained om he s udy, showing cell iabili y alues we e
470
main ained a ound 100 % o all he o mula ions assayed, hus indica ing he lack o
471
oxici y o he pa icles es ed a he concen a ions employed.
472
Inse Fig. 6 a ound he e
473
Fig. 6 Caco-2 cell iabili y (%) ob ained h ough he MTT assay a e incuba ion wi h NPs
474
LNPs plain anionic lipid nanopa icles; CTAB-LNPs CTAB-con aining plain ca ionic lipid
475
nanopa icles; CS-CTAB-LNPs chi osan-coa ed CTAB-con aining LNPs; SA-CTAB-LNPs
476
plain s ea ylamine- and CTAB- con aining lipid nanopa icles; CS-SA-CTAB-LNPs chi osan-
477
coa ed s ea ylamine- and CTAB-con aining LNPs; SA-LNPs plain s ea ylamine-con aining
478
LNPs; CS-SA-LNPs chi osan-coa ed s ea ylamine-con aining LNPs; PEG-LNPs PEG-coa ed
479
anionic LNPs; PLGA NPs; CS-PLGA NPs chi osan-coa ed PLGA NPs; PEG-PLGA NPs PEG-
480
coa ed PLGA NPs (e o ba s SD, n = 3).
481
Cell up ake
482
Caco-2 cells we e also used o simula e he gas oin es inal (GI) d ug ba ie o o al
483
deli e y (Zhang and Feng 2006) in up ake s udies. NPs coa ed wi h hyd ophilic
484
polyme s o moie ies such as CS and PEG ha e been epo ed in he li e a u e o display
485
highe in es inal anspo compa ed wi h ha o unmodi ied nanopa icles (des Rieux e
486
al. 2006; Du án-Loba o e al. 2014; Ma in-Bande as e al. 2013).
487
Fig. 7 shows simila le els o CS-coa ed LNPs and PLGA NPs (Fig. 7c, 7 , 7g), also
488
highe han he co esponding plain and PEG-coa ed o mula ions (Fig. 7). The esul s
489
a e in acco dance wi h p e ious li e a u e and accoun o a highe in e ac ion be ween
490
pa icles and cells due o he posi i e cha ges o chi osan molecules on he NPs su ace
491
(Du án-Loba o e al. 2014; Ga cia-Fuen es e al. 2005). Mo eo e , PEG-coa ed
492
22
o mula ions showed he lowes up ake (Fig. 7b, 7e, 7g), which could be a ibu ed o
493
he he s e ic impedimen exe ed by he PEG coa ing ha is known o p e en he
494
in e ac ion wi h cells, being he basis o i s “s eal h” p ope ies (G e e al. 2000;
495
Ma in-Bande as e al. 2013; Owens and Peppas 2006). I should be no ed ha hese
496
esul s seem o be in disag eemen wi h he highe anspo o PEG-coa ed PLA NPs
497
compa ed o plain PLA NPs p e iously epo ed in he li e a u e (Vila e al. 2004).
498
Howe e , al hough a mucoadhesion/chain pene a ion mechanism has been p oposed as
499
he basis o a highe anspo o PEG-coa ed NPs (Tobı o e al. 2000 , he same au ho s
500
ha e s a ed he s ill unclea ques ion o whe he PEG-coa ing o NPs inc eases
501
bioa ailabili y due o an imp o ed GI anspo o o a simply g ea e s abili y in GI
502
luids and blood s eam (Vila e al. 2004).
503
When compa ing bo h ypes o pa icles, plain and CS-coa ed PLGA and lipid NPs
504
showed simila cell up ake alues espec i ely. Howe e , PEG-coa ed LNPs showed
505
sligh ly highe up ake alues han he co esponding PLGA o mula ions (Fig. 7b, 7e,
506
7g). I should be no ed ha pa icle size is known o be an in luencing ac o in NPs
507
anspo h ough mucosa memb anes (F oehlich 2012; Vila e al. 2005; Vila e al.
508
2004) and he la ge size o coa ed PLGA NPs could be in luencing he lowe cell
509
in e naliza ion a he ime poin assayed in his s udy.
510
Based on he esul s, i could be concluded ha CS-coa ing o nanopa icles po en ially
511
p o ides he highe cell in e naliza ion a he GI le el, a p e ious and necessa y s ep o
512
eaching blood ci cula ion. Addi ional conside a ions such as NPs s abili y p io o
513
abso p ion and bioa ailabili y should be aken in o accoun when compa ing he gene al
514
pe o mance o bo h ypes o coa ings.
515
Inse Fig. 7 a ound he e
516
23
Fig. 7 Caco-2 up ake CLSM images and pa icle coun ing algo i hms. a) LNPs plain anionic
517
lipid nanopa icles; b) PEG-LNPs PEG-coa ed anionic LNPs; c) CS-CTAB-LNPs chi osan-
518
coa ed CTAB-con aining LNPs; d) PLGA NPs; e) PEG-PLGA NPs PEG-coa ed PLGA NPs; )
519
CS-PLGA NPs chi osan-coa ed PLGA NPs; g) numbe o pa icles pe cell (e o ba s SD, n =
520
3).
521
THP1 cell up ake
522
Up ake s udies in THP1 cells showed a common end o up ake PEG-coa ed << CS-
523
coa ed < plain NPs o polyme ic and lipid o mula ions. A ma ked dec ease o PEG-
524
coa ed NPs up ake compa ed o he co esponding plain and chi osan-coa ed
525
o mula ions was obse ed (Fig. 8), as expec ed and suppo ed by p e ious li e a u e
526
(Bocca e al. 1998), due o he hyd ophilic p ope ies ha he addi i e p o ides o he
527
su ace o pa icles (Owens and Peppas 2006; Vona bou g e al. 2006). Chi osan-coa ed
528
o mula ions showed also a dec ease in THP1 up ake wi h espec o plain o mula ions,
529
hough no as ma ked as in he case o PEG-coa ed o mula ions (Fig. 8). A lowe
530
phagocy ic up ake has been a ibu ed o he hyd ophilic p ope ies o chi osan as well
531
as i s posi i e cha ges (Sa men o e al. 2011), since he hyd ophobic su ace o pa icles
532
as well as nega i e su ace cha ges esembling bac e ia’s su ace a o clea ance
533
mechanisms wi hin he body (F oehlich 2012). In acco dance, plain o mula ions
534
p esen ed he highes THP1 up ake alues (Fig. 8).
535
Inse Fig. 8 a ound he e
536
Fig. 8 Quan i a i e analysis by low cy ome y o up ake in THP1 cells a e 120 min o
537
incuba ion wi h hodamine label pa icles. Plo s (a) LNPs; (b) PLGA; (c) CS-LNPs; (d) CS-
538
PLGA NPs; (e) PEG-LNPs; ( ) PEG-PLGA NPs. Pe cen age plo co esponds o o al e en s
539
included in R2 egion. Y-axis co esponds o numbe o coun s.
540
24
Howe e , a sligh ly inc ease in up ake could be obse ed in he case o CS- and PEG-
541
coa ed PLGA NPs wi h ega d o CS- and PEG-coa ed LNPs espec i ely, which could
542
be a ibu ed o he la ge size o PLGA o mula ions ob ained a e he deposi ion o
543
mul iple laye s o addi i e in he coa ing p ocess. Pa icle size has been epo ed o be a
544
key ac o in luencing he ex en o opsoniza ion and phagocy ic up ake (F oehlich
545
2012), speci ically in he case o PEG-coa ed PLGA NPs o he size ange s udied in
546
his wo k (Yang e al. 2012).
547
Acco ding o he esul s, PEG-coa ed NPs p o ide he highes p o ec ion agains
548
opsoniza ion and phagocy ic up ake and hus a highe chance o p olonged ci cula ion
549
in i o. Howe e , and as epo ed by p e ious wo ks (Ga cia-Fuen es e al. 2005), PEG
550
coa ing o LNPs did no lead o in i o imp o emen s, while CS-coa ing o he same
551
o mula ion did. I should be no ed ha in i o esul s do no always co ela e wi h in
552
i o da a, since he e a e many in luencing ac o s o he la e ha a e no p esen in in
553
i o expe imen s. Howe e , in i o da a allow ob aining a deepe unde s anding o he
554
pe o mance o he ca ie s ha is a key ac o owa ds he op imiza ion o unc ional
555
ca ie sys ems. Fu he s udies on he in e ac ion o hese pa icles wi h biological
556
su aces and he in luence o hei componen s and coa ing addi i es should be ca ied
557
ou co e ing a b oade ange o in i o condi ions, o ul ima ely be able o ully
558
unde s and hei beha iou in i o.
559
Conclusions
560
PLGA NPs wi h size o 320-420 nm in diame e , na ow size dis ibu ion and nega i e
561
ze a po en ial, and LNPs wi h 120- 60 nm in diame e wi h Pdi ≈ 0.3 and nega i e ze a
562
po en ial we e ob ained. Modi ica ion o NPs su ace wi h CS and PEG s ongly u ned
563
ZP o posi i e and nea neu ali y alues espec i ely. Elec on mic oscopy imaging
564
31
830
831
832
Fig. 1
833
834

32
835
Fig. 2
836
837
-40
-20
0
20
40
60
80
Ze a Po en ial (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
D ug eleased (%)
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
D ug eleased (%)
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
Viabili y (%)
CB13 concen a ion (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-selec in elease
(ng/mL)
C3a desA g
(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
Con ol (PBS solu ion)
0
99 ± 12
289 ± 10
11.3 ± 1.2
866
Table 2
867
Figu e 4b
Click he e o download high esolu ion image

Figu e 5
Click he e o download high esolu ion image
Figu e 6
Click he e o download high esolu ion image
Figu e 7
Click he e o download high esolu ion image
Figu e 8
Click he e o download high esolu ion 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
able
Click he e o download able: Table 1.doc

1
1
Sample
Haemolysis
(%)
sP-selec in elease
(ng/mL)
C3a desA g
(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
Con ol (PBS solu ion)
0
99 ± 12
289 ± 10
11.3 ± 1.2
2
able
Click he e o download able: Table 2.doc