pha maceu ics
A icle
Design o Polyme ic Nanocapsules o In anasal
Vaccina ion agains Mycobac e ium Tube culosis:
In luence o he Polyme ic Shell and
An igen Posi ioning
La a Diego-González 1,2, JoséC ecen e-Campo 3,* , Ma hew John Paul 4, Maha i Singh 5,
Rajko Reljic 4, Ma ía JoséAlonso 3,Á ica González-Fe nández 1,2 and
Rosana Simón-Vázquez 1,2,*
1Inmunología, Cen o de In es igaciones Biomédicas, CINBIO, Uni e sidade de Vigo,
Campus Uni e si a io Lagoas Ma cosende, 36310 Vigo, Spain; [email p o ec ed] (L.D.-G.);
[email p o ec ed] (Á.G.-F.)
2
Ins i u o de In es igaci
ó
n Sani a ia Galicia Su (IIS-GS), SERGAS-UVIGO, Es ada de Cla a Campoamo , 341,
36312 Vigo, PO, Spain
3
Depa men o Pha macology, Pha macy and Pha maceu ical Technology, School o Pha macy, Campus Vida,
Cen e o Resea ch in Molecula Medicine and Ch onic Diseases (CIMUS), IDIS Resea ch Ins i u e,
Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain; [email p o ec ed]
4Ins i u e o In ec ion and Immuni y, S Geo ge’s Medical School, London SW17 0RE, UK;
[email p o ec ed] (M.J.P.); [email p o ec ed] (R.R.)
5Lionex GmbH, 38126 B aunschweig, Ge many; [email p o ec ed]
*Co espondence: [email p o ec ed] (J.C.-C.); [email p o ec ed] (R.S.-V.)
Recei ed: 14 May 2020; Accep ed: 26 May 2020; Published: 28 May 2020
Abs ac :
Tube culosis (TB) is he leading cause o dea h om a single in ec ious mic oo ganism
and Bacillus Calme e Gue in (BCG), he only au ho ized accine, does no con e p o ec ion agains
pulmona y TB. Based on he hypo hesis ha mucosal p o ec ion could help o p e en he in ec ion
a he si e o en ance, he objec i e o his wo k was o de elop an in anasal accine agains
Mycobac e ium ube culosis (M b), he mic oo ganism ha causes TB. Ou app oach consis ed o
he use o polyme ic nanocapsules (NCs) wi h an oily co e and a polyme shell made o chi osan
(CS) o inulin/polya ginine (INU/pA g). The immunos imulan Imiquimod, a Toll-like ecep o -7
(TLR-7) agonis , was encapsula ed in he oily co e and a usion p o ein, o med by wo an igens
o M b, was abso bed ei he on o he NC su ace (CS:Ag and INU:pA g:Ag) o be ween wo
polyme laye s (INU:Ag:pA g) in o de o assess he in luence o he an igen posi ioning on he
immune esponse. Al hough CS NCs we e mo e immunos imulan han he INU/pA g NCs
in i o
,
he
in i o
expe imen s showed ha INU:pA g:Ag NCs we e he only p o o ype inducing an adequa e
immunoglobulin A (IgA) esponse. Mo eo e , a p e ious immuniza ion wi h BCG inc eased he
immune esponse o CS NCs bu , con e sely, dec eased o INU/pA g NCs. Fu he op imiza ion o
he an igen and he accina ion egime could p o ide an e icacious accine, using he INU:pA g:Ag
NC p o o ype as nanoca ie .
Keywo ds:
6 kDa ea ly sec e o y an igenic a ge (ESAT-6); 10 kDa cul u e il a e p o ein (CFP-10);
accina ion; Imiquimod; Toll-like ecep o -7 (TLR-7); an ibodies; cy okines; complemen sys em;
eac i e oxygen species (ROS)
Pha maceu ics 2020,12, 489; doi:10.3390/pha maceu ics12060489 www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2020,12, 489 2 o 22
1. In oduc ion
The use o accines o p e en se e e in ec ious diseases has been a his o ical landma k in medicine.
Howe e , some pa hogens emain elusi e o he de elopmen o an e icien accine agains hem, such
as he case o ube culosis (TB), he mos deadly in ec ious disease in he wo ld, caused by Mycobac e ium
ube culosis (M b) [
1
]. The BCG accine, con aining he Bacillus Calme e Gue in, which is he only
one licensed o da e o TB, p o ec s agains non-pulmona y TB in in an s, howe e , i is un eliable
in p o ec ing agains pulmona y TB, which accoun s o mos o he disease bu den wo ldwide [
2
].
App o ed accines based on li e-a enua ed o inac i a ed pa hogens p o ide a good immunogenici y
in gene al, bu he isk associa ed o hei adminis a ion is ele an . Fo ha eason, subuni accines
a e p e e ed due o hei inhe en sa e y, al hough hey show limi ed immunogenici y [
3
]. Mo eo e ,
he adju an s a ailable on he ma ke , mainly based on aluminum sal s, ha e ailed o induce an
e icien immune esponse agains some an igens, due o a biased o a supp essi e immune esponse,
among o he ac o s [4].
Fo hese easons, new s a egies o s imula e he immune sys em owa ds be e p o ec i e
esponses a e s ongly needed. In his sense, nano echnology o e s he possibili y o de elop mo e
powe ul accines. This is because he associa ion o an igens o nanoca ie s enables hei p o ec ion
agains deg ada ion and imp o es hei p esen a ion o he immune sys em [5,6].
Polyme - and lipid-based nanoca ie s a e among he mos widely used nanoca ie s o accine
de elopmen due o, among o he p ope ies, he i biocompa ibili y and biodeg adabili y, he capaci y
o some polyme s and lipids o in e ac wi h pa e n- ecogni ion ecep o s (PRRs) o cell memb anes,
and hei capaci y o enhance bo h humo al and cellula immune esponses [
5
,
7
–
10
]. In pa icula ,
polyme ic nanocapsules (NCs) ha e been shown o be p omising ca ie s o he deli e y o a a ie y
o an igens agains di e en pa hogens [11–13].
In mos accines, a balanced ype 1 T helpe / ype 2 T helpe (Th1/Th2) esponse is desi ed o igge
a wide- anging immune esponse and, consequen ly, p o ec i e e icacy [
8
,
14
]. The immunogenici y o
he nanosys ems can be u he enhanced by including small immunos imulan molecules in he pa icle
s uc u e [
4
]. In his sense, Imiquimod (IMQ) has been desc ibed as a good modula o o he inna e
immuni y and ac i a o o he Th1 immune esponse ia binding o he Toll-like ecep o -7 (TLR-7) on
an igen p esen ing cells (APCs). P e ious wo k om ou labo a o y has shown ha encapsula ion
o IMQ in chi osan (CS) NCs induced p o ec i e an ibody le els agains he ecombinan hepa i is B
su ace an igen (HB) in mice immunized by he in anasal (i.n.) ou e [
8
]. In e es ingly, he i.n. ou e
could also induce addi ional p o ec ion a he mucosal le el, wi h he p oduc ion o immunoglobulin
iso ype A (IgA) an ibodies and ac i a ion o local immune cells [
15
]. P omp , app op ia e mucosal
immune esponses could be e y help ul o neu alize pa hogens a hei main ou e o en ance, such
as in he case o M b, a oiding he de elopmen o he in ec ion al oge he .
Ha ing his backg ound in mind, he goal o his wo k was o de elop polyme ic NCs con aining
he immunos imulan IMQ and a usion p o ein an igen o he 6 kilodal ons (kDa) ea ly sec e o y
an igenic a ge (ESAT-6)and he10kDa Cul u eFil a eP o ein(CFP-10) agains M b o beadminis e ed
in anasally. To s udy he e ec o he polyme ic shell and an igen dis ibu ion on he immunogenici y
o an i.n. accine, we selec ed wo di e en NCs. CS and inulin/polya ginine (INU/pA g) we e selec ed
as polyme ic shell o he i s and second NC p o o ypes, espec i ely. Mo eo e , in he INU/pA g NCs,
he an igen was added on he su ace o he pA g polyme shell o be ween he wo polyme laye s o
assess he in luence o he an igen posi ioning on he immune esponse. In ac , he en apmen o he
an igen in a bilaye disposi ion o polyme ic NCs has ecen ly been shown o o e adequa e p o ec ion
and an enhanced immune esponse owa ds he associa ed an igen [11].
The biocompa ibili y and he immunos imulan p ope ies o he NCs we e es ed
in i o
wi h
di e en celllinesandhuman pe iphe albloodmononuclea cells(PBMCs). Finally, he immunogenici y
o he accine p o o ypes by he i.n. ou e was es ed ei he in naï e mice o in mice p e iously
immunized (subcu aneously, s.c.) wi h he BCG accine, o es he syne gy be ween he con en ional
BCG accine and he polyme ic NC accines.
Pha maceu ics 2020,12, 489 3 o 22
2. Ma e ials and Me hods
2.1. Ma e ials o he Syn hesis o he Nanocapsules (NCs)
Ul apu e CS hyd ochlo ide sal (CS.HCl) (Molecula weigh (Mw) 42.7 kilodal ons (kDa),
deace yla ion deg ee o 88%) was pu chased om Heppe Medical Chi osan GmbH (Saale, Ge many).
Inu ec
®
SL1 (25% modi ied inulin (INU) suspension in glyce ol) was a kind gi om C eaChem
(Tienen, Belgium).
PA g (Mw 24 kDa) was pu chased om PTS (Valencia, Spain) and Imiquimod was p o ided by
Fe e Heal hTech (Ba celona, Spain).
Sodium glycochola e and sodium chola e we e pu chased om Dex a (Reading, UK).
Miglyol
®
812 was dona ed by C eme Oleo GmbH & Co (Hambu g, Ge many) and linoleic acid
was p o ided by Ac os O ganics, The mo Fishe (Wal ham, MA, USA).
The 1,2-dis ea oyl-sn-glyce o-3-phosphoe hanolamine-N-[me hoxy (polye hylene glycol) 1000]
(18:O PE-PEG1000) was pu chased om A an i Pola Lipids Inc. (Alabas e , AL, USA).
Suc ose and ehalose we e pu chased om Aco a ma (Mad id, Spain).
ESAT-6/CFP-10 usion p o ein (ECH) was p o ided by Lionex Gmbh (B aunschweig, Ge many,
www.lionex.de).
All sol en s employed we e o analy ical g ade and supplied by Me ck O ganic sol en s
(
Mad id, Spain
). Endo oxin- ee wa e was collec ed in a Millipo e Ul apu e Wa e Sys em equipped
wi h a il e uni o endo oxin emo al.
2.2. Nanocapsules’ Syn hesis and Cha ac e iza ion
The NCs we e syn he ized by he sol en -displacemen me hod, as desc ibed p e iously [
16
,
17
].
Fo CS NCs, he o ganic phase was p epa ed wi h 125
µ
L o a mix u e o linoleic acid an Miglyol
®
812 (9.5:3, / a io) and 1 mg o IMQ, 20 mg o he PEGyla ed phosphoe hanolamine 18:0 PE-PEG1000
and 25
µ
L o an aqueous solu ion o 200 mg/mL sodium chola e in 5 mL o e hanol. The aqueous
phase con ained 5 mg o CS.HCl sal dissol ed in 10 mL o wa e . A e pou ing he o ganic phase
o e he aqueous phases, s i ing he suspension and e apo a ing he o ganic sol en in a o a apo
(Büchi, Swi ze land), he suspension was aken o a inal olume o 5 mL wi h ul apu e wa e .
Fo he INU/pA g NCs, he o ganic phase was o med by 34.5
µ
L o a mix u e o linoleic acid and
Miglyol
®
812 (6.5:1, / a io) and 1 mg o IMQ, 5 mL o e hanol, and 25
µ
L o an aqueous solu ion o
sodium glycochola e a 200 mg/mL. The aqueous phase was p epa ed by dissol ing 15 mg o Inu ec
SL1 in 10 mL o wa e . The NCs we e p epa ed as desc ibed abo e o he CS NCs. A e wa ds, 0.5 mL
o a pA g aqueous solu ion a 10 mg/mL was added o he INU NCs, be o e o a e he addi ion o
he an igen, o o m he INU:pA g:Ag and he INU:Ag:pA g NCs, espec i ely. The o mula ion was
shaken o 1 h a 300 pm in a he moblock (Eppendo The momixe R) o allow he adso p ion o he
posi i ely cha ged pA g o e he nega i ely cha ged INU co e by elec os a ic in e ac ions.
Lyophiliza ion s udies o he inal NC p o o ypes we e pe o med in he p esence o 5 and 10%
suc ose o ehalose, as c yop o ec an .
Pa icle size and polydispe si y index (PdI) o he o mula ions in ul apu e wa e we e
cha ac e ized by pho on co ela ion spec oscopy in a Ze asize Nano-S (Mal e n Ins umen s;
Mal e n, UK) a 25
◦
C. Ze a po en ial (Z-po en ial) was also measu ed in he same equipmen by lase
Dopple anemome y (LDA) using an app op ia e cell and 1 mM KCl as sol en .
The IMQ encapsula ed in he oily co e was de e mined a e ul acen i uga ion (30,000 pm, 1 h,
15
◦
C) o he NCs o sepa a e he unde na an , con aining he ee IMQ (C ), om he c eam, wi h
he encapsula ed IMQ (Ce). Besides, o al IMQ concen a ion (C ) was also measu ed in ini ial NCs.
All samples we e dilu ed 1:10 wi h MeOH o b eak he NCs and solubilize he IMQ. Subsequen ly,
he samples we e analyzed by HPLC, as desc ibed in [
8
]. A calib a ion cu e was also made up wi h
Pha maceu ics 2020,12, 489 4 o 22
s anda d solu ions o IMQ o de e mine he concen a ion in each sample. The encapsula ion e iciency
(%EE) and he d ug loading (%DL) we e quan i ied di ec ly acco ding o he o mula:
%EE =(Ce/C ) ×100, %DL =[IMQ (mg)/NCs (mg)] ×100 (1)
2.3. Adso p ion o he An igen on o he Nanocapsules and Quan i ica ion o he Adso bed P o ein
Fo he de elopmen o he model accine, a p elimina y sc eening was pe o med, adding
he ECH usion p o ein o he NCs a di e en a ios (polyme :an igen, w/w), o ind he maximum
an igen loading which did no comp omise he NCs colloidal s abili y. The an igen was added o
he IMQ-loaded NCs and he mix u e was incuba ed a oom empe a u e wi h cons an o bi al
shaking (300 pm) o one hou in a he moshake (Eppendo The momixe R) o allow he p o ein
adso p ion o he polyme su ace. The amoun o an igen adso bed on he su ace o IMQ-loaded NCs
(
CS:Ag and
INU:pA g:Ag) o en apped be ween wo polyme laye s (INU:Ag:pA g) was de e mined
by SDS-PAGE and Coomassie blue s aining. The NCs we e ul acen i uged (30,000 pm, 1 h, 15
◦
C) o
sepa a e ee (ECH ) om a ached p o ein (ECH.NCs) and an aliquo o bo h ac ions was loaded in a
10% polyac ylamide gel. A molecula ma ke was also loaded in a di e en lane o ollow he p o ein
sepa a ion and o check he ECH p o ein molecula weigh . A e unning he elec opho esis, he gel
was s ained and he densi y o he p o ein bands was quan i ied in a ChemiDoc equipmen using
he LabImage so wa e (Bio-Rad Labo a o ies, Inc, He cules, CA, USA). The pe cen age o abso bed
an igen (%AA) was calcula ed acco ding o he o mula:
%AA =ECH.NCs/(ECH.NCs +ECH ) (2)
2.4. Ma e ials o P o ein, Cell, and Animal S udies
ESAT-6 and CFP-10 an igens we e p o ided by Lionex Gmbh (B aunschweig, Ge many,
www.lionex.de).
Mouse monoclonal an ibody (mAb) agains human complemen ac o C3 and he spli p o ein
C3b (C3-C3b) was om Abcam, (Camb idge, UK) and polyclonal goa an i-mouse IgG an ibodies (Abs)
conjuga ed wi h alkaline phospha ase (AP) we e om Dako (Glos up, Denma k).
The 5-b omo-4-chlo o-3-indolyl-phospha e (BCIP)/ni o blue e azolium (NBT) subs a e
solu ion, 2
0
,7
0
-dichlo odihyd o luo escindiace a e (H
2
DCFDA), an i-mouse IgA, an i-mouse IgG,
and o-phenylenediamine dihyd ochlo ide (OPD) subs a e we e pu chased om Sigma-Ald ich Co.
and molecula weigh ma ke was om The mo Fishe Scien i ic, Spain.
Cell Ti e 96
®
AQueous One Solu ion Cell P oli e a ion Assay ki , 3-(4, 5-dime hyl hiazol-2-yl)-
5-(3-ca boxyme hoxyphenyl)-2-(4-sul ophenyl)-2H- e azolium (MTS), and endo oxin es we e om
P omega Bio ech Ib
é
ica, (Mad id, Spain) and Associa es o Cape Cod, Inc. (Falmou h, MA, USA),
espec i ely.
Pho bol 12-my is a e-13-ace a e (PMA) was om Abcam (Camb ige, UK), lipopolysaccha ide
(LPS) om In i oGen (Toulouse, F ance), and phy ohaemagglu inin (PHA) om Sigma-Ald ich Co.
The MILLIPLEX
®
MAP Ki used o cy okine p o ile cha ac e iza ion was p o ided by Millipo e,
Me ck KGaA (Da ms ad , Ge many).
2.5. Cells and Cul u e Condi ions
Fi e di e en cell lines om he Ame ican Type Cul u e Collec ion (ATCC) (Manassas, VA, USA)
we e used: Raw 264.7 (mouse mac ophages), THP-1 (human monocy es), A549 and NCI-H460 (human
lung epi helial cells), and HL-60 (human p omyeloblas cells).
The cells we e cul u ed in Roswell Pa k Memo ial Ins i u e medium (RPMI) supplemen ed wi h
10% ( / ) hea ed, inac i a ed e al bo ine se um (FBS) (PAA; Pasching, Aus ia), 2 mM glu amine and
100 U/mL o penicillin/s ep omycin, a 37
◦
C in 5% CO
2
a mosphe e. Cells we e dilu ed e e y wo o
h ee days, a e eaching a 70–80% o con luence.
Pha maceu ics 2020,12, 489 5 o 22
2.6. E alua ion o Bac e ial o Endo oxin Con amina ion
The absence o bac e ial con amina ion was e alua ed by seeding 100
µ
L o he samples on
Lu ia-Be ani (LB) aga pla es unde asep ic condi ions. The pla es we e incuba ed a 37
◦
C o 24–72 h.
Po en ial endo oxin con amina ion on he NC o mula ions was also assessed by he gel-clo assay,
ollowing he manu ac u e ’s ins uc ions.
2.7. Toxici y S udies on Mac ophages and Lung Epi helial Cells
Cell iabili y es s we e pe o med in wo mac ophage (RAW 264.7 and PMA-di e en ia ed
THP-1) and wo lung epi helial (A549 and NCI-H460) cell lines.
The colo ime ic MTS es was used o quan i y he changes on cell iabili y a e 24 h o incuba ion
wi h di e en concen a ions o he IMQ-loaded NCs.
The cells we e seeded on 96-well pla es a a densi y o 1.5
×
10
4
cells/well o THP-1 and RAW264.7,
and 6
×
10
3
and 7
×
10
3
cells/well o A549 and NCI-H460, espec i ely. THP-1 cells we e incuba ed
wi h pho bol 12-my is a e-13-ace a e (PMA) a 10 ng/mL o 24 h o induce hei di e en ia ion in o
mac ophages. The medium wi h PMA was eplaced wi h esh medium be o e adding he NCs.
The maximum concen a ion es ed o CS and INU/pA g NCs was 1440 and 545
µ
g/mL, espec i ely.
Se e al dilu ions (1:2) o each NC p o o ype we e also es ed in o de o de e mine he hal -maximun
inhibi o y concen a ion (IC50). Cul u e medium alone and wi h NCs was used o backg ound
sub ac ion and o disca d any in e e ence o he NCs in he abso bance. The pe cen age o cell
iabili y was calcula ed using he ollowing Equa ion:
%Cell iabili y =[(Abs Cells&NCs −Abs NCs)/(Abs Cell −Abs Medium)] ×100 (3)
2.8. P oduc ion o Reac i e Oxygen Species (ROS)
Fo he cha ac e iza ion o ROS elease, he p omyeloblas cell line HL-60 was used. The cells
we e seeded a a densi y o 1
×
10
5
cells/well in a 96-well pla e and incuba ed wi h he NCs du ing 1, 3,
6, and 14 h. The NCs we e es ed a h ee di e en concen a ions (50, 100, and 200
µ
g/mL o CS NCs
and 10, 50, and 100 µg/mL o INU:pA g NCs).
PMA a 20
µ
M was added o he cells as posi i e con ol and cells incuba ed only wi h cul u e
medium we e used as nega i e con ol. A e he co esponding incuba ion ime, H
2
DCFDA a 2.5
µ
M
was added o he cells and incuba ed o 30 min a 37
◦
C. A e di usion in o he cell and oxida ion
o 2
0
,7
0
-dichlo o luo escein (DCF) by ROS in a concen a ion-dependen a e, he cells we e washed
and he luo escence p oduced by DCF was measu ed in a Beckman Coul e FC500 low cy ome e
(Beckman Coul e , IN, USA). Cells we e also labelled wi h p opidium iodide (PI) o disc imina e li e
and dead popula ions.
2.9. Analysis o Complemen Ac i a ion
Complemen ac i a ion induced by he IMQ-loaded NCs in a pool o plasma om heal hy dono s
was assessed by measu ing he deg ada ion o he C3 ac o by Wes e n blo .
Equal olumes o plasma, PBS, and samples we e mixed and incuba ed a 37
◦
C o 1 h. Zymosan
a a inal concen a ion o 1 mg/mL and PBS we e used as posi i e and nega i e con ols, espec i ely.
The NCs we e es ed a h ee di e en concen a ions (50, 100, and 200
µ
g/mL o CS NCs and 10, 50,
and 100 µg/mL o INU/pA g NCs).
A e incuba ion, supe na an s we e collec ed and 2
µ
L we e mixed wi h sample bu e and loaded
on o a 10% SDS-PAGE gel and ans e ed o a Poly inylidene Di luo ide (PVDF) memb ane using he
T ansblo Semid y T ans e Equipmen (Bio-Rad Labo a o ies, Inc).
The PVDF memb anes we e blocked wi h 5% non a milk in T is-bu e ed saline in Tween 20
(TBS-T), and incuba ed sequen ially wi h mouse monoclonal an ibody (mAb) agains human C3-C3b
ollowed by polyclonal goa an i-mouse IgG an ibodies (Abs) conjuga ed wi h alkaline phospha ase
Pha maceu ics 2020,12, 489 6 o 22
(AP), bo h dilu ed 1:2000 in 5% non a milk in TBS-T. Each incuba ion was ca ied ou o 1 h a
oom empe a u e and he memb anes we e washed h ee imes wi h TBS-T be ween each incuba ion.
The bands o he in ac and spli C3 p o ein we e isualized adding a BCIP/NBT subs a e solu ion
ha eac ed wi h he AP and o med a da k p ecipi a e. The in ensi y o he bands was quan i ied
on he ChemiDoc (Bio-Rad Labo a o ies, Inc). The a io o spli /in ac p o ein was no malized o he
nega i e con ol.
2.10. Cy okine P o ile E alua ion
Cy okines’ elease was s udied in human PBMCs om h ee heal hy dono s. The PBMCs we e
ob ained om whole blood by densi y g adien cen i uga ion. A e cen i uga ion, cells we e washed
wi h PBS and seeded a a densi y o 1.75
×
10
6
cells/mL in 96-well U-bo om pla es. The cells we e
allowed o es o 24 h in he incuba o be o e adding he samples.
The NCs we e added a he same inal concen a ions as in he p e ious expe imen and he
pla e was incuba ed o ano he 24 h. As posi i e con ol, lipopolysaccha ide (LPS) a 1
µ
g/mL wi h
phy ohaemagglu inin (PHA) a 10
µ
g/mL was used and cells wi hou any ea men we e used as
nega i e con ol.
A e incuba ion wi h he NCs, he pla e was cen i uged a 1200 pm o 5 min and 100
µ
L o
supe na an pe well we e eco e ed and s o ed a −80 ◦C un il measu emen .
A MILLIPLEX
®
MAP Ki o 11 cy okines (g anulocy e monocy e-colony s imula ing ac o
(GM-CSF), in e e on (IFN)-
γ
, in e leukin (IL)-10, IL-12, IL-17, IL-1
β
, IL-2, IL-4, IL-5, IL-6, and
umo nec osis ac o (TNF)-
α
was used o measu e he concen a ion o hese cy okines in he
supe na an s collec ed om he PBMCs using a MAGPIX
®
ins umen (Millipo e, Me ck KGaA,
Da ms ad , Ge many) and ollowing he manu ac u e ’s ins uc ions. The MAGPIX
®
so wa e was
used o he analysis o he s anda d cu es and he samples.
Mouse IFN-
γ
, TNF-
α
, and IL-17 elease by he splenocy es o immunized animals a e an igen
ecall was moni o ed by enzyme-linked immunoso ben assay (ELISA) (In i ogen 88-7314-86,
88-7324-77, 88-7371-77, The moFishe Scien i ic, Loughbo ough, UK).
2.11. Immuniza ion Expe imen s
Female C57BL/6 mice we e ob ained om Cha les Ri e , UK, and we e be ween 8 and 12 weeks
o age be o e expe imen al use. In he g oups speci ied, mice we e subcu aneously immunized wi h
5×105
CFU BCG Pas eu (100
µ
L) o ehicle con ol. A e 12 weeks, in anasal boos e immuniza ions
we e gi en consis ing o 3x IMQ-loaded polyme ic NCs coa ed wi h 10
µ
g ECH in 50
µ
L pe animal pe
dose, o ehicle con ol. Doses we e gi en 2 weeks apa . These immuniza ions we e pe o med unde
ligh anes hesia. Th ee weeks ollowing he inal immuniza ion, animals we e culled by in ape i oneal
(i.p.) adminis a ion o 200 mg/kg pen oba bi one solu ion (Pen ojec
®
, Animalca e L d., Yo k, UK).
B onchoal eola luid was collec ed by passing 1 mL o PBS in o he lungs using a 22-gauge ca he e
posi ioned in he achea.
2.12. De ec ion o Speci ic An ibodies
An ibody le els we e quan i ied by ELISA. An igens [ECH, ESAT-6, and CFP-10] we e coa ed
on o a pla e a 2
µ
g/mL o e nigh , ollowed by blocking o 2 h wi h PBS con aining 1% bo ine se um
albumin (BSA). Se um samples we e loaded a a 1:10 dilu ion in PBS wi h 1% BSA and 4- old se ial
dilu ions we e made. B onchoal eola la age luid (BAL) samples we e loaded undilu ed and 3- old
se ial dilu ions we e pe o med. All samples we e incuba ed on he pla e in iplica e o 1 h a
37 ◦C
.
Le els o IgA (BAL) o IgG (se um) we e de ec ed using pe oxidase-conjuga ed an i-mouse IgA o
an i-mouse IgG and OPD subs a e. Pla es we e ead on a Tecan In ini e F200 P o pla e- eade a
450-nm abso bance.
Pha maceu ics 2020,12, 489 7 o 22
2.13. S a is ical Analysis
The G aphPad P ism 8 so wa e (G aphPad So wa e Inc., San Jose, CA, USA) was used o da a
ep esen a ion and s a is ical analysis. Each expe imen was pe o med a leas h ee imes (
n≥3
) and
he esul s o he di e en assays we e ep esen ed as mean
±
s anda d de ia ion (SD). T-s uden o
Mann–Whi ney es was pe o med o de e mine signi ican di e ences be ween nega i e con ol and
ea men s. In he g aphs, esul s a e indica ed as: * P
≤
0.05, ** P
≤
0.01, *** P
≤
0.001,
**** P ≤0.0001
.
3. Resul s
3.1. Physicochemical Cha ac e iza ion and S abili y o he Nanocapsules
Du ing his wo k, h ee di e en accine p o o ypes o polyme ic NCs we e de eloped. Fo he
i s p o o ype, CS was used as a polyme shell because o he good biocompa ibili y and he p o en
capaci y o CS-based nanoca ie s o elici mucosal and sys emic immune esponse agains a a ie y o
an igens a e i.n. adminis a ion [
8
,
18
–
20
]. The oily co e consis ed o a mix u e o linoleic acid and
Miglyol
®
812. PEGyla ed phosphoe hanolamine 18:0 and sodium chola e we e used as su ac an
and as co-su ac an , espec i ely. The an igen was abso bed on o he su ace by incuba ing he
an igen solu ion wi h he p e o med NCs. The adso p ion was p obably caused by a combina ion o
elec os a ic and hyd ophobic in e ac ions (Figu e 1and Table 1).
Pha maceu ics 2020, 12, x 7 o 22
Du ing his wo k, h ee di e en accine p o o ypes o polyme ic NCs we e de eloped. Fo he
i s p o o ype, CS was used as a polyme shell because o he good biocompa ibili y and he p o en
capaci y o CS-based nanoca ie s o elici mucosal and sys emic immune esponse agains a a ie y
o an igens a e i.n. adminis a ion [8,18–20]. The oily co e consis ed o a mix u e o linoleic acid and
Miglyol® 812. PEGyla ed phosphoe hanolamine 18:0 and sodium chola e we e used as su ac an
and as co-su ac an , espec i ely. The an igen was abso bed on o he su ace by incuba ing he
an igen solu ion wi h he p e o med NCs. The adso p ion was p obably caused by a combina ion o
elec os a ic and hyd ophobic in e ac ions (Figu e 1 and Table 1).
Fo he second p o o ype, pA g was selec ed as a polyme shell due o he pene a ing
p ope ies o his polypep ide, which could help c ossing he mucosal ba ie [21]. In he pA g NCs,
a i s laye o hyd ophobized INU and sodium glycochola e we e used o inc ease he s abili y o
he naoemulsion (NE), whose co e was again a mix u e o linoleic acid and Miglyol® 812.
A e wa ds, an igen was adso bed on o he pA g su ace. Howe e , he an igen could also be added
a he su ace o he INU be o e adding he pA g shell, en apping i in a sandwich-like a angemen
[11]. This enginee ing s a egy allowed ob aining wo di e en accine p o o ypes, one wi h he
an igen adso bed on he su ace (INU:pA g:Ag) and ano he one wi h he an igen p o ec ed by a
polyme ic bilaye (INU:Ag:pA g) (Figu e 1 and Table 1), which can in luence he accine
pe o mance, he eason why we conside ed i in e es ing o be s udied [11].
Figu e 1. Polyme ic accine p o o ypes designed o he de elopmen o an in anasal accine
agains he Mycobac e ium ube culosis usion p o ein ESAT-6/CFP-10 (ECH p o ein). In he oily co e,
Imiquimod, a TLR-7 agonis , was added as adju an o inc ease he immunogenici y o he usion
p o ein. Ag: An igen, CS: Chi osan, INU: Inulin, pA g: Polya ginine, TLR-7: Toll-like ecep o -7.
Table 1. Chemical composi ion o he CS and INU/pA g nanocapsules (NCs) de eloped in his wo k.
CS: Chi osan, INU: Inulin, pA g: Polya ginine.
Chemical Composi ion CS NCs INU/pA g NCs
Oil Miglyol / linoleic acid Miglyol/linoleic acid/glyce in
Adju an Imiquimod
Polyme Chi osan Modi ied inulin
Su ac an 18:0 PE-PEG1000
Co-su ac an Sodium chola e Sodium glycochola e
Second polyme ic shell - Polya ginine
An igen disposi ion Su ace Su ace/Polyme bilaye
To enhance he immunos imula o y p ope ies o he NCs, IMQ, a lipophilic immunos imulan ,
was inco po a ed in he oily co e o he nanosys ems. The IMQ-loaded polyme ic NCs, bo h o CS
Figu e 1.
Polyme ic accine p o o ypes designed o he de elopmen o an in anasal accine
agains he Mycobac e ium ube culosis usion p o ein ESAT-6/CFP-10 (ECH p o ein). In he oily co e,
Imiquimod, a TLR-7 agonis , was added as adju an o inc ease he immunogenici y o he usion
p o ein. Ag: An igen, CS: Chi osan, INU: Inulin, pA g: Polya ginine, TLR-7: Toll-like ecep o -7.
Table 1.
Chemical composi ion o he CS and INU/pA g nanocapsules (NCs) de eloped in his wo k.
CS: Chi osan, INU: Inulin, pA g: Polya ginine.
Chemical Composi ion CS NCs INU/pA g NCs
Oil Miglyol/linoleic acid
Miglyol/linoleic acid/glyce in
Adju an Imiquimod
Polyme Chi osan Modi ied inulin
Su ac an 18:0 PE-PEG1000
Co-su ac an Sodium chola e Sodium glycochola e
Second polyme ic shell - Polya ginine
An igen disposi ion Su ace Su ace/Polyme bilaye
Pha maceu ics 2020,12, 489 8 o 22
Fo he second p o o ype, pA g was selec ed as a polyme shell due o he pene a ing p ope ies
o his polypep ide, which could help c ossing he mucosal ba ie [
21
]. In he pA g NCs, a i s
laye o hyd ophobized INU and sodium glycochola e we e used o inc ease he s abili y o he
naoemulsion (NE), whose co e was again a mix u e o linoleic acid and Miglyol
®
812. A e wa ds,
an igen was adso bed on o he pA g su ace. Howe e , he an igen could also be added a he
su ace o he INU be o e adding he pA g shell, en apping i in a sandwich-like a angemen [
11
].
This enginee ing s a egy allowed ob aining wo di e en accine p o o ypes, one wi h he an igen
adso bed on he su ace (INU:pA g:Ag) and ano he one wi h he an igen p o ec ed by a polyme ic
bilaye (INU:Ag:pA g) (Figu e 1and Table 1), which can in luence he accine pe o mance, he eason
why we conside ed i in e es ing o be s udied [11].
To enhance he immunos imula o y p ope ies o he NCs, IMQ, a lipophilic immunos imulan ,
was inco po a ed in he oily co e o he nanosys ems. The IMQ-loaded polyme ic NCs, bo h o CS
and pA g p o o ypes, had a pa icle size abou 150 nm and a PdI lowe han 0.2 (Table 2). The su ace
cha ge was posi i e o CS and INU/pA g NCs and nega i e o INU NCs (Table 2), consis en wi h he
posi i e cha ge o CS and pA g polyme s and he nega i e cha ge o he chola e-s abilized INU NCs.
Table 2.
Physicochemical cha ac e iza ion, Imiquimod encapsula ion e iciency, and an igen abso p ion
o he di e en nanocapsules’ p o o ypes.
Nanocapsules Size (nm) Z (mV) IMQ %EE %AA
CS 145 ±14 +28 ±4.5 60 ±4 n.a.
CS:Ag 152 ±15 +8±10 n.a. 60 ±6
INU 158 ±7−37 ±4 69 ±6 n.a.
INU:Ag 136 ±7−36 ±6 - n.d.
INU/pA g 158 ±22 +20 ±1 - n.a.
INU:Ag:pA g 151 ±11 +27 ±8 - 55 ±5
INU:pA g:Ag 158 ±12 +31 ±7 - 30 ±10
IMQ EE: Imiquimod encapsula ion e iciency, AA: An igen abso bed on he NCs. Polydispe si y index (PdI)
≤
0.2
in all he size measu emen s; n.a.: No applicable; n.d.: No de e mined. Ag: An igen, CS: Chi osan, INU: Inulin,
pA g: Polya ginine.
The selec ed an igen was a usion p o ein o wo M b an igens, ESAT-6 and CFP-10, named ECH
p o ein, wi h a molecula weigh o app oxima ely 21.5 kDa and a heo e ical isoelec ic poin (pI) o
5.06. ESAT-6 and CFP-10 a e i ulence- ela ed M b p o eins which a e absen in he BCG accine [
22
].
The polyme : an igen a io 1:0.2 (w/w) was selec ed as he bes a io o bo h, CS and pA g NCs,
based on he SDS-PAGE and dynamic ligh sca e ing (DLS) s udies (da a no shown). The %AA
was es ima ed by SDS-PAGE (Table 2) and was abou 60, 55, and 30% o CS:Ag, INU:Ag:pA g, and
INU:pA g:Ag NCs, which co esponded o an an igen loading o 0.4, 1.1, and 0.55%, espec i ely.
The ee an igen ha was no bound o he NCs allowed an ini ial bu s elease ha could boos
he immune esponse [
23
]. Fo ha eason, ee an igen was no emo ed om he suspension and,
o immuniza ion s udies, he amoun o an igen injec ed was calcula ed acco ding o he ini ial
p o ein concen a ion.
The pe cen age o IMQ in he NCs was measu ed by HPLC (as desc ibed in Sec ion 2.2) and
simila encapsula ion e iciency was ound o CS and INU/pA g NCs (Table 2). The encapsula ion
e iciency was abou 60–70% in bo h cases, which co esponded wi h a d ug loading o 4.2% o CS
NCs and 13% o INU/pA g NCs, espec i ely.
The nonloaded CS NCs we e s able in suspension a 4
◦
C o a leas 6 weeks, as shown by
he size and Z-po en ial measu emen s (Figu e 2). Howe e , he an igen-loaded NCs we e s able
only one week (Supplemen a y Figu e S1) due o, likely, he neu aliza ion o he CS NCs’ su ace
cha ge a e addi ion o he ECH usion p o ein (Table 2). Fo his eason, o all he expe imen s
wi h an igen-adso bed CS NCs, he an igen was added in si u and incuba ed one hou be o e he
adminis a ion o he o mula ion.
Pha maceu ics 2020,12, 489 9 o 22
Pha maceu ics 2020, 12, x 9 o 22
econs i u ion, he lyophilized NCs showed a size, a PdI, and a Ze a-po en ial simila o he alues
obse ed a e hei lyophiliza ion. Opposi ely, he size and PdI o he CS:Ag NCs inc eased a e he
lyophiliza ion, and he use o ehalose o a lowe suc ose pe cen age p oduced simila esul s (da a
no shown).
Figu e 2. Size (ba s), PdI, and Ze a-po en ial (lines and symbols) o Imiquimod-loaded CS (A and B),
INU:Ag:pA g and INU:pA g:Ag (C and D) nanocapsules s o ed a 4 °C a di e en ime poin s. Due
o he educed s abili y o he CS nanocapsules a e an igen adso p ion (Figu e S1), he nanocapsules
we e es ed in he absence o he an igen. Size and PdI we e measu ed in ul apu e wa e and
Ze a-po en ial in 1 mM KCl. Ag: An igen, CS: Chi osan, INU: Inulin, pA g: Polya ginine.
In o de o be able o d aw adequa e conclusions om he in i o expe imen s, he s abili y o
he NCs was es ed in comple e cul u e medium by eco ding size and PdI a 4 di e en ime poin s
(Figu e 3). CS:Ag and INU:Ag:pA g NCs we e s able a all he ime poin s eco ded (4 h), whe eas
he s abili y o INU:pA g:Ag NCs was limi ed o less han 30 min, bu p esumably long enough o
allow pa icle in e naliza ion and d ainage o he lymph nodes, which usually akes place in he i s
minu es a e adminis a ion [24].
Hence, he p o ec ion o he an igen by he pA g shell inc eased he s abili y o he INU/pA g
NCs, in compa ison wi h he adso p ion o he an igen in o he su ace o he NCs, in physiological
medium. Con e sely, CS NCs wi h he an igen on he su ace we e e y s able.
Figu e 3. S abili y o Imiquimod-loaded CS:Ag (A), INU:Ag:pA g, and INU:pA g:Ag (B)
nanocapsules on RPMI cul u e medium supplemen ed wi h 10% FBS and incuba ed a 37 °C. G aphs
Figu e 2.
Size (ba s), PdI, and Ze a-po en ial (lines and symbols) o Imiquimod-loaded CS (
A
,
B
),
INU:Ag:pA g and INU:pA g:Ag (
C
,
D
) nanocapsules s o ed a 4
◦
C a di e en ime poin s. Due o he
educed s abili y o he CS nanocapsules a e an igen adso p ion (Figu e S1), he nanocapsules we e
es ed in he absence o he an igen. Size and PdI we e measu ed in ul apu e wa e and Ze a-po en ial
in 1 mM KCl. Ag: An igen, CS: Chi osan, INU: Inulin, pA g: Polya ginine.
The INU/pA g NCs, bo h wi h he an igen p o ec ed by he pA g shell (INU:Ag:pA g) o wi h
he an igen on he su ace (INU:pA g:Ag), we e s able o h ee weeks a 4
◦
C (Figu e 2). A e his
pe iod, he NCs showed a endency o unde go coalescence (da a no shown). To inc ease hei s abili y,
he an igen-loaded CS and INU/pA g NCs we e lyophilized wi h 5 o 10% suc ose o ehalose as
c yop o ec an . Among all o hem, he use o 10% suc ose p o ed o be e ec i e o p ese e he
INU/pA g NCs du ing he lyophiliza ion p ocess and u he econs i u ion in wa e , bu no o he
CS:Ag NCs (Supplemen a y Figu e S2). The lyophilized INU/pA g NCs we e easily econs i u ed by
adding wa e and immedia ely o exing he ial o wo minu es. A e econs i u ion, he lyophilized
NCs showed a size, a PdI, and a Ze a-po en ial simila o he alues obse ed a e hei lyophiliza ion.
Opposi ely, he size and PdI o he CS:Ag NCs inc eased a e he lyophiliza ion, and he use o
ehalose o a lowe suc ose pe cen age p oduced simila esul s (da a no shown).
In o de o be able o d aw adequa e conclusions om he
in i o
expe imen s, he s abili y o
he NCs was es ed in comple e cul u e medium by eco ding size and PdI a 4 di e en ime poin s
(Figu e 3). CS:Ag and INU:Ag:pA g NCs we e s able a all he ime poin s eco ded (4 h), whe eas he
s abili y o INU:pA g:Ag NCs was limi ed o less han 30 min, bu p esumably long enough o allow
pa icle in e naliza ion and d ainage o he lymph nodes, which usually akes place in he i s minu es
a e adminis a ion [24].
Hence, he p o ec ion o he an igen by he pA g shell inc eased he s abili y o he INU/pA g
NCs, in compa ison wi h he adso p ion o he an igen in o he su ace o he NCs, in physiological
medium. Con e sely, CS NCs wi h he an igen on he su ace we e e y s able.
Pha maceu ics 2020,12, 489 16 o 22
Pha maceu ics 2020, 12, x 16 o 22
low in all he g oups. In con as , TNF-α was no de ec ed in any o he g oups es ed (da a no
shown). Hence, he polyme ic NC accines we e able o induce IFN-γ and IL-17 elease, a Th1 and a
Th17 ac i a o o cy okines, espec i ely, bu only agains he ESAT-6 an igen. The CFP-10 an igen
induced only a weak p oduc ion o IFN-γ in animals immunized wi h he INU:pA g:Ag NCs.
Figu e 9. IL-17 and IFN-γ elease om splenocy es o immunized mice a e an an igen in i o ecall
expe imen . The splenocy es we e incuba ed wi h CFP-10, ESAT-6, o he ESAT-6/CFP-10 usion
p o ein (ECH) and he supe na an s we e collec ed o cy okine measu emen and we e compa ed o
he non-s imula ed (Uns im) samples. Mice p e iously immunized wi h BCG (+BCG) o no ,
ecei ed PBS (as nega i e con ol), CS:Ag, INU:pA g:Ag, o INU:Ag:pA g as accine p o o ypes.
IL-17: in e leukin 17, IFN-γ: in e e on gamma, Ag: An igen, CS: Chi osan, INU: Inulin, pA g:
Polya ginine. S a is ical signi icance: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
4. Discussion
Th ee accine p o o ypes we e designed and de eloped du ing his wo k, using CS o
INU/pA g NCs as an igen nanoca ie s. IMQ was inco po a ed in he oily co e as an addi ional
adju an . The IMQ-loaded CS:Ag, INU:pA g:Ag, and INU:Ag:pA g NCs showed a simila pa icle
size o abou 150 nm, app op ia e o nasal adminis a ion. In his sense, o an e icien deli e y
h ough he mucosal ou es, i was s ablished ha he pa icle size should be smalle han he mucus
mesh size [38]. This easily explains why NPs, in gene al, pe o med be e han mic opa icles [39–
43]. Howe e , in he nanome ic ange, in i o da a has shown ha e y small nanome ic size (30
nm) may be de imen al compa ed o la ge ones (200 nm) [44]. Besides, no only he pa icle size,
bu also he composi ion, play a c i ical ole when using NPs o nasal adminis a ion. In 1998,
Alonso’s g oup epo ed he impo ance o PEG o inc ease he mucope mea ion o polyes e -based
NPs [45], and, a e ha , many o he s udies con i med he impo ance o an adequa e PEG coa ing
o a o he di usion ac oss he mucus laye [46,47].
On he o he hand, he nanome ic size o he de eloped NCs was also adequa e o ha e a
p e e en ial in e naliza ion by dend i ic cells (DCs) [6,48]. DCs induce a be e T-cell s imula ion
han o he APCs, such as mac ophages and B-cells, and play a ele an ole in pa hogen cap u e and
induc ion o a p ima y immune esponse. Fo ha eason, pa icle accines y o a ge hese cells
[49]. Mo eo e , he NCs had a low (CS:Ag) o high (INU:pA g:Ag and INU:Ag:pA g) posi i e
cha ge, which is also use ul o he in e ac ion wi h he nega i ely cha ged mucin ollowing
in e naliza ion in he mucosa [47,50].
The selec ed an igen was a usion p o ein (ECH) o wo well-known M b an igens, ESAT-6 and
CFP-10. Bo h p o eins belong o he egion o di e ence (RD1) o M b, which is absen om all
s ains o BCG and is ela ed wi h he i ulence o he M b and M. bo is s ains. In ac , he
Figu e 9.
IL-17 and IFN-
γ
elease om splenocy es o immunized mice a e an an igen
in i o
ecall
expe imen . The splenocy es we e incuba ed wi h CFP-10, ESAT-6, o he ESAT-6/CFP-10 usion p o ein
(ECH) and he supe na an s we e collec ed o cy okine measu emen and we e compa ed o he
non-s imula ed (Uns im) samples. Mice p e iously immunized wi h BCG (+BCG) o no , ecei ed PBS
(as nega i e con ol), CS:Ag, INU:pA g:Ag, o INU:Ag:pA g as accine p o o ypes. IL-17: in e leukin
17, IFN-
γ
: in e e on gamma, Ag: An igen, CS: Chi osan, INU: Inulin, pA g: Polya ginine. S a is ical
signi icance: * P ≤0.05, ** P ≤0.01, *** P ≤0.001.
4. Discussion
Th ee accine p o o ypes we e designed and de eloped du ing his wo k, using CS o INU/pA g
NCs as an igen nanoca ie s. IMQ was inco po a ed in he oily co e as an addi ional adju an .
The IMQ-loaded CS:Ag, INU:pA g:Ag, and INU:Ag:pA g NCs showed a simila pa icle size o abou
150 nm, app op ia e o nasal adminis a ion. In his sense, o an e icien deli e y h ough he
mucosal ou es, i was s ablished ha he pa icle size should be smalle han he mucus mesh size [
38
].
This easily explains why NPs, in gene al, pe o med be e han mic opa icles [
39
–
43
]. Howe e ,
in he nanome ic ange,
in i o
da a has shown ha e y small nanome ic size (30 nm) may be
de imen al compa ed o la ge ones (200 nm) [
44
]. Besides, no only he pa icle size, bu also he
composi ion, play a c i ical ole when using NPs o nasal adminis a ion. In 1998, Alonso’s g oup
epo ed he impo ance o PEG o inc ease he mucope mea ion o polyes e -based NPs [
45
], and, a e
ha , many o he s udies con i med he impo ance o an adequa e PEG coa ing o a o he di usion
ac oss he mucus laye [46,47].
On he o he hand, he nanome ic size o he de eloped NCs was also adequa e o ha e a
p e e en ial in e naliza ion by dend i ic cells (DCs) [
6
,
48
]. DCs induce a be e T-cell s imula ion
han o he APCs, such as mac ophages and B-cells, and play a ele an ole in pa hogen cap u e and
induc ion o a p ima y immune esponse. Fo ha eason, pa icle accines y o a ge hese cells [
49
].
Mo eo e , he NCs had a low (CS:Ag) o high (INU:pA g:Ag and INU:Ag:pA g) posi i e cha ge, which
is also use ul o he in e ac ion wi h he nega i ely cha ged mucin ollowing in e naliza ion in he
mucosa [47,50].
The selec ed an igen was a usion p o ein (ECH) o wo well-known M b an igens, ESAT-6 and
CFP-10. Bo h p o eins belong o he egion o di e ence (RD1) o M b, which is absen om all s ains o
BCG and is ela ed wi h he i ulence o he M b and M. bo is s ains. In ac , he ESAT-6/CFP-10 p o ein
complex could be ela ed wi h mac ophage apop osis du ing TB in ec ion [
22
]. The ECH ecombinan
p o ein is a po en immune ac i a o o bo h humo al and cellula immune esponses. Mo eo e ,
he i.n. adminis a ion o he ecombinan p o ein in mice, in combina ion wi h and adju an , p omo ed
he cellula immune esponse in compa ison wi h he s.c. o in amuscula (i.m.) immuniza ion
Pha maceu ics 2020,12, 489 17 o 22
ou es ha induced highe an ibody and lowe cellula immune esponses [
51
]. Besides, i has been
ecen ly desc ibed ha inna e immune cells, monocy es/mac ophages, and na u al kille (NK) cells can
also be ained h ough accina ion, pa hogen coloniza ion, o e en p e ious in ec ions [
52
]. These
ained cells display s onge ac i a ion a e a second exposu e o he mic obial molecules. Hence,
ac i a ion o a b oad se o immune cells could be he key o igh hose pa hogens ha ha e a complex
in ec ion cycle.
Based on hese esul s, he objec i e o his wo k was o in es iga e he po en ial o wo di e en
polyme ic NC, made o wo ca ionic polyme s, CS and pA g, as ca ie s o he i.n. deli e y o he ECH
usion p o ein, and i a p e ious boos wi h he BCG accine could imp o e he immune esponse by
aining he inna e immuni y. To inc ease he adju ancy o he NCs, IMQ, a TLR-7 binding agonis ,
was encapsula ed in he oily co e o he NCs. The binding o his agonis o TLR-7 in APCs induces
he elease o p o-in lamma o y cy okines, such as IFN-
α
, TNF-
α
, and IL-12, which, in u n, p omo e
di e en ia ion o imma u e Th cells in o Th1 e ec o cells [37].
The elease o TNF-
α
, IL-6, and IL-10 has been p e iously desc ibed o IMQ-loaded CS NCs,
bu no o emp y CS NCs [
10
], and i can be associa ed o he in e naliza ion o he NCs and he
s imula iono heTLR-7 ecep o inducedby heIMQ,as p e iouslydesc ibed o hisimmunos imulan
molecule [
37
]. The di e ences in cy okine concen a ion ound be ween he CS and he INU/pA g NCs
could be due o di e ences in he in e naliza ion o he NCs o in he elease o he IMQ. Low cy okine
le els induced by IMQ-loaded CS NCs in i o we e also desc ibed in a p e ious wo k [11].
The s abili y o he o mula ions a s o age condi ions (4
◦
C) was good o all he p o o ypes, o
he NCs be o e he addi ion o he an igen, in he case o CS NCs, bu u he lyophiliza ion s udies
should be ca ied ou o a inal p o o ype. In ac , he lyophiliza ion o he INU/pA g p o o ypes
using 10% suc ose, as c yop o ec an , showed an e icien pa icle eco e y a e esuspension o he
lyophilized o mula ion in wa e .
CS and INU/pA g NCs showed a good cy ocompa ibili y, al hough he oxici y was lowe o CS
s. INU/pA g NCs. These esul s we e in line wi h p e ious indings ega ding he highe oxici y
o pA g NCs han CS NCs [
5
,
11
]. On he o he hand, INU NCs, wi hou he pA g laye , showed
conside ably lowe oxici ies [
53
,
54
]. In addi ion, he highe IMQ con en in he INU/pA g NCs could
also be ela ed wi h he inc eased oxici y [10].
Besides a highe cy ocompa ibili y, CS NCs showed also a supe io immunogenici y
in i o
based
on he induc ion o cy okine and ROS elease and complemen ac i a ion, all o hem mechanisms ha
a e ela ed wi h an imp o ed s imula ion o he inna e and adap i e immuni y and associa ed e icacy
in i o [5,26,31,32].
Howe e , om he
in i o
expe imen s, i can be concluded ha INU:pA g:Ag NCs we e he
only p o o ype able o induce speci ic IgA an ibodies agains he ESAT-6 an igen in mice immunized
h ee imes in anasally wi h he NCs. Mo eo e , he INU:pA g:Ag and he CS:Ag NCs induced
high le els o IgG an ibodies agains he ESAT-6 and CFP-10 p o eins, and IFN-
γ
and IL-17 elease in
splenocy es e-s imula ed wi h ESAT-6 o he usion p o ein. In con as , p o ec ion o he an igen in a
bilaye disposi ion (INU:Ag:pA g), despi e inc easing he s abili y o he NCs and he an igen loading
compa ed o he INU:pA g:Ag NCs, educed signi ican ly he immune esponse. The placemen o
he an igen on he su ace inc eased p obably B-cell ac i a ion, as desc ibed p e iously [
55
,
56
]. Ye ,
he polyme ic composi ion played also a signi ican ole because he CS:Ag NCs ailed o induce
speci ic IgA an ibodies al hough he an igen loading was also highe han o he INU:pA g:Ag.
In e es ingly, he p iming o he animals wi h he BCG accine (s.c. injec ed) only inc eased
o main ained he immune esponse induced by he CS:Ag NCs, while, in gene al, i dec eased o
in e e ed wi h he esponse induced by he INU/pA g p o o ypes. In ag eemen wi h p e ious s udies,
combining di e en ou es o immuniza ion can in e e e on he mucosal immune esponse [
57
].
In gene al, p iming a he mucosal le el and boos ing h ough a sys emic ou e has shown o induce a
highe mucosal p o ec ion. Howe e , his should be es ed on indi idual p o o ypes because, in he
Pha maceu ics 2020,12, 489 18 o 22
case o he CS NCs, he p iming wi h BCG did no in e e e in he immune esponse induced by he NCs
alone, and e en inc eased he cy okine elease in e-s imula ed splenocy es om accina ed animals.
The s udies o he immune esponse agains he ECH usion p o ein and he indi idual componen s
showed ha ESAT-6 was a be e an igen han CFP-10. The ESAT-6 p o ein was able o induce some
speci ic mucosal IgA p oduc ion in animals accina ed wi h he INU:pA g:Ag NCs, and IL-17 and
INF-
γ
elease in splenocy es om animals immunized wi h he h ee accine p o o ypes a e an igen
ecall, while he CFP-10 p o ein induced mino o unde ec able le els. Al hough Th17 and Th1 linages
a e di e en ly ac i a ed by ESAT-6, i has been shown in mice and human a de elopmen al plas ici y
o he Th17 cells ha enable a shi om dominan IL-17 sec e ion o dominan IFN-
γ
exp ession
cells [
58
,
59
]. Hence, a combined immune esponse in ol ing Th2 and ei he independen Th1 and
Th17 and/o Th1-Th17 cells was elici ed by he polyme ic NCs agains he ESAT-6 an igen, based on
an ibodies’ de ec ion in animal se um and BAL, and cy okine elease (IFN-
γ
and IL-17) in splenocy es
a e he an igen ecall expe imen . In con as , a clea Th2- ype immune esponse was obse ed o
he CFP-10 an igen, based on an ibody p oduc ion.
The mucosal p o ec ion agains ESAT-6, an M b-speci ic an igen ha is no p esen in he BCG
accine, could help in he neu aliza ion o he pa hogen a he ou e o en ance. Howe e , o achie e a
good immune p o ec ion o mos o he popula ion o he an igens a e needed. A ecen compu a ional
s udy on he immunogenici y po en ial o 10 an igens om M b p edic ed ha R 2608 could be he
mos e icien one, based on he p omiscui y o he epi opes o bind di e se human leukocy e an igen
(HLA) molecules ha a e equen in he popula ion o h ee high-TB-bu den coun ies [60].
Besides, o a oid he inhibi ion o he immune esponse induced by he INU:pA g:Ag NCs in
animals p e iously immunized wi h he BCG accine, o he accina ion egimes should be es ed,
such as he p iming h ough he i.n. ou e wi h he NCs and he boos ing h ough he s.c. ou e wi h
he BCG accine.
5. Conclusions
Two di e en IMQ-loaded NCs, wi h a CS o an INU/pA g polyme ic shell, we e syn hesized
o he de elopmen o an i.n. model accine con aining a ecombinan usion p o ein (ECH) de i ed
om he ESAT-6 and CFP-10 an igen o M b. The accine con aining INU:pA g:Ag NCs was he mos
immunogenic p o o ype agains he ECH usion p o ein. Fu he op imiza ion o he NCs o inc ease
he cellula esponse migh p o ide an e icien an igen ca ie o he de elopmen o new i.n. accines
o igh pa hogens a ec ing he mucosa and ai ways.
Due o he in e e ence o he BCG p iming on he immune esponse, he use o INU:pA g:Ag NCs
o p iming and, a e wa ds, a boos wi h he INU:pA g:Ag NCs o he con en ional BCG accine (s.c.)
could imp o e he esul s obse ed o his p o o ype. In addi ion, an igen op imiza ion should also
be conside ed o ob ain a mo e e icien accine agains M b because CFP-10 induced lowe cellula
and humo al immune esponses han he ESAT-6 p o ein.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h p://www.mdpi.com/1999-4923/12/6/489/s1.
Figu e S1. Size (ba s), PdI (lines and symbols) (A), and Z-po en ial (B) o he chi osan (CS) nanocapsules wi h
di e en a ios o an igen (Ag) adso bed on he su ace a h ee di e en ime poin s (0, 4, and 7 days). Size and PdI
we e measu ed in ul apu e wa e and Z-po en ial in 1 mM KCl. Figu e S2. Size (ba s), PdI (lines and symbols) (A),
and Z-po en ial (B) o he CS:Ag, INU:Ag:pA g and INU:pA g:Ag nanocapsules be o e and a e lyophiliza ion
wi h 10% suc ose as c yop o ec an . Size and PdI we e measu ed in ul apu e wa e and Z-po en ial in 1 mM
KCl. Ag: An igen, CS: Chi osan, INU: Inulin, pA g: Polya ginine. Figu e S3. Absence o ROS elease in HL-60
cells incuba ed wi h he CS o he INU/pA g nanocapsules a ou di e en concen a ions o 1 h (
le panel
) and
3 h ( igh panel). Cells incuba ed wi h RPMI medium alone (C
−
) o wi h zymosan (C+) we e used as nega i e
and posi i e con ols, espec i ely. Figu e S4. ROS elease in A549 cells incuba ed wi h he CS o he INU/pA g
nanocapsules a wo di e en concen a ions o 1 h (le panel) and 3 h ( igh panel). Cells incuba ed wi h RPMI
medium alone (C−) o wi h zymosan (C+) we e used as nega i e and posi i e con ols, espec i ely.
Au ho Con ibu ions:
Concep ualiza ion, R.S.-V.,
Á
.G.-F., J.C.-C., and M.J.A.; Me hodology, L.D.-G., R.S.-V.,
J.C.-C., and M.J.P.; Valida ion, L.D.-G., R.S.-V., and M.J.P.; Fo mal Analysis, L.D.-G., R.S.-V., J.C.-C., and M.J.P.;
In es iga ion, L.D.-G., R.S.-V., J.C.-C., and M.J.P.; Resou ces, A.G.-F., M.J.A., R.R., M.S., and R.S.-V.; Da a Cu a ion,
L.D.-G., R.S.-V., and M.J.P.; W i ing—O iginal D a P epa a ion, R.S.-V., J.C.-C., and M.J.P.; W i ing—Re iew &
Pha maceu ics 2020,12, 489 19 o 22
Edi ing,
Á
.G.-F., M.J.A., and R.R.; Visualiza ion, R.S.-V., J.C.-C.,
Á
.G.-F., and M.J.P.; Supe ision, R.S.-V., J.C.-C.,
Á
.G.-F., and M.J.A.; P ojec Adminis a ion, R.S.-V., J.C.-C.,
Á
.G.-F., and M.J.A.; Funding Acquisi ion,
Á
.G.-F.,
M.J.A., M.S., and R.R. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This wo k was suppo ed by he EU Ho izon2020 Elici ing Mucosal Immuni y in Tube culosis (EMI-TB)
p ojec [G an Numbe 643558], Xun a de Galicia: G upo de Re e encia Compe i i o (unde G an ED431C
2016/041 and ED431C 2017/09), cen o singula de in es igaci
ó
n de Galicia (acc edi a ion 2019-2022) and he EU
(Eu opean Regional De elopmen Fund-ERDF)—Re . ED431G2019/06.
Acknowledgmen s:
We would like o acknowledge he excellen echnical suppo om Bel
é
n Cues a Reguei o,
Balbina Fe n
á
ndez Ma
í
nez and Me cedes Pele ei o Olmedo. L.D.-G. and R.S.-V. acknowledge a ellowship ( e .
ED481A-2018/294) and a pos doc o al con ac ( e . ED481D-2017/017), espec i ely, om he Xun a de Galicia
(plan I2C), Spain.
Con lic s o In e es :
The au ho s ha e no ele an a ilia ions o inancial in ol emen wi h any o ganiza ion
o en i y wi h a inancial in e es in o inancial con lic wi h he subjec ma e o ma e ials discussed in he
manusc ip . This includes employmen , consul ancies, hono a ia, s ock owne ship o op ions, expe es imony,
g an s o pa en s ecei ed o pending, o oyal ies. The unde s had no ole in he design o he s udy; in he
collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip , o in he decision o publish
he esul s.
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