1
LL37 LOADED NANOSTRUCTURED LIPID CARRIERS (NLC): A NEW
STRATEGY FOR THE TOPICAL TREATMENT OF CHRONIC WOUNDS
I xaso Ga cia-O uea, Ga azi Gainzaa,c, Cecilia Gi baud, Rod igo Alonsod, José Ja ie
Agui ee, José Luis Ped aza,b, Manoli Iga uaa,b and Rosa Ma ia He nandeza,b,*
a NanoBioCel G oup, Labo a o y o Pha maceu ics, School o Pha macy, Uni e si y o he Basque Coun y
(UPV/EHU), Paseo de la Uni e sidad 7, Vi o ia-Gas eiz 01006, Spain.
b Biomedical Resea ch Ne wo king Cen e in Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN).
Vi o ia-Gas eiz, Spain.
c Biop axis Resea ch AIE, Miñano, Vi o ia-Gas eiz
d Depa men o Immunology, Mic obiology and Pa asi ology, School o Pha macy, Uni e si y o he Basque
Coun y (UPV/EHU), Paseo de la Uni e sidad 7, Vi o ia-Gas eiz 01006, Spain
e Hospi al Uni e si a io de Ála a (HUA) Txago i xu, Vi o ia-Gas eiz, 01009, Spain
*Co esponding au ho : P o . Rosa Ma ia He nandez
Labo a o y o Pha maceu ics, Uni e si y o he Basque Coun y
School o Pha macy, Paseo de la Uni e sidad, 7
01006 – Vi o ia-Gas eiz, Spain
Telephone: +34 945013095
Fax: +34 945013040
E-mail: osa.he [email protected]
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Eu opean Jou nal o Pha maceu ics and Biopha maceu ics
108 : 310-316 (2016), which has been published in inal o m a h ps://doi.o g/10.1016/j.ejpb.2016.04.006. © 2016 Else ie unde CC
BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
2
ABSTRACT
The LL37 is a human an imic obial pep ide which no only has a b oad spec um o
an imic obial ac i i y, bu i has also been p o ed o modula e wound healing by
pa icipa ing in angiogenesis, epi helial cell mig a ion and p oli e a ion, and immune
esponse. In his wo k, LL37 has been encapsula ed in nanos uc u ed lipid ca ie s
(NLCs), p oduced by he mel -emulsi ica ion me hod, in o de o imp o e i s
e ec i eness. The cha ac e iza ion o he NLC-LL37 showed a mean size o 270 nm, a
ze a po en ial o -26 mV and an encapsula ion e iciency o 96.4%. The cy o oxici y
assay pe o med in Human Fo eskin Fib oblas s demons a ed ha he NLC-LL37 did
no a ec cell iabili y. Mo eo e , he in i o bioac i i y assay e idenced ha he
pep ide emained ac i e a e he encapsula ion, since he NLC-LL37 e e sed he
ac i a ion o he mac ophages induced by LPS in he same way as he LL37 in solu ion.
In addi ion, he in i o an imic obial assay e ealed he NLC-LL37 ac i i y agains E.
coli. The e ec i eness o he nanopa icles was assessed in a ull hickness wound
model in db/db mice. The da a demons a ed ha NLC-LL37 signi ican ly imp o ed
healing compa ed o he same concen a ion o he LL37 solu ion in e ms o wound
closu e, eepi helisa ion g ade and es o a ion o he in lamma o y p ocess. O e all,
hese indings sugges a p omising po en ial o he NLC-LL37 o mula ion o ch onic
wound healing.
KEYWORDS
LL37, an imic obial pep ide, lipid nanopa icles, NLC (nanos uc u ed lipid ca ie s),
ch onic wounds
3
1. INTRODUCTION
The incidence o su e ing non-healing ch onic wounds has exponen ially inc eased
wi h aging o popula ion oge he wi h he esul an como bidi ies o diabe es, enous
insu iciency and associa ed ch onic diseases. I has been es ima ed ha 1-2% o he
popula ion in he de eloped coun ies would expe ience a ch onic wound in hei
li e ime, ep esen ing 2% o he Eu opean heal h budge acco ding o he Uni ed
Na ions [1,2].
Cu aneous wound healing is an o de ly and imely epa a i e p ocess, designed o
es o e he skin ba ie unc ion and homeos asis. I is accomplished by egula ed
p ocesses ha o e lap in space and ime, including an ini ial in lamma o y esponse, a
p oli e a i e phase and a inal emodelling phase [3,4]. Ch onic wounds ail o p oceed
in his subsequen phases, s agna ing in a pe manen in lamma o y s a e in which he
wound do no cu e. This in lamma ion main ains a cons an in il a ion o mac ophages
and neu ophils, which p oduce an excessi e amoun o collagenases, p o eases and
eac i e oxygen species ha deg ade healing media o s and hampe he o ma ion o he
ex acellula ma ix and new epi helia. In addi ion, hese lesions a e usually in ec ed and
emain open o ex ended pe iods o ime, occasionally endange ing he li e o he
pa ien [1,5].
Cu en ad ances in wound ca e ha e ocused on inding new ea men s o wound
healing, such as he adminis a ion o healing media o s o wound epai . In his ega d,
one o he s a egies ha is gaining a no o ious in e es is he adminis a ion o he
human an imic obial pep ide LL37. I should be no ed ha he down egula ion o LL37
has been associa ed wi h an inc eased isk in su e ing ch onic ulce s [6].
The LL37 has been de ec ed in an inac i e p e o m (hCAP18) in se e al immune and
de mal-epi helial cells. A e a skin inju y, hCAP18 is eleased o he ex acellula
en i onmen due o cell deg anula ion, leading o i s ac i a ion in o he LL37 pep ide
[4,7]. I has been p o en ha his molecule modula es wound healing by ac i a ing
angiogenesis [8,9] and epi helial cell mig a ion and p oli e a ion [10,11]. Fu he mo e,
i has demons a ed a b oad spec um o an imic obial, an i i al and an i ungal ac i i y
[12-15].
In addi ion, he LL37 p esen s an immunomodula o y e ec , since i is chemo ac ic o
monocy es, neu ophils and dend i ic cells [13]. The LL37 also shows he abili y o
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neu alise he p oin lama o y esponses exe ed by mac ophages ( e y impo an in
ch onic wounds) by i s union o lipopolysaccha ide (LPS) [13,16].
The in i o wound healing s udies conduc ed o da e, ha e equi ed high doses and
dosing equencies o ee LL37 because o he apid deg ada ion o he pep ide [15,17]
o gene he apy [18] o achie e imp o ed healing. Wi h he aim o o e coming hese
limi a ions, Che eddy and cols. encapsula ed he LL37 pep ide in o PLGA
nanopa icles ob aining a signi ican ly imp o emen in he wound healing ac i i y a e
in ade mal adminis a ion [19]. In line wi h his app oach, he encapsula ion o LL37
in o nanos uc u ed lipid ca ie s (NLC) is p esen ed as a p omising al e na i e o
op imise he adminis a ion o he LL37 in e ms o dose, deli e y pa e n, and sa e y.
The NLCs, besides o p o ec ing he encapsula ed pep ide agains he deg ada ion o
he p o eases, can be adminis e ed h ough he opical ou e, he eby educing he
sys emic e ec s due o a lowe sys emic bioa ailabili y o he d ug. Fu he mo e, he
NLCs allow a sus ained elease o he d ugs a he si e o ac ion and p esen an
excellen biocompa ibili y, making hem a sui able op ion o he opical ea men o
ch onic wounds [20-22].
Thus, he aim o ou s udy is he de elopmen o a opical o mula ion based on LL37
loaded-NLC (NLC-LL37) o he ea men o ch onic wounds. In i o s udies we e
ca ied ou o de e mine he biocompa ibili y o he o mula ion and he ac i i y agains
LPS o he encapsula ed LL37. In addi ion, an imic obial es s we e unde aken in E.
coli o analyse he e icacy o NLC-LL37 agains bac e ia. Finally, he wound healing
ac i i y o he NLC-LL37 was e alua ed in i o in a ull hickness wound model in
db/db mice.
2. MATERIALS AND METHODS
2.1 NLC-LL37 p epa a ion
The NLC-LL37 we e p epa ed ough he mel emulsi ica ion me hod and ollowing he
p ocedu e p e iously desc ibed by ou g oup [23,24]. B ie ly, a wa m aqueous phase
(hea ed a 40 °C, 1 min) con aining 3 ml o wa e , 20 mg o Poloxame 188 (Pan eac,
Spain) and 40 mg o Tween® 80 (Pan eac, Spain) was added o a mel ed lipid phase
con aining 200 mg o he solid lipid P eci ol® ATO 5 (Ga e ossé Spain, Spain) and 20
mg o he liquid lipid Miglyol® 812N (Sasol Ge many GmbH). Then, 50 µL o an 80
mg/mL LL37 (95.0% pu e, Caslo ApS, DK) aqueous solu ion was added and
5
immedia ely a e , he mix u e was emulsi ied o 15 s a 50 W (B anson® 250 soni ie ,
CT, USA). The esul ing emulsion was s o ed a 4°C o allow he e-c ys allisa ion o
he lipid o he NLC o ma ion. On he ollowing day, he pa icles we e collec ed
using a 100-kDa molecula weigh cu -o cen i ugal il e uni (Amicon, “Ul acel-
100k”, Millipo e, Spain) a 2,500 mp o 10 minu es and washed h ee imes wi h
MillliQ wa e . Finally, he NLC suspension was eeze d ied wi h he c yop o ec an
ehalose (Sigma-Ald ich, Spain) in a inal concen a ion o 15% (w/w) o he weighed
lipid. The a ge loading o LL-37 in NLC-LL37 was 2% (w/w).
2.2 Nanopa icle cha ac e isa ion
The mean pa icle size (Z-a e age diame e ) and he polydispe si y index (PDI) we e
measu ed by Dynamic Ligh Sca e ing (DLS), and he ze a po en ial was de e mined
h ough Lase Dopple mic o-elec opho esis (Mal e n® Ze asize Nano ZS, Model
Zen 3600; Mal e n ins umen s L d., UK). The measu emen medium o ze a po en ial
was wa e (pH 5.6), and he measu ed elec opho e ic mobilil y was con e ed in o ze a
po en ial h ough Smoluchowski app oxima ion. Each assay was pe o med in iplica e
a e nanopa icles lyophilisa ion. Nanopa icle mo phology was examined by
ansmission elec on mic oscopy (TEM, Philips EM208S).
The encapsula ion e iciency (EE) o he NLC-LL37 was assessed indi ec ly by
measu ing he ee LL-37 (non-encapsula ed) in he supe na an ob ained a e he
il a ion/cen i uga ion p ocess desc ibed in Sec ion 2.1. The amoun o ee LL-37 was
quan i ied using a comme cially a ailable ELISA ki o LL-37 (human LL-37 ELISA
ki , Hycul ® bio ech, Ne he lands). The EE (%) was de e mined using he ollowing
equa ion (1):
𝐸𝐸 %𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐿𝐿37 𝐹𝑟𝑒𝑒 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐿𝐿37
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐿𝐿37 100 1
2.3 In i o cell cul u e s udies
2.3.1 Cell cul u e
Human Fo eskin Fib oblas s (HFF) (ATCC, Manassas, USA) we e cul u ed on
Dulbeccos’s modi ied Eagle’s medium (DMEM) (ATCC, Manassas, USA)
supplemen ed wi h 15% ( / ) e al bo ine se um (FBS), 1% ( / ) L-glu amine and 1%
( / ) penicillin-s ep omycin.
6
The RAW 264.7 cell line (Mu ine Mac ophages; ATCC, Manassas, USA) was cul u ed
on a speci ic g ow h medium DMEM/F-12, Glu aMAX™ Supplemen (Gibco®/Li e
echnologies, Spain) supplemen ed wi h 10% ( / ) FBS and 1% ( / ) penicillin-
s ep omycin.
The cell lines we e main ained a 37°C in a humidi ied incuba o wi h a 5% CO2
a mosphe e. The cell passages we e pe o med e e y 2-3 days depending on he cell
line.
2.3.2 Cell iabili y s udies
The e ec o he NLC-LL37 on cell iabili y was assayed using HFF cells. 1000
cells/well we e seeded in o 96-well cul u e pla es and incuba ed 24 h o allow cell
a achmen . Then, he medium was eplaced by se ial concen a ions o NLC-LL37
(co esponding o 5000-50 ng o LL37/mL) and emp y NLC esuspended in 1% se um-
supplemen ed-DMEM. A e 48 h o incuba ion, he cell iabili y was measu ed by
adding 10 µL o CCK-8 eagen (Sigma-Ald ich, Sain Louise, USA) o he wells. The
mix u e was incuba ed o 4 h and he abso bance was hen ead a 450 nm and a 650
nm as he e e ence wa eleng h. The abso bance was di ec ly p opo ional o he
numbe o li ing cells in he cul u e.
2.3.3 Inhibi ion o mac ophages ac i a ion induced by LPS
This assay was conduc ed in he RAW 264.7 cell line. 105 cells/well we e seeded in o a
96-well cul u e pla e and incuba ed o 24 h o allow cell a achmen . Then, he medium
was eplaced by he ollowing samples (all o he samples we e esuspended in 1 %
se um-supplemen ed DMEM con aining 20 ng/ml o LPS p io o hei inco po a ion
in o he cell cul u e): (i) 5000 ng/ml o ee LL37, (ii) he concen a ion o NLC-LL37
equi alen o 5000 ng/ml o LL37 and (iii) he same concen a ion o emp y NLC. As
nega i e con ol 1% se um-supplemen ed medium was used and as posi i e con ol 1%
se um-supplemen ed medium wi h 20 ng/ml o LPS.
A e 6 h o incuba ion, he supe na an o he wells was collec ed o quan i y he TNF-α
eleased om he mac ophages using a comme cially a ailable ELISA ki (Mu ine
TNF- α Elisa De elopmen ki , Pep oTech). The esul s we e displayed as he
pe cen age o abso bance alue o each g oup compa ed o hose ob ained in he
nega i e con ol.
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2.4 An imic obial assay
In o de o es he an imic obial ac i i y o he o mula ions, Esche ichia coli ATCC
25922 was g own o e nigh a 37°C in Muelle Hin on b o h (Conda, P onadisa, Spain).
Then, he bac e ial suspension was dilu ed o 105 CFU/ml. F ee LL37 (20 µg/ml), NLC-
LL37 ( he concen a ion co esponding o 20 µg/ml LL37) and emp y NLC ( he same
nanopa icle concen a ions as he NLC-LL37 g oup) we e incuba ed wi h 1 ml o his
bac e ial suspension a 37°C wi h gen le agi a ion. In addi ion, 1 ml o he bac e ial
suspension alone was also incuba ed, o use i as con ol.
A 4 h o incuba ion, aliquo s we e aken om each suspension, dilu ed in PBS and 100
µl we e inocula ed in Muelle Hin on aga pla es (Conda/P onadisa, Spain). The pla es
we e incuba ed o 24 h a 37°C and subsequen ly, he colony o ming uni s we e
de e mined as he o al numbe o colonies g own on each pla e. Th ee independen
expe imen s we e pe o med. The an ibac e ial e ec was assessed as he pe cen age o
dea h cells compa ed o he con ol.
2.5 In i o wound healing s udy
2.5.1. Animals
Fo he in i o expe imen , 16 eigh week-old male db/db (BKS.Cg-m+/+Lep db/J) mice
we e used (Jan ie labo a o ies, Sain Be he in Cedex, F ance). E e y expe imen was
conduc ed acco ding o he p o ocols app o ed by he Ins i u ional E hical Commi ee
o Animal Expe imen a ion o he Uni e si y o he Basque Coun y (P ocedu e
numbe : CEBA/243/2012/HERNANDEZ MARTIN). Animals we e housed in
indi idual cages ha we e main ained on a 12 h ligh -da k cycle, and we e gi en
s anda d oden chow and wa e ad libi um.
2.5.2 Wound healing assay
The wound healing assay was pe o med adap ing he p ocedu e desc ibed by Michaels
e al. [25]. Human main healing mechanism is h ough g anula ion issue o ma ion and
e-epi helisa ion, while mice’s one is h ough wound con ac ion. In o de o mimic
human healing p ocess and a oid mice’s main p ocess, wo silicone ings o 1 cm in
diame e we e su u ed on each side o he midline using a 3-0 nylon su u e (A agó,
Spain), a e anes he ising he mice and emo ing hei do sal hai . Then, wo ull
hickness wounds o 8 mm in diame e and ex ending h ough he panniculus ca nosus
we e c ea ed using a punch biopsy ool (Acu-Punch, Acude m, USA). A e he
8
adminis a ion o he ea men s, he wounds we e co e ed wi h one laye o pe ola um
gauze (Tegade m® 3M) and wo laye s o adhesi e.
The mice we e di ided in 4 g oups (n=4): (i) un ea ed con ol, (ii) 6 µg o ee LL37,
(iii) 6 µg o LL37 encapsula ed in o NLC-LL37 and (i ) 2 µg o LL37 encapsula ed in o
NLC-LL37. T ea men s, p e iously esuspended in 10 µL o MilliQ wa e , we e
adminis e ed opically allowing hem o sp ead o e he wound bed, on day 1 and 4
a e he wound induc ion. On day 8, he mice we e sac i iced h ough CO2 inhala ion.
2.5.3 E alua ion o wound healing
The e ec i eness o he ea men s was e alua ed by measu ing he wound a ea (px2) on
days 1, 4 and 8 a e he su ge y. Those days, pho og aphs o he wounds we e aken
using a digi al came a (Lumix FS16, Panasonic®, Spain), and he wound a ea was
assessed wi h an image analysis p og amme (ImageJ®, Biopho onics Facili y,
Uni e si y o McMas e , Canada). The wound closu e was exp essed as he pe cen age
o he ini ial wound a ea.
2.5.4 His ological analysis o wound healing
A e he sac i ice o he mice, he wound and su ounding issue (~1 cm) we e excised
and ixed in 3.7% pa a o maldehyde o 24 h. Then he issues we e bisec ed, embedded
in pa a in, and sec ioned in 5 µm hick laye s. The samples we e p ocessed by H&E
s aining o wound healing e alua ion and by Masson ich ome s aining o collagen
deposi ion e alua ion.
The e-epi helisa ion p ocess was assessed ollowing he scale es ablished by Sinha e
al., 2003 [26]. The sco e o each wound was de e mined semi-quan i a i ely gi ing o
each wound a alue wi hin a ange om 0 o 4: 0, e-epi helisa ion a he ma gin o he
wound; 1, e-epi helisa ion co e ing less han hal o he wound; 2, e-epi helisa ion
co e ing mo e han hal o he wound; 3, e-epi helisa ion co e ing he en i e wound
wi h i egula hickness, and 4, e-epi helisa ion co e ing he en i e wound wi h no mal
hickness.
The esolu ion o he in lamma o y p ocess and wound ma u i y was de e mined
acco ding o he scale desc ibed by Co an e al., 2000 [27]. 1, acu e in lamma ion, wi h
he o ma ion o ib in clo and mig a ion o leucocy es and polynuclea neu ophils; 2,
di use acu e in lamma ion, wi h he p edominance o g anula ion issue o ma ion and
angiogenesis and wi h ba ely p esence o pyogenic memb ane; 3, ch onic in lamma ion,
9
wi h he p esence o g anula ion issue and wi h ib oblas p oli e a ion and 4, esolu ion
and healing, disappea ance o ch onic in lamma ion, al hough occasionally ound cells
can be ound.
The deposi ion o collagen was de e mined ollowing he scale desc ibed by Gal e al,
2006. [28]: 0, absen o collagen; 1, mild con en o collagen; 2, mode a e con en o
collagen and 3, ma ked con en o collagen.
2.5.5 Immunohis ochemis y
In o de o assess neoangiogenesis in he wound bed, immunohis ochemical s udies
we e pe o med using a speci ic monoclonal an i-CD31 an ibody (JC70 clon, 760-4378,
Ven ana-Roche). 5 µm hick issue slides we e incuba ed wi h he p ima y an ibody o
36 min a 37°C, and subsequen ly he laye s we e ea ed wi h Ul a iew Uni e sal
DAB de ec ion ki (760-500, Ven ana-Roche) o isualize he an igen. A e wa ds, he
numbe o blood essels was coun ed pe ield.
2.6 S a is ical analyses
All o he da a a e exp essed as he mean ± s anda d de ia ion. Based on he esul o
he no mali y es and he Le ene es o he homogenei y o a iances, he means we e
compa ed h ough one-way ANOVA, wi h he subsequen applica ion o S uden -
Newman-Keuls pos -hoc; o h ough Mann-Whi ney U es . All he s a is ical
calcula ions we e pe o med using SPSS 22.0.01 (SPSS®, INC., Chicago, IL, USA).
3. RESULTS AND DISCUSSION
3.1 Nanopa icle cha ac e isa ion
As illus a ed in Table 1 he NLC-LL37 and emp y NLC showed simila mean size,
howe e , he NLC-LL37 exhibi ed a sligh ly highe size: 273.6± 27.64 nm and 220.6 ±
5.48 nm, espec i ely. The polydispe si y index (PDI) was below 0.4 in bo h
o mula ions, indica ing a s able polydispe se sys em. The analysis o he ze a po en ial
e ealed ha bo h o mula ions p esen ed a simila su ace cha ge o abou -31 mV in
he case o NLC-LL37 and abou -26 mV in he case o emp y NLC. In addi ion, he
NLC-LL37 p esen ed high encapsula ion e iciency (96.40 % ± 0.41) and a pep ide
loading o 16.76±0.07 µg LL37/mg nanopa icle.
Acco ding o he TEM pho og aphs o he nanopa icles, hey showed a simila size o
ha ob ained wi h DLS (Fig. 1).
16
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18
Fig. 1. TEM pho og aphs o NLC-LL37 and NLC-B. The scale ba indica es 200 nm.
Fig. 2. Cell iabili y a e NLC ea men . The esul s a e gi en as he mean % o li ing
cells ela i e o he con ol ± SD. Con ols a e C (cells wi hou any addi ion) and DMSO
(cells a e addi ion o DMSO).
19
Fig. 3. Inhibi ion o he ac i a ion o he mac ophages. The esul s a e gi en as he
mean % o TNF-α p oduc ion ela i e o he con ol ± SD. ** signi ican ly g ea e han
emp y NLC and C+ (p<0.01). Con ols a e C- (cells wi hou any addi ion) and C+
(cells a e he addi ion o LPS).
Fig. 4. An imic obial assay. The esul s a e gi en as he mean pe cen age o dea h
bac e ia ela i e o he con ol ± SD. *** p<0.001 be ween he h ee g oups.
Fig 5. In i o wound closu e. (A) Wound closu e ep esen ed as he pe cen age o he
educ ion o ini ial a ea. * Signi ican ly g ea e han un ea ed g oup (p<0.05); ○
signi ican ly g ea e han un ea ed g oup (p>0.05), + signi ican ly g ea e han he es
o he g oups (p<0.05). (B) Wound images.
20
Fig 6. His ological analysis. (A) Reepi helisa ion g ade. ** Signi ican ly g ea e han
he es o he g oups (p>0.01). (B) G ade o esolu ion o in lamma ion. **
Signi ican ly g ea e han NLC-LL37 low dose (p<0.05); ● signi ican ly g ea e han
un ea ed g oup (p<0.05). (C) Collagen deposi ion. (D) Numbe o new essels in
immunohis ochemically s ained issue slides. All he esul s a e shown as mean ±SD.
Table 1.Cha ac e isa ion o NLCs: Mean size, PDI, ze a po en ial, EE and pep ide
loading. Da a a e shown as he means ± SD.
Size (nm) PDI Ze a
Po en ial(mV) EE % Pep ide loading
(µg LL37/mg NLC)
NLC-LL37 273.6 ± 27.64 0.31 ± 0.06 -31.63 ± 1.94 96.40 ± 0.41 16.76±0.07
Emp y NLC 220.6 ± 5.48 0.27 ± 0.03 -26.10 ± 0.53
