Starch-Chitosan Polyplexes: A Versatile Carrier System for Anti-Infectives and Gene Delivery
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polyme s
A icle
S a ch-Chi osan Polyplexes: A Ve sa ile Ca ie
Sys em o An i-In ec i es and Gene Deli e y
Hanzey Yasa 1,2,†, Duy-Khie Ho 1,2,†, Chia a De Rossi 1, Jenni e He mann 1, Sa ah Go don 1ID ,
B igi a Lo e z 1,* and Claus-Michael Leh 1,2 ID
1Helmhol z Ins i u e o Pha maceu ical Resea ch Saa land (HIPS), Helmhol z Cen e o In ec ion
Resea ch (HZI), Saa land Uni e si y, D-66123 Saa b ücken, Ge many;
Hanzey.Y[email p o ec ed] (H.Y.); [email p o ec ed] (D.-K.H.);
[email p o ec ed] (C.D.R.); jenni e [email p o ec ed] (J.H.);
[email p o ec ed] (S.G.); [email p o ec ed] (C.-M.L.)
2Depa men o Pha macy, Saa land Uni e si y, D-66123 Saa b ücken, Ge many
*Co espondence: [email p o ec ed]; Tel.: +49-681-98806-1030
† These au ho s con ibu ed equally o his wo k.
Recei ed: 8 Decembe 2017; Accep ed: 27 Feb ua y 2018; Published: 1 Ma ch 2018
Abs ac :
Despi e he eno mous po en ial o nanomedicine, he sea ch o ma e ials om enewable
esou ces ha balance bio-medical equi emen s and enginee ing aspec s is s ill challenging.
This s udy p oposes an easy me hod o make nanopa icles composed o oxidized s a ch and chi osan,
bo h isola ed om na u al biopolyme s. The ca e ul adjus men o C/N a io, polyme concen a ion
and molecula weigh allowed o uning o pa icle cha ac e is ics. The sys em’s ca ie capabili y
was assessed bo h o an i-in ec i es and o nucleic acid. Highe s a ch con en polyplexes we e
ound o be sui able o high encapsula ion e iciency o ca ionic an i-in ec i es and p ese ing
hei bac e icidal unc ion. A ca ionic ca ie was ob ained by coa ing he anionic polyplex wi h
chi osan. Coa ing allowed o a minimal amoun o ca ionic polyme o be employed and acili a ed
plasmid DNA loading bo h wi hin he pa icle co e and on he su ace. T ans ec ion s udies showed
encou aging esul , app oxima ely 5% o A549 cells wi h epo e gene exp ession. In summa y,
s a ch-chi osan complexes a e sui able ca ie s wi h p omising pe spec i es o pha maceu ical use.
Keywo ds:
polyme ic nanopa icles; enewable polysaccha ides; anionic s a ch; ca ionic an i-in ec i es;
ans ec ion
1. In oduc ion
Nanopa icula e ca ie sys ems ep esen a well es ablished pla o m o accina ion and
ea men o se e e diseases, such as in ec ion and cance , by p o ec ing ac i e agen s, p e en ing bu s
elease kine ics, p o iding he po en ial o enhance c ossing o biological ba ie s and imp o ing local
d ug deli e y [
1
–
4
]. Howe e , he selec ion o ma e ials o excipien s o nanomedical applica ions
emains challenging due o s ic equi emen s o he ield. Such ma e ials should be biocompa ible
and biodeg adable, sa e and a he same ime p o ide good d ug loading capaci y as well as a
po en ial o ca y di e se bioac i e agen s [
3
]. Mo eo e , o la ge scale p oduc ion, he used ma e ials
should be en i onmen ally iendly, and able o be manu ac u ed by acile p ocesses. In ecen
yea s, a a ie y o polyme ic ma e ials de i ed om na u al biopolyme s ha e been syn hesized
and in es iga ed o o mula e ehicles o deli e bioac i e molecules. These molecules ha e been
embedded inside he polyme ic ma ix o adso bed on o he colloidal su ace [
5
] by ei he physical
in e ac ion (e.g., elec os a ic complexa ion) o chemical modi ica ion. Ne e heless, he numbe o
biodeg adable and biocompa ible polyme s which a e u he compa ible wi h wa e (as a sol en
sui able o pha maceu ical use) and can o m nanopa icles wi h a high and e sa ile ac i e agen
Polyme s 2018,10, 252; doi:10.3390/polym10030252 www.mdpi.com/jou nal/polyme s
Polyme s 2018,10, 252 2 o 21
encapsula ion capaci y a e s ill limi ed. Hence, he p oduc ion o excipien s o nanomedicine wi h a
balance be ween pha maceu ical equi emen s and enginee ing aspec s as well as a unable po en ial o
d ug deli e y has gained conside able a en ion. In pa icula , na u al and modi ied polysaccha ides
such as chi osan, algina e, s a ch and dex in, and hei syn he ic de i a i es, ha e been conside ed
as e icien candida es o d ug ca ie sys ems [
1
,
6
,
7
]. Howe e , achie ing a consis en and obus
p oduc ion o polysaccha ide nanopa icles is challenging due o he he e ogeneous physicochemical
p ope ies o na u al and syn he ic polyme s. In addi ion, depending on he ac i es o be deli e ed
and he ou e o adminis a ion, di e en p o ocols a e needed [
8
] o p epa e polysaccha ide-based
polyme ic nanopa icles [
9
,
10
]. Thus, he chosen polyme s need o be app op ia ely ailo ed, chemically
modi ied and op imized o quali y o a ge ed applica ions [1].
Among na u al polysaccha ides, s a ch and chi osan ha e many p omising p ope ies. S a ch is a
biocompa ible and biodeg adable polysaccha ide, which is deg aded by
α
-amylase, and a ailable a
ela i ely low cos . I has been widely used in able s and capsules, e.g., as a binde o diluen [
11
].
Sligh ly modi ied de i a i es o s a ch wi h ac ional molecula weigh s ha e p e iously been s udied
as a pla o m o o mula e homogenous ca ie sys ems o gene deli e y [
12
]. O he esea che s
ha e also s udied s a ch-based pa icula e sys ems o d ug deli e y [
13
–
15
]. Chi osan is simila ly
biodeg adable and biocompa ible, and has been in es iga ed and widely used in pha maceu ical
esea ch o d ug [
16
,
17
], p o ein [
18
] and nucleic acid deli e y, and o accina ion pu poses [
19
–
21
].
I has also been used as a biomedical ma e ial o a i icial skin and wound healing bandages [
22
]
as a biodeg adable polysaccha ide [
23
]. Mo eo e , chi osan has good biocompa ibili y as es ed in
humans [
24
]. Yamada e al. [
12
] has epo ed he p epa a ion o anionic s a ch de i a i es by mild
chemical modi ica ion, and he sepa a ion o di e en molecula weigh s by a ac ional cu -o p o ocol,
which was la e aimed o ans ec ion s udy. The esea ch showed p omising pe spec i es o s a ch
de i a i es as d ug ca ie sys em. Howe e , he cha ge media ed complexa ion o ac ional s a ch
de i a i es was no ully explo ed in ha s udy; he ca ie capaci y o such sys em hus emains o
be in es iga ed.
In ligh o hese ad an ages, he aim o his wo k was o p oduce e sa ile and lexible
nanoca ie s using bo h s a ch and chi osan, wi h a acile and o ganic sol en - ee p epa a ion me hod
combining he ad an ages o hese wo polyme s in o a ca ie sys em. The in es iga ed sys ems we e
composed o s a ch de i a i es o molecula weigh (M
w
) >100 kDa o wi h M
w
ange o 30–100 kDa,
and oligochi osan M
w
5 kDa o P o asan M
w
90 kDa as chi osan de i a i es. A wide ange o
molecula weigh s was used o achie e complex s abili y. We also explo ed he design space o he
sys em o ob ain pa icles wi h high colloidal s abili y as well as unable su ace cha ge and size. Thus,
he a ied p oduc ion pa ame e s o s a ch-chi osan polyplexes (Scheme 1A) we e: (i) mola a io o
ca boxyla e and amine unc ional g oups (C/N a io) o s a ch and chi osan, espec i ely; (ii) polyme
concen a ion; and (iii) coun e polyme ype. The loading capaci y and e sa ili y o hese simple
ca ie s was hen in es iga ed using ob amycin and colis in as clinically ele an models o small
molecule and pep ide an i-in ec i es espec i ely [
25
,
26
], as well as nucleic acids (plasmid DNA).
Fu he mo e, o imp o e encapsula ion capaci y, we coa ed he s a ch-chi osan polyplexes wi h an
addi ional chi osan (P o asan) laye (Scheme 1B), and explo ed he loading capaci y o he esul ing
nanopa icles. Coa ing he polyplexes enabled d ug loading on he su ace o pa icles, which led
o a be e encapsula ion pa icula ly in he case o he u ilized nucleic acids. This app oach also
c ea es he u he po en ial o o mula ing a mul i unc ional deli e y sys em. The no el app oach o
s a ch-chi osan-based complex-coace a ion sugges ed in his s udy is a s aigh o wa d and p omising
echnique o p epa e e sa ile ca ie sys ems wi h po en ial in nanomedicine applica ions. The e o e,
we unde ook p elimina y s udies o design, syn hesis, and o mula ion o such ca ie sys ems,
and explo ed hei lexibili y and capaci y o encapsula ing selec ed model mac omolecula d ugs.
Polyme s 2018,10, 252 3 o 21
Polyme s 2018, 10, x FOR PEER REVIEW 3 o 21
Scheme 1. Illus a ion o d ug- ee (plain) s a ch-chi osan polyplex-p epa a ion.
2. Expe imen al Sec ion
2.1. Ma e ials
As aw ma e ial, pa ially hyd olyzed po a o s a ch (Mw o 1300 kDa), which was a kind gi
om AVEBE (Veendam, The Ne he lands), was used. Selec i e oxida ion o he p ima y alcohol on
s a ch was pe o med o inc ease wa e solubili y and ob ain an anionic cha ge. The oxida ion
p ocedu e and molecula weigh ac iona ion o h ee Mw samples (5, 30–100, and >100 kDa) was
conduc ed in acco dance wi h he p o ocol o Yamada e al. [12]. The ob ained s a ch de i a i es had
an oxida ion deg ee o 45%. The Mw ac ion >100 kDa is used unless s a ed o he wise and is e med
“anionic s a ch” in all u he desc ip ions.
Chi osan oligosaccha ide lac a e (oligochi osan; Mw 5 kDa), poly inyl alcohol (PVA; Mowiol® 4-
88), sodium hyd oxide, i luo oace ic acid (TFA), ace oni ile and ace ic acid we e pu chased om
Sigma-Ald ich (Da ms ad , Ge many). Tob amycin sul a e sal and colis in sul a e sal we e used as
ecei ed also om Sigma-Ald ich. Ul apu e chi osan chlo ide sal (P o asan UP CL113; Mw ~90 kDa,
deace yla ion deg ee 75–90%) was ob ained om FMC Biopolyme AS No aMa ix (Sand ika,
No way). Pu i ied wa e was p oduced by a Milli-Q wa e pu i ica ion sys em om Me ck Millipo e
(Da ms ad , Ge many). O-Ph halaldehyde (OPA), 2-me cap oe hanol, phospho ungs ic acid (PTA)
and bo ic acid we e used as pu chased om Sigma-Ald ich.
Aga ose SERVA o DNA Elec opho esis o esea ch g ade was bough om Se a (Heidelbe g,
Ge many). E hidium b omide solu ion (10 mg/mL), hepa in sodium sal om po cine in es inal
mucosa, 3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide) (MTT eagen ), T i on™ X-
100, dime hyl sul oxide (DMSO) and Dulbecco’s phospha e bu e ed saline solu ion (PBS) we e
ob ained om Sigma-Ald ich. Gibco Hanks’ balanced sal solu ion (HBSS) bu e was pu chased
om The mo Fishe Scien i ic (Da ms ad , Ge many). A549 cells (human lung ca cinoma cell line,
No. ACC 107) we e ob ained om DSMZ GmbH (B aunschweig, Ge many). Cell cul u e medium
(RPMI 1640) was pu chased om PAA labo a o ies GmbH (Pasching, Aus ia) and supplemen ed
wi h 10% e al cal se um (FCS, Sigma-Ald ich). Plasmid DNA (pDNA) encoding o he luo escen
p o ein AmCyan was bough om Clon ech Labo a o ies, Inc. (pAmCyan 1-N1, Moun ain View, CA,
USA). The plasmid was p opaga ed in Esche ichia coli DH5α and isola ed wi h Qiagen EndoF ee
Plasmid Mega Ki (Qiagen, Hilden, Ge many) o ob ain pDNA o cell cul u e quali y. je PRIME®
Scheme 1. Illus a ion o d ug- ee (plain) s a ch-chi osan polyplex-p epa a ion.
2. Expe imen al Sec ion
2.1. Ma e ials
As aw ma e ial, pa ially hyd olyzed po a o s a ch (M
w
o 1300 kDa), which was a kind gi om
AVEBE (Veendam, The Ne he lands), was used. Selec i e oxida ion o he p ima y alcohol on s a ch
was pe o med o inc ease wa e solubili y and ob ain an anionic cha ge. The oxida ion p ocedu e
and molecula weigh ac iona ion o h ee M
w
samples (5, 30–100, and >100 kDa) was conduc ed in
acco dance wi h he p o ocol o Yamada e al. [
12
]. The ob ained s a ch de i a i es had an oxida ion
deg ee o 45%. The M
w
ac ion >100 kDa is used unless s a ed o he wise and is e med “anionic
s a ch” in all u he desc ip ions.
Chi osan oligosaccha ide lac a e (oligochi osan; M
w
5 kDa), poly inyl alcohol (PVA; Mowiol
®
4-88),
sodium hyd oxide, i luo oace ic acid (TFA), ace oni ile and ace ic acid we e pu chased om
Sigma-Ald ich (Da ms ad , Ge many). Tob amycin sul a e sal and colis in sul a e sal we e used
as ecei ed also om Sigma-Ald ich. Ul apu e chi osan chlo ide sal (P o asan UP CL113; M
w
~90 kDa, deace yla ion deg ee 75–90%) was ob ained om FMC Biopolyme AS No aMa ix (Sand ika,
No way). Pu i ied wa e was p oduced by a Milli-Q wa e pu i ica ion sys em om Me ck Millipo e
(Da ms ad , Ge many). O-Ph halaldehyde (OPA), 2-me cap oe hanol, phospho ungs ic acid (PTA) and
bo ic acid we e used as pu chased om Sigma-Ald ich.
Aga ose SERVA o DNA Elec opho esis o esea ch g ade was bough om Se a (Heidelbe g,
Ge many). E hidium b omide solu ion (10 mg/mL), hepa in sodium sal om po cine in es inal
mucosa, 3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide) (MTT eagen ), T i on
™
X-100,
dime hyl sul oxide (DMSO) and Dulbecco’s phospha e bu e ed saline solu ion (PBS) we e ob ained
om Sigma-Ald ich. Gibco Hanks’ balanced sal solu ion (HBSS) bu e was pu chased om The mo
Fishe Scien i ic (Da ms ad , Ge many). A549 cells (human lung ca cinoma cell line, No. ACC 107)
we e ob ained om DSMZ GmbH (B aunschweig, Ge many). Cell cul u e medium (RPMI 1640) was
pu chased om PAA labo a o ies GmbH (Pasching, Aus ia) and supplemen ed wi h 10% e al cal
se um (FCS, Sigma-Ald ich). Plasmid DNA (pDNA) encoding o he luo escen p o ein AmCyan was
bough om Clon ech Labo a o ies, Inc. (pAmCyan 1-N1, Moun ain View, CA, USA). The plasmid
Polyme s 2018,10, 252 4 o 21
was p opaga ed in Esche ichia coli DH5
α
and isola ed wi h Qiagen EndoF ee Plasmid Mega Ki
(Qiagen, Hilden, Ge many) o ob ain pDNA o cell cul u e quali y. je PRIME
®
ans ec ion eagen
was pu chased om Polyplus- ans ec ion (Illki ch, F ance). Rhodamine Ricinus communis agglu inin I
(RGA I) was ob ained om Vec o Labo a o ies. 4
0
,6-diamidino-2-phenylindole (DAPI) was pu chased
om Li e Technologies (Da ms ad , Ge many).
2.2. P epa a ion, Op imiza ion and Cha ac e iza ion o S a ch–Chi osan Co e Polyplexes
2.2.1. P epa a ion and Op imiza ion o S a ch-Chi osan Co e Polyplexes (CP)
S a ch-chi osan co e polyplexes (CP) we e p epa ed by sel -assembly o anionic s a ch de i a i es
and chi osan de i a i es in aqueous medium. CP cha ac e is ics, including hei : (i) su ace p ope ies;
(ii) size; and (iii) physicochemical s abili y we e a ied by: (i) he molecula weigh o u ilized anionic
s a ch and chi osan de i a i es; (ii) polyme concen a ion; and (iii) mola a io o ca boxyla e (COONa)
o amine (NH
2
) g oups (C/N a io) in oxidized s a ch and chi osan, espec i ely. The polyplex
o mula ion p ocedu e is desc ibed in Scheme 1A. B ie ly, a solu ion o anionic s a ch was p epa ed
in Milli-Q wa e a a de ined concen a ion, while he u ilized chi osan de i a i e was solubilized in
0.02 M ace ic acid, ollowed by pH adjus men o 5.5. The assembly in o CP o oxidized s a ch and i s
coun e excipien occu ed by he addi ion o an app op ia e amoun o s a ch polyme solu ion in o
he p e-wa med solu ion o chi osan de i a i e, ollowed by 2 min o o exing and 1 h incuba ion
a oom empe a u e. To p epa e anionic co e polyplexes (anCP), anionic s a ch (M
w
o >100 kDa)
and oligochi osan (M
w
o 5 kDa) we e employed a a ious C/N a ios, anging om 50:1 o 10:1 and
u he o 1:1, designed o op imize he o mula ion and s abili y o he polyplexes. Ca ionic co e
polyplexes (ca ionic CP) we e p epa ed by co-assembly o nega i e s a ch (M
w
o 30–100 kDa) and
P o asan (M
w
o 90 kDa) ha ing a highe amoun o posi i ely cha ged amine g oups. The op imal
C/N a io was iden i ied by in es iga ing he a ios o 1:30, 1:10 and 1:1. All samples wi h a sol en
pH- alue o 5.5 we e cha ac e ized by dynamic ligh sca e ing (DLS), using a Ze asize Nano om
Mal e n Ins umen s (UK) o ob ain hyd odynamic size, polydispe si y index (PDI), and using lase
Dopple elocime y o ob ain
ζ
-po en ial. All samples we e p epa ed a leas in h ee di e en ba ches.
2.2.2. P epa a ion and Op imiza ion o P o asan Coa ed CP (cCP)
Ano he app oach aken o u he imp o e he loading capaci y o s a ch-chi osan ca ie s was
o p epa e coa ed polyplexes wi h a u he laye o P o asan on anCP. The op imized coa ing me hod
is desc ibed b ie ly as ollowing: anCP we e p epa ed as desc ibed and hen coa ed wi h an addi ional
laye o posi i ely cha ged P o asan, by an associa ion o amine unc ional g oups o he chi osan and
he anionic su ace o he anCP (Scheme 1B). The coa ing solu ion was p epa ed by dissol ing 3 mg o
P o asan in 1 mL PVA 2% (w/ ) solu ion, which was hen dilu ed wi h Milli-Q wa e o a 1.5 mg/5 mL
concen a ion o coa ing. A 500
µ
L olume (6.6 mg/mL) o anCP was added d opwise o he p epa ed
P o asan solu ion, which was con inuously s i ed o 30 min a 150 pm. This was ollowed by
incuba ion a oom empe a u e o 3 h p io o cha ac e iza ion. The esul ing P o asan-coa ed anCP
(cCP, c= 0.87 mg/mL) we e kep o u he s udies. Samples we e p epa ed in a leas h ee di e en
ba ches. All pa icle samples we e cha ac e ized o hei hyd odynamic size, PDI and
ζ
-po en ial.
This me hod was also applied o in es iga e he physicochemical s abili y o anCP and ca ionic CP
unde s o age condi ions o 4 ◦C o 27 days.
2.2.3. pH-S abili y o D ug-F ee CP and cCP
The colloidal s abili y o anCP and cCP a di e en pH alues was in es iga ed by incuba ing
pa icle suspensions a pH alues o 3.5, 4.0, 4.5, 5.5, 6.0, 7.5 and 8.0, all wi hin he physiologically- ele an
ange. Samples we e analyzed o ob ain hyd odynamic size, PDI, and
ζ
-po en ial, a e p ede e mined
incuba ion imes (30 min, 1 h, 3 h and 24 h). The pH- alue was adjus ed ollowing polyplex p epa a ion
a pH 5.5 (as desc ibed abo e) by using ei he 0.02 M ace ic acid solu ion o 1 M NaOH solu ion.
Polyme s 2018,10, 252 5 o 21
All expe imen s we e conduc ed in iplica es wi h n= 3, and esul s exp essed as mean
±
s anda d
de ia ion (SD).
2.2.4. Mo phology
The mo phology o all p oduced polyplexes was isualized by ansmission elec on mic oscopy
(TEM, JEM 2011, JEOL, S And ews, UK). Be o e he TEM isualiza ion, 8.7
µ
g/10
µ
L o polyplexes
we e added on a coppe g id (ca bon ilms on 400 mesh coppe g ids, Plano GmbH, We zla , Ge many)
and incuba ed o 10 min o allow an adhesion o polyplexes o he su ace. The excess was emo ed,
and polyplexes we e u he s ained wi h 0.5% (w/ ) PTA o imp o e he con as o TEM images.
2.2.5. Cy o oxici y S udy: MTT Assay
A549 cells we e seeded in a 96 well pla e a a densi y o 1
×
10
5
cells pe well, in 200
µ
L o RPMI
cell cul u e medium supplemen ed wi h 10% FCS. Cells we e g own o 4 days p io o he conduc ion
o he assay o allow o app oxima ely 95% cell con luency. On Day 4, CP and cCP samples we e
dilu ed wi h a sui able amoun o RPMI medium (wi hou FCS) o achie e es concen a ions o 5, 10,
40, 70, 100, 200 and 500
µ
g/mL. Cells we e hen washed wice wi h 200
µ
L HBSS bu e (pH 7.4), and
polyplex samples we e added o cells in iplica e. Cells incuba ed wi h only RPMI medium we e used
as a nega i e con ol (de e mined o esul in 100% cell iabili y) and cells ea ed wi h 1% T i on
™
X-100 in RPMI medium we e used as posi i e con ol (designa ed as 0% cell iabili y). All samples
we e incuba ed wi h cells o 4 h, on a ho izon al shake wi h ca e ul shaking a 150 pm a 37
◦
C and
5% CO
2
. Subsequen ly, he supe na an was emo ed, and cells we e washed once wi h HBSS. Then,
200
µ
L o he MTT- eagen (5 mg/mL) in HBSS was applied o each well and u he incuba ed o
4 h wi h gen le shaking. The supe na an was hen emo ed and DMSO was immedia ely added o
achie e cell lysis. Cells we e incuba ed in DMSO o 15 min unde ca e ul shaking and p o ec ed om
ligh . The abso bance o each well a 550 nm was hen measu ed wi h a pla e eade (In ini e
®
200 P o,
TECAN, Männedo , Swi ze land). The pe cen age o iable cells was calcula ed in compa ison o
nega i e and posi i e con ols as desc ibed by Na ee e al. [27].
2.3. Ca ionic An i-In ec i e Loaded anCP
2.3.1. P epa a ion and Op imiza ion o Ca ionic An i-In ec i e Loaded anCP
Iso he mal Ti a ion Calo ime y
Two ele an an i-in ec i es we e used o es he loading capaci y o anCP. Tob amycin was
used as an example o a ca ionic small molecule an ibio ic ha ing a molecula weigh o 467.5 Da,
and colis in (polymyxin E) was used as an example o a pep ide an ibio ic wi h a molecula weigh o
1267.5 Da (Scheme 2A).
In e ac ion be ween anionic s a ch and he ca ionic an i-in ec i es ob amycin and colis in
was in es iga ed by iso he mal i a ion calo ime y (ITC) using a NanoITC 2G (TA Ins umen s,
New Cas le, DE, USA). The pu pose o such measu emen was o op imize excipien o ca go a io
in d ug loaded ca ie p oduc ion. B ie ly, all d ug and anionic s a ch solu ions we e p epa ed in
milli-Q wa e . A 25 mM solu ion o ob amycin o colis in was p epa ed in a 250
µ
L sy inge and used
o sa u a e 1.5 mL o anionic s a ch a a concen a ion o 0.1 mM illed in he sample cell. Following
an ini ial delay o 300 s, 250
µ
L o d ug solu ion was epea edly injec ed in o he sample cell wi h a
spacing o 500 s be ween injec ions, and a a e e ence powe o 10
µ
Cal/s. The inal he mog am and
he modynamic pa ame e s we e p oduced by sub ac ing he hea o dilu ion o ei he ob amycin
o colis in (25 mM in 1.5 mL milli-Q wa e ), ollowed by i ing using he One Se o Si es model in
he da a analysis so wa e NanoAnalyze. The ee ene gy o binding (
∆
G) was calcula ed using he
equa ion
∆
G=
∆
H
−
T
∆
S, whe e
∆
His he en halpy change, Tis empe a u e (Kel in), and
∆
Sis he
change in en opy. All measu emen s we e pe o med a 25 ◦C.
Polyme s 2018,10, 252 6 o 21
P epa a ion and Op imiza ion o Ca ionic An i-In ec i e Loaded anCP
Bo h ob amycin and colis in we e loaded using he same p ocedu e, du ing o ma ion o anCP,
employing a ious C/N a ios, as ollows: (i) 1 mg ob amycin o 3 mg colis in was incuba ed wi h an
app op ia e amoun o anionic s a ch solu ion o 2 h; and (ii) p e-wa med chi osan solu ion a pH 5.5
was added, and coace a ion was achie ed by o ex mixing (2 min).
The an i-in ec i e loaded anCP suspension was hen cen i uged a 13,000
×
gand 4
◦
C o 20 min
a leas wice and allowed o equilib a e a 4
◦
C o e nigh be o e conduc ing u he expe imen s. In all
expe imen s he supe na an p oduced by cen i uga ion was collec ed o d ug loading quan i ica ion.
Loading Quan i ica ion
The deg ee o an i-in ec i e loading in anCP was de e mined using an indi ec quan i ica ion
me hod (d ug amoun inside anCP = ini ial d ug amoun
−
d ug amoun in he supe na an ). Colis in
was quan i ied by high-pe o mance liquid ch oma og aphy (HPLC), while ob amycin was quan i ied
based on a p o ocol o de ec ion o aminoglycosides [28], as de ailed below.
HPLC Analysis
The HPLC analysis was pe o med on a Dionex Ul iMa e 3000 sys em (The mo-Fische Scien i ic,
D eieich, Ge many) equipped wi h LPG-3400 SD pump, WPS-3000 au osample , DAD3000 de ec o ,
and TCC-3000 column o en. Ch omeleon so wa e (Ch omeleon 6.80 SP2 build 9.68, The mo Scien i ic
Dionex, D eieich, Ge many) was used o da a analysis. A column se o LiCh osphe
®
100 RP-18 (5
µ
m)
LiCh oCART
®
125-4, consis ing o a 125 mm
×
4 mm LiCh osphe 100/RP-18 column (Me ck-Hi achi,
Da ms ad , Ge many) wi h a LiCh osphe 100/RP-18 gua d column (5
µ
m) (Me ck-Hi achi, Da ms ad ,
Ge many) a 30
◦
C was used as s a iona y phase o all subs ances. A g adien me hod was used
s a ing wi h 20% A, inc easing o 50% A wi hin 2 min, and holding o 1.5 min (A = ace oni ile,
B = 0.1% TFA solu ion in wa e ). Be o e injec ion, he samples we e il e ed h ough a cellulose ace a e
0.2
µ
m memb ane. The low a e was 1.0 mL/min, and he injec ion olume was 50
µ
L. A calib a ion
cu e was cons uc ed using eigh di e en concen a ions o colis in in wa e , anging om 0.2 mg/mL
o 0.005 mg/mL (
2
= 0.9955). All 8 s anda ds we e measu ed 5 imes, and a pe cen ela i e s anda d
de ia ion (% RSD) o less han 3.9% was calcula ed. The un ime was 6 min, and a e en ion ime o
3.6 min and 3.9 min was obse ed o colis in A and colis in B, espec i ely. As colis in is a mix u e
o wo main ac ions, colis in A and colis in B, bo h we e quan i ied o de e mine colis in loading.
The de ec ion wa eleng h was 210 nm o colis in A and 214 nm o colis in B.
Aminoglycoside De ec ion P o ocol
The p oduc luo escence o ob amycin eac ed wi h a luo escen eagen was measu ed a
344/450 nm (Ex/Em) using a Tecan mic opla e eade ollowing a published me hod [
28
]. To p epa e
he eagen solu ion, a 0.2 g amoun o OPA eagen was dissol ed in a mix u e o 1 mL me hanol,
19 mL bo ic acid 0.4 M a pH 10.4, and 0.4 mL o 14.3 M 2-me cap oe hanol. A 2 mL o he esul ing
mix u e was hen dilu ed wi h 16 mL me hanol be o e use. A calib a ion cu e was cons uc ed using
i e di e en concen a ions o ob amycin in wa e (0.04–0.005 mg/mL, 2= 0.9976).
In bo h cases, he encapsula ion e iciency (EE) and he d ug loading a e (LR) we e calcula ed
acco ding o he ollowing equa ions:
EE =
Weigh o encapsula ed d ug in nanopa icles
Ini ial amoun o d ug in he sys em ×100
LR =
Weigh o d ug in nanopa icles
Weigh o nanopa icles ×100
(1)
whe e “weigh o nanopa icles” was calcula ed as weigh o polyme ic ma e ial + weigh o
encapsula ed d ug in nanopa icles.
Polyme s 2018,10, 252 7 o 21
Each sample was assayed a leas in iplica e, and esul s a e epo ed as he mean ±SD.
D ug Release S udy
Tob amycin o colis in elease p o iles om ob amycin loaded anCP o colis in loaded anCP was
pe o med in PBS (pH 7.4) a 37
◦
C. B ie ly, ei he ob amycin loaded anCP o colis in loaded anCP was
dilu ed in PBS o ha e inal ob amycin o colis in concen a ion a 10% (w/w) and loaded in o dialysis
memb ane (MWCO 1 kDa, Spec um Labs, Rancho Domiguez, CA, USA) in he case o ob amycin,
o dialysis memb ane (MWCO 3.5–5 kDa, Spec um Labs, USA) in he case o colis in. A e ha ,
he whole sys em was pu in o 20 mL PBS and placed on a shake a 400 pm a 37
◦
C. The concen a ion
o eleased d ug was analyzed by collec ing samples om he supe na an du ing he pe iod om
1 h o 24 h. The amoun o colis in and ob amycin we e de e mined by HPLC and aminoglycoside
de ec ion p o ocol, espec i ely. The olume was kep cons an by e illing wi h an iden ical olume
o PBS. The cumula i e eleased d ug (%) was calcula ed (mean
±
SD o n= 3). Th ee independen
expe imen s we e conduc ed in iplica es, and esul s exp essed as he mean
±
s anda d de ia ion (SD).
2.3.2. Minimum Inhibi o y Concen a ion (MIC) Assay
The an imic obial p ope ies o anCP, an i-in ec i e loaded anCP, and ee d ugs we e pe o med
by s anda d mic ob o h dilu ion assays wi h Esche ichia coli (DH5
α
) and Pseudomonas ae uginosa
(PA14) in 96 well pla es. A suspension o E. coli o P. ae uginosa p epa ed om mid log cul u es in
Muelle -Hin on b o h o Lysogeny B o h medium (a 25
◦
C) was i s dilu ed o OD
600
(abso p ion
a 600 nm) 0.01, which co esponds o app oxima ely 5
×
10
6
CFU/mL (CFU, colony- o ming uni s).
Polyplex samples (anCP, d ug-loaded anCP), ee d ug solu ion and PBS as con ol we e hen added
o bac e ia-con aining wells by se ial dilu ion o e a ange o 0.03–64
µ
g/mL. A e incuba ion o
16 h a 37
◦
C, inhibi o y concen a ion (IC) IC
90
alues we e de e mined by sigmoidal cu e i ing o
abso p ion alues (600 nm) ha we e measu ed on a Tecan mic opla e eade . The expe imen s we e
conduc ed in duplica e.
2.4. P epa a ion o pDNA Loaded cCP
Plasmid DNA pAmCyan was inco po a ed in o he polyplexes o e alua e he po en ial o he
ca ie sys em wi h espec o nucleic acid ac i es. A a io o amine g oups (chi osan) o phospha e
g oups (pDNA) o 20/1 was chosen and is e e ed o as N/P a io. The p epa a ion was pe o med
in h ee s eps: i s , an app op ia e amoun o pAmCyan was added o a solu ion o anionic s a ch
and mixed ho oughly. A 1 mL olume o his pAmCyan-s a ch solu ion was added o 1 mL o
oligochi osan solu ion (650
µ
g/mL) and mixed immedia ely by o ex o 15 s. A u he incuba ion o
1 h a oom empe a u e was hen ca ied ou , leading o he o ma ion o pAmCyan-loaded anCP. In he
second s ep, he pAmCyan loaded anCP we e coa ed by P o asan as desc ibed in Sec ion 2.2, o o m
pAmCyan-loaded cCP. In he hi d s ep, a u he laye o pAmCyan was applied o pAmCyan-loaded
cCP (1:30 w/w) esul ing in pAmCyan double loaded cCP (Scheme 2B). The pDNA encapsula ion
e iciency o each s ep was analyzed by pelle ing he samples down and measu ing he abso bance o
unbound pDNA (a 260/280 nm wi h NanoD op Spec opho me e ) emaining in he supe na an a e
cen i uga ion o 30 min a 24,400
×
g. Thus, he amoun o bound pDNA was examined indi ec ly.
The p oduc s o each s ep we e cha ac e ized o ob ain hyd odynamic size, PDI, and
ζ
-po en ial,
and hei mo phology was obse ed by TEM.
2.4.1. De e mina ion he Complexa ion o pAmCyan in S a ch-Chi osan Polyplexes
Complexa ion and s abili y o pAmCyan in s a ch-chi osan polyplexes was e alua ed by a
gel e a da ion assay using aga ose gel elec opho esis. Fu he , o acili a e DNA agmen a ion,
he endonuclease BamHI was used, which linea izes he plasmid, and hepa in addi ion o cause
he elease o pDNA om he complex. Polyplexes con aining 500 ng o pDNA pe sample om
each s ep o he o mula ion p ocess we e i s diges ed wi h 0.5
µ
L BamHI o 2 h a 37
◦
C wi h
Polyme s 2018,10, 252 8 o 21
shaking. A e wa d, 3
µ
L (30 mg/mL) hepa in was added o solu ions o diges ed polyplexes,
incuba ed o 15 min a oom empe a u e and hen mixed wi h 2
µ
L o o ange DNA loading dye
(6
×
; The mo Fishe Scien i ic, Wal ham, MA, USA). These mix u es we e hen loaded in o 0.75%
(w/ ) aga ose gel con aining 5
µ
L o e hidium b omide and un o 60 min a 50 V in 0.5
×
TBE-bu e .
The isualiza ion o he bands was pe o med wi h a UV illumina o , Fusion FX7 imaging sys em om
Peqlab (E langen, Ge many).
2.4.2. In Vi o T ans ec ion S udies in A549 Cells
To es he e iciency o he pAmCyan loaded polyplexes,
in i o
ans ec ion s udies we e
pe o med in A549 cells. B ie ly, A549 cells we e seeded in 24-well pla es, a a densi y o 25
×
10
4
cells
pe well in 500
µ
L o RPMI cell cul u e medium wi h 10% FCS. Cells we e g own o 2 days
o each a cell con luency o 60–70%. Polyplexes o he pAmCyan double loaded ca ie sys em
(see Sec ion 2.4) con aining 1
µ
g o pAmCyan (polyplex concen a ion ~60
µ
g/mL) we e p epa ed
wi h a a io o 1:30, 1:50 and 1:100 be ween pDNA:polyplexes in 500
µ
L o HBSS bu e . Then,
cells we e washed wice wi h HBSS bu e and incuba ed wi h he polyplexes o 6 h. A e 6 h
o incuba ion, polyplexes we e emo ed and eplaced wi h RPMI con aining 10% FCS. Cells we e
u he g own o 2, 3 and 4 days o iden i y he ime poin o maximum epo e gene exp ession.
Fo compa ison, he comme cially a ailable ans ec ion eagen je PRIME
®
was used as posi i e
con ol. Cells ea ed wi h pAmCyan- ee cCP and cell cul u e medium alone we e used as nega i e
con ols. Fo con ocal lase scanning mic oscope (CLSM; Leica TCS SP 8, Leica, We zla , Ge many)
isualiza ion, cell memb anes we e s ained using RGA I (15
µ
g/mL), and cell nuclei we e s ained
wi h DAPI (0.1
µ
g/mL). Samples we e hen ixed wi h 3% pa a o maldehyde and s o ed a 4
◦
C un il
analysis. All images we e acqui ed using a 25
×
wa e imme sion objec i e a 1024
×
1024 esolu ion
and u he p ocessed wi h LAS X so wa e (LAS X 1.8.013370, Leica Mic osys ems, Leica, Ge many).
The pe cen age e iciency o ans ec ed cells was quan i ied using low cy ome y (BD LSRFo essa
™
Cell Analyze , Biosciences, Heidelbe g, Ge many). Fi y housand cells pe sample we e coun ed by
he cy ome e and da a we e analyzed using FlowJo so wa e (FlowJo 7.6.5, FlowJo LLC, Ashland,
OR, USA). Th ee independen expe imen s we e pe o med in iplica es, and esul s exp essed as he
mean ±s anda d de ia ion (SD).
Polyme s 2018, 10, x FOR PEER REVIEW 8 o 21
elease o pDNA om he complex. Polyplexes con aining 500 ng o pDNA pe sample om each
s ep o he o mula ion p ocess we e i s diges ed wi h 0.5 µL BamHI o 2 h a 37 °C wi h shaking.
A e wa d, 3 µL (30 mg/mL) hepa in was added o solu ions o diges ed polyplexes, incuba ed o 15
min a oom empe a u e and hen mixed wi h 2 µL o o ange DNA loading dye (6×; The mo Fishe
Scien i ic, Wal ham, MA, USA). These mix u es we e hen loaded in o 0.75% (w/ ) aga ose gel
con aining 5 µL o e hidium b omide and un o 60 min a 50 V in 0.5× TBE-bu e . The isualiza ion
o he bands was pe o med wi h a UV illumina o , Fusion FX7 imaging sys em om Peqlab
(E langen, Ge many).
2.4.2. In Vi o T ans ec ion S udies in A549 Cells
To es he e iciency o he pAmCyan loaded polyplexes, in i o ans ec ion s udies we e
pe o med in A549 cells. B ie ly, A549 cells we e seeded in 24-well pla es, a a densi y o 25 × 104 cells
pe well in 500 µL o RPMI cell cul u e medium wi h 10% FCS. Cells we e g own o 2 days o each
a cell con luency o 60–70%. Polyplexes o he pAmCyan double loaded ca ie sys em (see Sec ion
2.4) con aining 1 µg o pAmCyan (polyplex concen a ion ~60 µg/mL) we e p epa ed wi h a a io o
1:30, 1:50 and 1:100 be ween pDNA:polyplexes in 500 µL o HBSS bu e . Then, cells we e washed
wice wi h HBSS bu e and incuba ed wi h he polyplexes o 6 h. A e 6 h o incuba ion, polyplexes
we e emo ed and eplaced wi h RPMI con aining 10% FCS. Cells we e u he g own o 2, 3 and 4
days o iden i y he ime poin o maximum epo e gene exp ession. Fo compa ison, he
comme cially a ailable ans ec ion eagen je PRIME® was used as posi i e con ol. Cells ea ed
wi h pAmCyan- ee cCP and cell cul u e medium alone we e used as nega i e con ols. Fo con ocal
lase scanning mic oscope (CLSM; Leica TCS SP 8, Leica, We zla , Ge many) isualiza ion, cell
memb anes we e s ained using RGA I (15 µg/mL), and cell nuclei we e s ained wi h DAPI (0.1
µg/mL). Samples we e hen ixed wi h 3% pa a o maldehyde and s o ed a 4 °C un il analysis. All
images we e acqui ed using a 25× wa e imme sion objec i e a 1024 × 1024 esolu ion and u he
p ocessed wi h LAS X so wa e (LAS X 1.8.013370, Leica Mic osys ems, Leica, Ge many). The
pe cen age e iciency o ans ec ed cells was quan i ied using low cy ome y (BD LSRFo essa™
Cell Analyze , Biosciences, Heidelbe g, Ge many). Fi y housand cells pe sample we e coun ed by
he cy ome e and da a we e analyzed using FlowJo so wa e (FlowJo 7.6.5, FlowJo LLC, Ashland,
OR, USA). Th ee independen expe imen s we e pe o med in iplica es, and esul s exp essed as
he mean ± s anda d de ia ion (SD).
Scheme 2. Illus a ion o s a ch-chi osan polyplex-p epa a ion o d ug-loaded polyplexes.
Scheme 2. Illus a ion o s a ch-chi osan polyplex-p epa a ion o d ug-loaded polyplexes.
Polyme s 2018,10, 252 9 o 21
3. Resul s and Discussion
3.1. P epa a ion and Cha ac e iza ion o D ug-F ee S a ch-Chi osan Polyplexes
This s udy ep esen s an ex ension in compa ison o he pa icle p epa a ion app oach o
Ba hold e al. [
29
], in which he la ge poly dispe si y index modi ied s a ch was employed o colloidal
o ma ion. Fu he mo e, he chemical modi ica ion in ha epo ed s udy, which was used o p oduce
ca ionic s a ch de i a i e, esul ed in un a o ably addi ional syn hesis s ep. Al hough he pa icle
p epa a ion was well es ablished, he lack o ca ionic s eng h due o an ob iously low con e ing yield
o ca ionic s a ch syn hesis limi ed he ca ie capaci y o anionic ne cha ge ac i es o such sys em.
Thus, we used ac ionally modi ied s a ch de i a i es o ha e be e con ol o colloidal s abili y,
and di e en molecula weigh chi osan de i a i es as s ong coun e excipien o he polyplexes
p oduce. Bo h excipien s a e polysaccha ides and he e o e ha e a o able cha ac e is ics wi h espec
o biological sa e y, biocompa ibili y and biodeg adabili y. The simple p oduc ion o polyplexes using
hese excipien s has he pe spec i e o be eadily up-scaled. In he i s se ies o p epa a ions, we
s udied he plain polyme ic complexes by combining bo h excipien s in aqueous solu ion, wi h he
elec os a ic in e ac ion be ween opposi e cha ges o he indi idual polyme s esul ing in polyplex
sel -assembly. Du ing he op imiza ion o his p ocess, a ious combina ions o ypes o polyme s,
C/N a io, and ini ial polyme solu ion concen a ion we e in es iga ed o ind a s able and na ow
size dis ibu ion o he p oduced colloidal s uc u es (de ails o he op imiza ion can be ound in
he Supplemen a y Ma e ials, Tables S1 and S2). The bes o se e al s able polyplex o mula ions
was p oduced using a C/N a io o 10:1, u ilizing anionic s a ch and oligochi osan. S a ch-chi osan
polyplexes we e ob ained wi h an anionic su ace cha ge e idenced by a
ζ
-po en ial o a ound
−
30 mV.
The size o polyplexes could be a ied om 150 nm o 350 nm by changing o polyme concen a ion,
wi h a na ow PDI (<0.3) in all cases. The impac o polyme concen a ion on polyplex size was
expec ed and al eady desc ibed o compa able sys ems [
30
,
31
]. Sphe ical polyplex mo phology was
isualized using TEM (Figu e 1A).
Polyme s 2018, 10, x FOR PEER REVIEW 9 o 21
3. Resul s and Discussion
3.1. P epa a ion and Cha ac e iza ion o D ug-F ee S a ch-Chi osan Polyplexes
This s udy ep esen s an ex ension in compa ison o he pa icle p epa a ion app oach o
Ba hold e al. [29], in which he la ge poly dispe si y index modi ied s a ch was employed o
colloidal o ma ion. Fu he mo e, he chemical modi ica ion in ha epo ed s udy, which was used
o p oduce ca ionic s a ch de i a i e, esul ed in un a o ably addi ional syn hesis s ep. Al hough he
pa icle p epa a ion was well es ablished, he lack o ca ionic s eng h due o an ob iously low
con e ing yield o ca ionic s a ch syn hesis limi ed he ca ie capaci y o anionic ne cha ge ac i es
o such sys em. Thus, we used ac ionally modi ied s a ch de i a i es o ha e be e con ol o
colloidal s abili y, and di e en molecula weigh chi osan de i a i es as s ong coun e excipien o
he polyplexes p oduce. Bo h excipien s a e polysaccha ides and he e o e ha e a o able
cha ac e is ics wi h espec o biological sa e y, biocompa ibili y and biodeg adabili y. The simple
p oduc ion o polyplexes using hese excipien s has he pe spec i e o be eadily up-scaled. In he
i s se ies o p epa a ions, we s udied he plain polyme ic complexes by combining bo h excipien s
in aqueous solu ion, wi h he elec os a ic in e ac ion be ween opposi e cha ges o he indi idual
polyme s esul ing in polyplex sel -assembly. Du ing he op imiza ion o his p ocess, a ious
combina ions o ypes o polyme s, C/N a io, and ini ial polyme solu ion concen a ion we e
in es iga ed o ind a s able and na ow size dis ibu ion o he p oduced colloidal s uc u es (de ails
o he op imiza ion can be ound in he Supplemen a y Ma e ials, Tables S1 and S2). The bes o
se e al s able polyplex o mula ions was p oduced using a C/N a io o 10:1, u ilizing anionic s a ch
and oligochi osan. S a ch-chi osan polyplexes we e ob ained wi h an anionic su ace cha ge
e idenced by a ζ-po en ial o a ound −30 mV. The size o polyplexes could be a ied om 150 nm o
350 nm by changing o polyme concen a ion, wi h a na ow PDI (<0.3) in all cases. The impac o
polyme concen a ion on polyplex size was expec ed and al eady desc ibed o compa able sys ems
[30,31]. Sphe ical polyplex mo phology was isualized using TEM (Figu e 1A).
Figu e 1. T ansmission elec on mic oscope (TEM) images o d ug- ee s a ch-chi osan polyplexes
s ained by 0.5% phospho ungs ic acid solu ion: (A) d ug- ee anCP; and (B) d ug- ee cCP.
Re e sing he C/N a io o 1:10, and using s a ch (Mw 30–100 kDa) and P o asan (Mw ~90 kDa)
esul ed in a swi ch o he su ace cha ge om anionic o ca ionic ( u he e med as ca ionic CP),
wi h a ζ-po en ial o a ound +40 mV. The size o pa icles a ied om 214.3 nm o app oxima ely 400
nm depending on he polyme concen a ion and C/N a io (Supplemen a y Ma e ials, Tables S1 and
S2). As bo h anCP and ca ionic CP sys ems o med as a esul o a ac i e o ces o polyme
unc ional g oups, u he agg ega ion o sys ems o e ime may po en ially occu ; he physical
s abili y o he polyplexes was he e o e s udied o e a ime cou se wi h s o age a 4 °C. The colloidal
cha ac e is ics o bo h, anCP and ca ionic CP, emained s able o 27 days wi h a PDI o ~0.18 and a
ζ-po en ial o −30 mV and +35 mV o anCP and ca ionic CP, espec i ely (Supplemen a y Ma e ials,
Figu e S1). Consequen ly, he u ilized p epa a ion p ocess ep esen s a s aigh o wa d app oach o
he o mula ion o e sa ile nanopa icles.
Figu e 1.
T ansmission elec on mic oscope (TEM) images o d ug- ee s a ch-chi osan polyplexes
s ained by 0.5% phospho ungs ic acid solu ion: (A) d ug- ee anCP; and (B) d ug- ee cCP.
Re e sing he C/N a io o 1:10, and using s a ch (M
w
30–100 kDa) and P o asan (M
w
~90 kDa)
esul ed in a swi ch o he su ace cha ge om anionic o ca ionic ( u he e med as ca ionic CP),
wi h a
ζ
-po en ial o a ound +40 mV. The size o pa icles a ied om 214.3 nm o app oxima ely
400 nm depending on he polyme concen a ion and C/N a io (Supplemen a y Ma e ials, Tables S1
and S2). As bo h anCP and ca ionic CP sys ems o med as a esul o a ac i e o ces o polyme
unc ional g oups, u he agg ega ion o sys ems o e ime may po en ially occu ; he physical
s abili y o he polyplexes was he e o e s udied o e a ime cou se wi h s o age a 4
◦
C. The colloidal
cha ac e is ics o bo h, anCP and ca ionic CP, emained s able o 27 days wi h a PDI o ~0.18 and a
ζ
-po en ial o
−
30 mV and +35 mV o anCP and ca ionic CP, espec i ely (Supplemen a y Ma e ials,
Polyme s 2018,10, 252 16 o 21
3.3. Loading o anCP wi h High-Mw pDNA
A model plasmid DNA encoding a luo escen dye (pAmCyan) was u he inco po a ed in o he
ca ie sys em in a h ee-s ep p ocedu e (co e o ma ion, P o asan coa ing and pDNA complexa ion),
o demons a e he abili y o he polyplexes o deli e a b oad spec um o ca gos. The h ee-s ep
p ocedu e also lead o an inc ease o nucleic acid encapsula ion wi hin he polyplexes, p o ec ing
nucleic acids om enzyma ic deg ada ion. P oduced pAmCyan loaded polyplexes we e again ound
o ha e a sphe ical s uc u e (Figu e 5C,D). The physiochemical cha ac e is ics o all in e media e
and inal polyplexes in his s epwise p oduc ion can be ound in Table 2. Each subsequen s ep
in he p epa a ion p ocedu e esul s in a dense complexa ion, wi h he pAmCyan double loaded
cCP showing he smalles size and mos na ow size dis ibu ion (lowes PDI alue). Fu he mo e,
he
ζ
-po en ial was obse ed o swi ch om nega i e o posi i e a e coa ing wi h P o asan, wi h
a u he sligh dec ease a e complexa ion wi h nega i ely cha ged pAmCyan. The addi ional
complexa ion wi h pAmCyan esul ed in a 15% highe encapsula ion e iciency in compa ison o he
in e media e s ep 2 (Table 2). Addi ionally, aga ose gel elec opho esis (Figu e 6, le ) elucida es ha
no pDNA could un h ough he gel, which indica es ha pDNA is s ongly complexed wi hin he
polyplexes. Only u he ea men wi h BamHI and hepa in causes pDNA elease as seen h ough
he bands (Figu e 6, igh ). Fu he mo e, pDNA loaded anCP and pDNA associa ed on he su ace o
polyplexes (pDNA double loaded cCP) allow an easie in e cala ion o E B and as e elease wi h
hepa in, whe eas pDNA loaded cCP is densely packed impeding pDNA elease as no ee pDNA
bands can be obse ed in he gel.
Polyme s 2018, 10, x FOR PEER REVIEW 16 o 21
3.3. Loading o anCP wi h High-Mw pDNA
A model plasmid DNA encoding a luo escen dye (pAmCyan) was u he inco po a ed in o
he ca ie sys em in a h ee-s ep p ocedu e (co e o ma ion, P o asan coa ing and pDNA
complexa ion), o demons a e he abili y o he polyplexes o deli e a b oad spec um o ca gos.
The h ee-s ep p ocedu e also lead o an inc ease o nucleic acid encapsula ion wi hin he polyplexes,
p o ec ing nucleic acids om enzyma ic deg ada ion. P oduced pAmCyan loaded polyplexes we e
again ound o ha e a sphe ical s uc u e (Figu e 5C,D). The physiochemical cha ac e is ics o all
in e media e and inal polyplexes in his s epwise p oduc ion can be ound in Table 2. Each
subsequen s ep in he p epa a ion p ocedu e esul s in a dense complexa ion, wi h he pAmCyan
double loaded cCP showing he smalles size and mos na ow size dis ibu ion (lowes PDI alue).
Fu he mo e, he ζ-po en ial was obse ed o swi ch om nega i e o posi i e a e coa ing wi h
P o asan, wi h a u he sligh dec ease a e complexa ion wi h nega i ely cha ged pAmCyan. The
addi ional complexa ion wi h pAmCyan esul ed in a 15% highe encapsula ion e iciency in
compa ison o he in e media e s ep 2 (Table 2). Addi ionally, aga ose gel elec opho esis (Figu e 6,
le ) elucida es ha no pDNA could un h ough he gel, which indica es ha pDNA is s ongly
complexed wi hin he polyplexes. Only u he ea men wi h BamHI and hepa in causes pDNA
elease as seen h ough he bands (Figu e 6, igh ). Fu he mo e, pDNA loaded anCP and pDNA
associa ed on he su ace o polyplexes (pDNA double loaded cCP) allow an easie in e cala ion o
E B and as e elease wi h hepa in, whe eas pDNA loaded cCP is densely packed impeding pDNA
elease as no ee pDNA bands can be obse ed in he gel.
Figu e 6. Gel e a da ion assay using aga ose gel elec opho esis o plain and pDNA (pAmCyan)
inco po a ed polyplexes o all h ee p epa a ion s eps in compa ison wi h naked pDNA (undiges ed
pDNA) and diges ed pDNA (pDNA + BamHI).
Po en ial o Polyplexes o pDNA Deli e y
Using nanopa icles as a non- i al deli e y sys em o gene he apy ep esen s a signi ican
challenge, as nanoca ie s need o c oss se e al biological ba ie s while p ese ing he unc ionali y
o ca ied pDNA. pDNA condensed inside he nanoca ie s mus su i e he acidic condi ions inside
he lysosomes and escape he lysosomal compa men in o de o c oss he nuclea memb ane [43].
Cu en knowledge o polyme ic ans ec ion sys ems sugges s ha a good pH-bu e ing capaci y (a
p ocess known as he “p o on sponge e ec ”) [47] is an impo an ac o in he achie emen o
endosomal escape. He e, he po en ial o s a ch–chi osan polyplexes o nucleic acid deli e y was
explo ed by in i o ans ec ion s udies using A549 cells. Th ee di e en a ios be ween
pDNA:polyplexes ha e been s udied o in es iga e he bes ans ec ion a e. While 1:50 and 1:100
show no signi ican ans ec ion (da a no shown), 1:30 media ed success ul ans ec ion, wi h he
highes epo e gene exp ession obse ed a e 48 h wi h 5% o ans ec ed cells. In compa ison,
je PRIME® as posi i e con ol had a highe ans ec ion e iciency (45%) a e 48 h, which apidly
Figu e 6.
Gel e a da ion assay using aga ose gel elec opho esis o plain and pDNA (pAmCyan)
inco po a ed polyplexes o all h ee p epa a ion s eps in compa ison wi h naked pDNA (undiges ed
pDNA) and diges ed pDNA (pDNA + BamHI).
Po en ial o Polyplexes o pDNA Deli e y
Using nanopa icles as a non- i al deli e y sys em o gene he apy ep esen s a signi ican
challenge, as nanoca ie s need o c oss se e al biological ba ie s while p ese ing he unc ionali y o
ca ied pDNA. pDNA condensed inside he nanoca ie s mus su i e he acidic condi ions inside he
lysosomes and escape he lysosomal compa men in o de o c oss he nuclea memb ane [
43
]. Cu en
knowledge o polyme ic ans ec ion sys ems sugges s ha a good pH-bu e ing capaci y (a p ocess
known as he “p o on sponge e ec ”) [
47
] is an impo an ac o in he achie emen o endosomal
escape. He e, he po en ial o s a ch–chi osan polyplexes o nucleic acid deli e y was explo ed
by
in i o
ans ec ion s udies using A549 cells. Th ee di e en a ios be ween pDNA:polyplexes
ha e been s udied o in es iga e he bes ans ec ion a e. While 1:50 and 1:100 show no signi ican
ans ec ion (da a no shown), 1:30 media ed success ul ans ec ion, wi h he highes epo e gene
exp ession obse ed a e 48 h wi h 5% o ans ec ed cells. In compa ison, je PRIME
®
as posi i e
Polyme s 2018,10, 252 17 o 21
con ol had a highe ans ec ion e iciency (45%) a e 48 h, which apidly dec eased o 30% a e
72 h and o 25% a e 96 h (Figu e 7). The compa a i ely lowe ans ec ion e iciency o he
polyplexes may be due o a high s abili y o condensed pDNA, leading o an incomple e elease
o pDNA inside he cy oplasmic compa men [
48
,
49
]. Fu he imp o emen o he ans ec ion
e iciency would p esumably be achie able by addi ion o endosomal escape moie ies [
50
,
51
], o wi h
chi osan de i a i es (e.g., ime hyla ion o amino acid conjuga ion) [
52
,
53
]. Howe e , such e icacy
imp o emen s o en impac he biocompa ibili y. Thus, op imiza ion be ween sa e y and e icacy
should be pe o med o a selec ed nucleo ide ype, a ge applica ion, and deli e y ou e, since ca ie
s abili y, cellula up ake, and unc ional e icacy a e highly dependen on all hese ac o s.
Polyme s 2018, 10, x FOR PEER REVIEW 17 o 21
dec eased o 30% a e 72 h and o 25% a e 96 h (Figu e 7). The compa a i ely lowe ans ec ion
e iciency o he polyplexes may be due o a high s abili y o condensed pDNA, leading o an
incomple e elease o pDNA inside he cy oplasmic compa men [48,49]. Fu he imp o emen o
he ans ec ion e iciency would p esumably be achie able by addi ion o endosomal escape
moie ies [50,51], o wi h chi osan de i a i es (e.g., ime hyla ion o amino acid conjuga ion) [52,53].
Howe e , such e icacy imp o emen s o en impac he biocompa ibili y. Thus, op imiza ion
be ween sa e y and e icacy should be pe o med o a selec ed nucleo ide ype, a ge applica ion,
and deli e y ou e, since ca ie s abili y, cellula up ake, and unc ional e icacy a e highly
dependen on all hese ac o s.
Figu e 7. (A) Rep esen a i e con ocal images o A549 cells ans ec ed wi h pAmCyan double loaded
pAmCyan by using je PRIME® as posi i e con ol and only cell cul u e medium as nega i e con ol.
T ans ec ion was analyzed wi h CLSM a e 48 h, 72 h, and 96 h. G een luo escence e eals cells
success ully ans ec ed wi h he polyplexes while hei mo phology emains consis en wi h non-
ans ec ed cells ( ed: cell memb ane; blue: cell nucleus; scale ba 50 µm). (B) The ans ec ion e iciency
was u he quan i ied using low cy ome y, which indica ed he highes amoun o ans ec ion a e
48 h o pAmCyan double loaded cCP. (C)Rep esen a i e g aphs ob ained wi h low cy ome e .
Figu e 7.
(
A
) Rep esen a i e con ocal images o A549 cells ans ec ed wi h pAmCyan double
loaded pAmCyan by using je PRIME
®
as posi i e con ol and only cell cul u e medium as nega i e
con ol. T ans ec ion was analyzed wi h CLSM a e 48 h, 72 h, and 96 h. G een luo escence e eals
cells success ully ans ec ed wi h he polyplexes while hei mo phology emains consis en wi h
non- ans ec ed cells ( ed: cell memb ane; blue: cell nucleus; scale ba 50
µ
m). (
B
) The ans ec ion
e iciency was u he quan i ied using low cy ome y, which indica ed he highes amoun o
ans ec ion a e 48 h o pAmCyan double loaded cCP. (
C
) Rep esen a i e g aphs ob ained wi h
low cy ome e .
Polyme s 2018,10, 252 18 o 21
4. Conclusions
In his wo k, we p oduced a lexible, s aigh o wa d and o ganic sol en - ee p ocedu e o
he manu ac u e o nanoca ie sys ems based on he na u al, biodeg adable and biocompa ible
polysaccha ides s a ch and chi osan. S a ch and chi osan de i a i es o di e en M
w
anges we e
combined by adjus ing he mola a io o ca boxyl and amine unc ional g oups, polyme concen a ion
and coun e polyme ype o ob ain a deli e y sys em wi h unable p ope ies including su ace cha ge
and size. Co e polyplexes (CP) we e buil by complex coace a ion o anionic s a ch (M
w
~100 kDa)
wi h posi i ely cha ged chi osan de i a i es (M
w
~5 kDa) in aqueous solu ion. The polyplexes wi h
he bes colloidal p ope ies we e ob ained a a mola a io o ca boxyl and amine g oups o 10:1.
The nega i ely cha ged co e polyplexes emained s able on s o age o o e 27 days. We u he ocused
on op imizing anionic CPs by coa ing hem wi h an addi ional laye o chi osan (P o asan, M
w
~90 kDa).
Cell iabili y es ing o anCPs and cCPs indica ed a low le el o cy o oxici y accep able o use in
biological sys ems, and colloidal s abili y a di e en es ed pH alues. The de eloped anCP sys em
u he showed good ca ie p ope ies, allowing o high encapsula ion e iciency (>90%) o ca ionic
pep ide (colis in) and small molecule ( ob amycin) an i-in ec i es wi hou comp omising an imic obial
ac i i y. Mo eo e , he ca ionic polyplexes, cCP, allowed o double encapsula ion o plasmid DNA
(pAmCyan) o in acellula deli e y as con i med by gel e a da ion assay, and acili a ing in- i o
ans ec ion in A549 cells.
S a ch-chi osan polyplexes show high lexibili y o designing mul i unc ional ca ie s, in which
o example he co e polyplexes can encapsula e an i-in ec i es, while he ou e coa ing laye could
be used o inco po a e o he componen s like enzymes o nucleases (e.g., deoxy ibonuclease I) o
enhance d ug pene a ion h ough bio ilms o mucus [
30
]. Fo gene he apy pu poses he inne
polyplex can be used o ca y and p o ec plasmid DNA, while he su ace could be deco a ed wi h a
second polynucleo ide.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h p://www.mdpi.com/2073-4360/10/3/252/s1,
Figu e S1: Physicochemical s abili y o s a ch-chi osan CP, in which anCP was p oduced wi h C/N a io 10/1,
and ca CP was p oduced wi h C/N a io 1/10, upon s o age (4
◦
C). The pa icles we e dilu ed in o Milli-Q
wa e a each ime poin o he measu emen o size, PDI and
ζ
-po en ial. N= 3, n= 3, mean
±
SD; Figu e S2:
Physicochemical s abili y o s a ch-chi osan anCP and cCP a di e en pH alues anging om 3.5 o 8.0, a e
30 min and 1 h incuba ion. The ini ial pH- alue o he samples was 5.5. N= 3, n= 3, mean
±
SD; Figu e S3:
Cumula i e elease o ob amycin om ob amycin loaded anCP, and colis in om colis in loaded anCP pe o med
in PBS a 37
◦
C. N= 3, n= 3, mean
±
SD; Table S1: Summa y o s a ch-chi osan CP cha ac e is ics ob ained by
a ying polyme ypes, polyme concen a ion, and C/N mola a io. N> 3, n= 3, mean
±
SD; Table S2: Summa y
o s a ch-chi osan CP cha ac e iza ion wi h op imal C/N a io a ied by change o polyme concen a ion. N> 3,
n= 3, mean
±
SD; Table S3: Summa y o anionic CP (anCP) and P o asan coa ed anCP (cCP) cha ac e is ics,
in which anCP was p oduced wi h pa ame e s, namely C/N a io 10/1, and polyme concen a ion a 6.5 mg/mL.
N> 3, n= 3, mean
±
SD; Table S4: Summa y o ob amycin-loaded anCP cha ac e is ics achie ed by a ia ion
o C/N a io and polyme concen a ion. N> 3, n= 3, mean
±
SD; Table S5: Summa y o colis in-loaded anCP
cha ac e is ics esul ing om a ia ion o polyme concen a ion. N> 3, n= 3, mean
±
SD; Table S6: Summa y o
d ug loading quan i ica ion o ob amycin-loaded anCP. N> 3, n= 3, mean
±
SD; Table S7: Summa y o d ug
loading quan i ica ion o colis in-loaded anCP. N> 3, n= 3, mean ±SD.
Acknowledgmen s:
The au ho s hank Xabie Mu gia Es e e, Flo ian G ä , and A ianna Cas oldi o ui ul
discussions; Pe a König and Jana Wes hues o suppo and handling o cell cul u es; and Vik o ia Schmi o
bac e ia cul u e. This p ojec has ecei ed unding om he Eu opean Union F amewo k P og amme o Resea ch
and Inno a ion Ho izon 2020 (2014–2020) unde he Ma ie Sklodowska-Cu ie G an Ag eemen No. 642028.
Au ho Con ibu ions:
Hanzey Yasa and Duy-Khie Ho con ibu ed equally in his s udy in which hey ini ia ed
he esea ch idea, concei ed and designed all expe imen s. Hanzey Yasa and Duy-Khie Ho syn hesized and
p epa ed molecula weigh ac ionalized anionic s a ch, and op imized pa icles p epa ing p ocess o s a ch
and chi osan (including nega i e, posi i e and coa ed su ace pa icles), as well as in es iga ed s abili y o all
pa icles a s o age condi ion and di e en pH en i onmen s. Hanzey Yasa u he s udied cy o oxici y (by MTT
assay) and plasmid (pAmCyan) loading capaci y (by a gel e a da ion assay) o he pa icles, and pe o med
ans ec ion s udy on A549 cell line by CLSM and quan i ica ion me hod using low cy ome y. Duy-Khie Ho
u he s udied and op imized ca ionic an i-in ec i es ( ob amycin and colis in) loading capaci y (by iso he mal
i a ion calo ime y) o he pa icles, and pe o med he minimum inhibi o y concen a ion (MIC) assay on E. coli
and P. ae uginosa. Hanzey Yasa and Duy-Khie Ho analyzed all he da a and w o e he manusc ip wi h an equal
manne . Chia a De Rossi isualized he de eloped pa icles by using TEM, pe o med HPLC, and con ibu ed
Polyme s 2018,10, 252 19 o 21
he expe ise in imaging and analyzing he ans ec ion s udy by using CLSM. Jenni e He mann con ibu ed
he expe ise in bac e ia s udy and analyzed he da a. Sa ah Go don, B igi a Lo e z and Claus-Michael Leh
supe ised Hanzey Yasa and Duy-Khie Ho, ini ia ed he p ojec and ha e been esponsible o he o e all
scien i ic app oach. All au ho s con ibu ed wi h hei scien i ic inpu o he w i en manusc ip .
Con lic s o In e es : The au ho s decla e no compe ing inancial in e es .
Abb e ia ions
CP co e polyplexes
anCP anionic co e polyplexes
ca ionic CP (o ca CP) ca ionic co e polyplexes
cCP coa ed polyplexes
Re e ences
1.
Kang, B.; Opa z, T.; Land es e , K.; Wu m, F.R. Ca bohyd a e nanoca ie s in biomedical applica ions:
Func ionaliza ion and cons uc ion. Chem. Soc. Re . 2015,44, 8301–8325. [C ossRe ] [PubMed]
2.
Bachmann, M.F.; Jennings, G.T. Vaccine deli e y: A ma e o size, geome y, kine ics and molecula pa e ns.
Na . Re . Immunol. 2010,10, 787–796. [C ossRe ] [PubMed]
3.
Abed, N.; Cou eu , P. Nanoca ie s o an ibio ics: A p omising solu ion o ea in acellula bac e ial
in ec ions. In . J. An imic ob. Agen s 2014,43, 485–496. [C ossRe ] [PubMed]
4.
D’Angelo, I.; Con e, C.; Mi o, A.; Quaglia, F.; Unga o, F. Pulmona y d ug deli e y: A ole o polyme ic
nanopa icles? Cu . Top. Med. Chem. 2015,15, 386–400. [C ossRe ] [PubMed]
5.
Mahapa o, A.; Singh, D.K. Biodeg adable nanopa icles a e excellen ehicle o si e di ec ed in- i o deli e y
o d ugs and accines. J. Nanobio echnol. 2011,9, 55. [C ossRe ] [PubMed]
6.
Baldwin, A.D.; Kiick, K.L. Polysaccha ide-modi ied syn he ic polyme ic bioma e ials. Biopolyme s
2010
,94,
128–140. [C ossRe ] [PubMed]
7.
Wiks öm, J.; Elomaa, M.; Sy äjä i, H.; Kuokkanen, J.; Ylipe ula, M.; Honkakoski, P.; U i, A.
Algina e-based mic oencapsula ion o e inal pigmen epi helial cell line o cell he apy. Bioma e ials
2008,29, 869–876. [C ossRe ] [PubMed]
8.
Hans, M.L.; Lowman, A.M. Biodeg adable nanopa icles o d ug deli e y and a ge ing. Cu . Opin. Solid
S a e Ma e . Sci. 2002,6, 319–327. [C ossRe ]
9.
Azzam, T.; Eliyahu, H.; Shapi a, L.; Linial, M.; Ba enholz, Y.; Domb, A.J. Polysaccha ide? Oligoamine Based
Conjuga es o Gene Deli e y. J. Med. Chem. 2002,45, 1817–1824. [C ossRe ] [PubMed]
10.
Sim, H.J.; Thambi, T.; Lee, D.S. Hepa in-based empe a u e-sensi i e injec able hyd ogels o p o ein deli e y.
J. Ma e . Chem. B 2015,3, 8892–8901. [C ossRe ]
11.
Builde s, P.F.; A hewoh, M.I. Pha maceu ical applica ions o na i e s a ch in con en ional d ug deli e y.
S a ch S ä ke 2016,68, 864–873. [C ossRe ]
12.
Yamada, H.; Lo e z, B.; Leh , C.-M. Design o s a ch-g a -PEI polyme s: An e ec i e and biodeg adable
gene deli e y pla o m. Biomac omolecules 2014,15, 1753–1761. [C ossRe ] [PubMed]
13.
Mahmoudi Naja i, S.H.; Baghaie, M.; Asho i, A. P epa a ion and cha ac e iza ion o ace yla ed s a ch
nanopa icles as d ug ca ie : Cip o loxacin as a model. In . J. Biol. Mac omol.
2016
,87, 48–54. [C ossRe ]
[PubMed]
14.
Balmayo , E.R.; Ba an, E.T.; Aze edo, H.S.; Reis, R.L. Injec able biodeg adable s a ch/chi osan deli e y
sys em o he sus ained elease o gen amicin o ea bone in ec ions. Ca bohyd . Polym.
2012
,87, 32–39.
[C ossRe ]
15.
San ande -O ega, M.J.; S aune , T.; Lo e z, B.; O ega-Vinuesa, J.L.; Bas os-González, D.; Wenz, G.;
Schae e , U.F.; Leh , C.M. Nanopa icles made om no el s a ch de i a i es o ansde mal d ug deli e y.
J. Con ol. Release 2010,141, 85–92. [C ossRe ] [PubMed]
16.
Be nkop-Schnu ch, A.; Dunnhaup , S. Chi osan-based d ug deli e y sys ems. Eu . J. Pha m. Biopha m.
2012
,
81, 463–469. [C ossRe ] [PubMed]
17.
G enha, A.; Gomes, M.E.; Rod igues, M.; San o, V.E.; Mano, J.F.; Ne es, N.M.; Reis, R.L. De elopmen o
new chi osan/ca ageenan nanopa icles o d ug deli e y applica ions. J. Biomed. Ma e . Res. Pa A
2010
,
92, 1265–1272. [C ossRe ] [PubMed]
Polyme s 2018,10, 252 20 o 21
18.
Gan, Q.; Wang, T. Chi osan nanopa icle as p o ein deli e y ca ie —Sys ema ic examina ion o ab ica ion
condi ions o e icien loading and elease. Colloids Su . B Bioin e aces
2007
,59, 24–34. [C ossRe ] [PubMed]
19.
Wen, Z.-S.; Xu, Y.-L.; Zou, X.-T.; Xu, Z.-R. Chi osan nanopa icles ac as an adju an o p omo e bo h Th1 and
Th2 immune esponses induced by o albumin in mice. Ma . D ugs
2011
,9, 1038–1055. [C ossRe ] [PubMed]
20.
De Campos, A.M.; Sánchez, A.; Alonso, M.J. Chi osan nanopa icles: A new ehicle o he imp o emen o
he deli e y o d ugs o he ocula su ace. Applica ion o cyclospo in A. In . J. Pha m.
2001
,224, 159–168.
[C ossRe ]
21.
Van de Lubben, I.M.; Ve hoe , J.C.; an Aels , A.C.; Bo cha d, G.; Junginge , H.E. Chi osan mic opa icles
o o al accina ion: P epa a ion, cha ac e iza ion and p elimina y
in i o
up ake s udies in mu ine Peye ’s
pa ches. Bioma ials 2001,22, 687–694. [C ossRe ]
22.
Dodane, V.; Vili alam, V.D. Pha maceu ical applica ions o chi osan. Pha m. Sci. Technol. Today
1998
,1,
246–253. [C ossRe ]
23.
Onishi, H.; Machida, Y. Biodeg ada ion and dis ibu ion o wa e -soluble chi osan in mice. Bioma e ials
1999
,
20, 175–182. [C ossRe ]
24. Aspden, T.J.; Mason, J.D.; Jones, N.S.; Lowe, J.; Skaug ud, O.; Illum, L. Chi osan as a nasal deli e y sys em:
The e ec o chi osan solu ions on
in i o
and
in i o
mucocilia y anspo a es in human u bina es and
olun ee s. J. Pha m. Sci. 1997,86, 509–513. [C ossRe ] [PubMed]
25.
Elbo n, J.S.; Va ai e, A.-L.; Fukushima, A.; Aballea, S.; Khemi i, A.; Moo e, C.; Medic, G.; Hemels, M.E.H.
Compa ison o Inhaled An ibio ics o he T ea men o Ch onic Pseudomonas ae uginosa Lung In ec ion in
Pa ien s wi h Cys ic Fib osis: Sys ema ic Li e a u e Re iew and Ne wo k Me a-analysis. Clin. The .
2016
,38,
2204–2226. [C ossRe ] [PubMed]
26.
Elbo n, S.; Va ai e, A.-L.; Fukushima, A.; Aballéa, S.; Khemi i, A.; Moo e, C.; Medic, G.; Hemels, M. E icacy
and sa e y o inhaled an ibio ics o ch onic pseudomonas in ec ion in cys ic ib osis: Ne wo k me a-analysis.
Eu . Respi . J. 2016,48, PA4863. [C ossRe ]
27.
Na ee, N.; Schneide , M.; Schae e , U.F.; Leh , C.-M. Rele ance o he colloidal s abili y o chi osan/PLGA
nanopa icles on hei cy o oxici y p o ile. In . J. Pha m. 2009,381, 130–139. [C ossRe ] [PubMed]
28.
Benson, J.R.; Ha e, P.E. O-ph halaldehyde: Fluo ogenic de ec ion o p ima y amines in he picomole ange.
Compa ison wi h luo escamine and ninhyd in. P oc. Na l. Acad. Sci. USA
1975
,72, 619–622. [C ossRe ]
[PubMed]
29.
Ba hold, S.; Kle ing, S.; Ta ne , J.; de Souza Ca alho-Woda z, C.; Lepel ie , E.; Lo e z, B.; Leh , C.-M.
P epa a ion o nanosized coace a es o posi i e and nega i e s a ch de i a i es in ended o pulmona y
deli e y o p o eins. J. Ma e . Chem. B 2016,4, 2377–2386. [C ossRe ]
30.
Deacon, J.; Abdelghany, S.M.; Quinn, D.J.; Schmid, D.; Megaw, J.; Donnelly, R.F.; Jones, D.S.;
Kissenp ennig, A.; Elbo n, J.S.; Gilmo e, B.F.; e al. An imic obial e icacy o ob amycin polyme ic
nanopa icles o Pseudomonas ae uginosa in ec ions in cys ic ib osis: Fo mula ion, cha ac e isa ion and
unc ionalisa ion wi h do nase al a (DNase). J. Con ol. Release 2015,198, 55–61. [C ossRe ] [PubMed]
31.
Dul, M.; Paluch, K.J.; Kelly, H.; Healy, A.M.; Sasse, A.; Tajbe , L. Sel -assembled ca ageenan/p o amine
polyelec oly e nanoplexes-In es iga ion o c i ical pa ame e s go e ning hei o ma ion and cha ac e is ics.
Ca bohyd . Polym. 2015,123, 339–349. [C ossRe ] [PubMed]
32.
Rado ic-Mo eno, A.F.; Lu, T.K.; Puscasu, V.A.; Yoon, C.J.; Lange , R.; Fa okhzad, O.C. Su ace
cha ge-swi ching polyme ic nanopa icles o bac e ial cell wall- a ge ed deli e y o an ibio ics. ACS Nano
2012,6, 4279–4287. [C ossRe ] [PubMed]
33.
Mandzy, N.; G ulke, E.; D u el, T. B eakage o TiO
2
agglome a es in elec os a ically s abilized aqueous
dispe sions. Powde Technol. 2005,160, 121–126. [C ossRe ]
34.
Jonassen, H.; Kjoniksen, A.-L.; Hio h, M. S abili y o chi osan nanopa icles c oss-linked wi h
ipolyphospha e. Biomac omolecules 2012,13, 3747–3756. [C ossRe ] [PubMed]
35.
Hona y, S.; Zahi , F. E ec o Ze a Po en ial on he P ope ies o Nano-D ug Deli e y Sys ems—A Re iew
(Pa 2). T op. J. Pha m. Res. 2013,12, 265–273. [C ossRe ]
36.
Yoo, M.K.; Sung, Y.K.; Chong, S.C.; Young, M.L. E ec o polyme complex o ma ion on he cloud-poin o
poly(N-isop opyl ac ylamide) (PNIPAAm) in he poly(NIPAAm-co-ac ylic acid): Polyelec oly e complex
be ween poly(ac ylic acid) and poly(allylamine). Polyme 1997,38, 2759–2765. [C ossRe ]
37.
Fan, W.; Yan, W.; Xu, Z.; Ni, H. Fo ma ion mechanism o monodispe se, low molecula weigh chi osan
nanopa icles by ionic gela ion echnique. Colloids Su . B Bioin e aces
2012
,90, 21–27. [C ossRe ] [PubMed]
Polyme s 2018,10, 252 21 o 21
38.
Shu, X.; Zhu, K. The in luence o mul i alen phospha e s uc u e on he p ope ies o ionically c oss-linked
chi osan ilms o con olled d ug elease. Eu . J. Pha m. Biopha m. 2002,54, 235–243. [C ossRe ]
39.
Ensign, L.M.; Cone, R.; Hanes, J. Nanopa icle-based d ug deli e y o he agina: A e iew. J. Con ol. Release
2014,190, 500–514. [C ossRe ] [PubMed]
40.
E ans, D.F.; Pye, G.; B amley, R.; Cla k, A.G.; Dyson, T.J.; Ha dcas le, J.D. Measu emen o gas oin es inal
pH p o iles in no mal ambulan human subjec s. Gu 1988,29, 1035–1041. [C ossRe ] [PubMed]
41.
Melis, G.B.; Ibba, M.T.; S e i, B.; Ko sonis, P.; Ma a, V.; Paole i, A.M. Ruolo del pH come modula o e
dell’equilib io isiopa ologico aginale. Min. Ginecol. 2000,52, 111–121.
42.
Fische , D.; Li, Y.; Ahlemeye , B.; K iegls ein, J.; Kissel, T.
In i o
cy o oxici y es ing o polyca ions: In luence
o polyme s uc u e on cell iabili y and hemolysis. Bioma ials 2003,24, 1121–1131. [C ossRe ]
43.
F ohlich, E. The ole o su ace cha ge in cellula up ake and cy o oxici y o medical nanopa icles. In . J.
Nanomed. 2012,7, 5577–5591. [C ossRe ] [PubMed]
44.
Maiz, L.; Gi on, R.M.; Ol ei a, C.; Quin ana, E.; Lamas, A.; Pas o , D.; Can on, R.; Mensa, J. Inhaled an ibio ics
o he ea men o ch onic b onchopulmona y Pseudomonas ae uginosa in ec ion in cys ic ib osis: Sys ema ic
e iew o andomised con olled ials. Expe Opin. Pha maco he .
2013
,14, 1135–1149. [C ossRe ] [PubMed]
45.
Ba goni, A.; Ca alli, R.; Za a, G.P.; Funda o, A.; Capu o, O.; Gasco, M.R. T ansmucosal anspo
o ob amycin inco po a ed in solid lipid nanopa icles (SLN) a e duodenal adminis a ion o a s.
Pa II—Tissue dis ibu ion. Pha macol. Res. 2001,43, 497–502. [C ossRe ] [PubMed]
46.
Wallace, S.J.; Li, J.; Na ion, R.L.; P anke d, R.J.; Velko , T.; Boyd, B.J. Sel -assembly beha io o colis in and
i s p od ug colis in me hanesul ona e: Implica ions o solu ion s abili y and solubiliza ion. J. Phys. Chem. B
2010,114, 4836–4840. [C ossRe ] [PubMed]
47.
Akinc, A.; Thomas, M.; Klibano , A.M.; Lange , R. Explo ing polye hylenimine-media ed DNA ans ec ion
and he p o on sponge hypo hesis. J. Gene Med. 2005,7, 657–663. [C ossRe ] [PubMed]
48.
T uong, N.P.; Jia, Z.; Bu gess, M.; Payne, L.; McMillan, N.A.J.; Mon ei o, M.J. Sel -ca alyzed deg adable
ca ionic polyme o elease o DNA. Biomac omolecules 2011,12, 3540–3548. [C ossRe ] [PubMed]
49.
Ha ono, S.B.; Phuoc, N.T.; Yu, M.; Jia, Z.; Mon ei o, M.J.; Qiao, S.; Yu, C. Func ionalized la ge po e
mesopo ous silica nanopa icles o gene deli e y ea u ing con olled elease and co-deli e y. J. Ma e .
Chem. B 2014,2, 718–726. [C ossRe ]
50.
T uong, N.P.; Gu, W.; P asadam, I.; Jia, Z.; C aw o d, R.; Xiao, Y.; Mon ei o, M.J. An in luenza i us-inspi ed
polyme sys em o he imed elease o siRNA. Na . Commun. 2013,4, 1902. [C ossRe ] [PubMed]
51.
Sanz, V.; Coley, H.M.; Sil a, S.R.P.; McFadden, J. P o amine and Chlo oquine Enhance Gene Deli e y and
Exp ession Media ed by RNA-W apped Single Walled Ca bon Nano ubes. J. Nanosci. Nano echnol.
2012
,12,
1739–1747. [C ossRe ] [PubMed]
52.
Kean, T.; Ro h, S.; Thanou, M. T ime hyla ed chi osans as non- i al gene deli e y ec o s: Cy o oxici y and
ans ec ion e iciency. J. Con ol. Release 2005,103, 643–653. [C ossRe ] [PubMed]
53.
Zheng, H.; Tang, C.; Yin, C. Explo ing ad an ages/disad an ages and imp o emen s in o e coming gene
deli e y ba ie s o amino acid modi ied ime hyla ed chi osan. Pha m. Res.
2015
,32, 2038–2050. [C ossRe ]
[PubMed]
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