nanoma e ials
A icle
Plasma Su ace Polyme ized and Bioma ke
Conjuga ed Bo on Ni ide Nanopa icles
o Cance -Speci ic The apy: Expe imen al
and Theo e ical S udy
Eliza e a S. Pe myako a 1, Liubo Yu. An ipina 1,2 , Philipp V. Ki yukhan se -Ko nee 1,
And ey M. Ko alskii 1, Jose Polˇcak 3,4 , An on Manakho 1, K is ina Yu. Gudz 1,
Pa el B. So okin 1and Dmi y V. Sh ansky 1,*
1Na ional Uni e si y o Science and Technology “MISIS”, Leninsky p ospec 4, 119049 Moscow, Russia;
[email p o ec ed] (E.S.P.); [email p o ec ed] (L.Y.A.);
[email p o ec ed] (P.V.K.-K.); [email p o ec ed] (A.M.K.);
[email p o ec ed] (A.M.); [email p o ec ed] (K.Y.G.); [email p o ec ed] (P.B.S.)
2Labo a o y o New Ma e ials Simula ion, FSBI Technological Ins i u e o Supe ha d and No el Ca bon
Ma e ials, 7a Tsen alnaya s ee , T oi sk, 108840 Moscow, Russia
3CEITEC-Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Technická3058/10,
61600 B no, Czech Republic; [email p o ec ed].cz
4Ins i u e o Physical Enginee ing, B no Uni e si y o Technology, Technicka 2896/2,
61669 B no, Czech Republic
*Co espondence: [email p o ec ed]; Tel.: +7-499-236-6629
Recei ed: 21 Oc obe 2019; Accep ed: 19 No embe 2019; Published: 21 No embe 2019


Abs ac :
A new low-p essu e plasma-based app oach o ac i a e he su ace o BN nanopa icles
(BNNPs) in o de o acili a e he a achmen o ola e acid (FA) molecules o cance -speci ic he apy
is desc ibed. Plasma ea men o BNNPs (BNNPs
PT
) was pe o med in a adio equency plasma
eac o using e hylene and ca bon dioxide monome s. The ca boxyl g oups deposi ed on he
su ace o BNNPs
PT
we e ac i a ed by N,N’-dicyclohexylca bodiimide (DCC) and pa icipa ed in
he condensa ion eac ion wi h e hylene diamine (EDA) o o m a hin amino-con aining laye
(EDA-BNNP
PT
). Then, he DCC-ac i a ed FA was co alen ly bonded wi h BNNPs
PT
by a chemical
eac ion be ween amino g oups o EDA-BNNPs
PT
and ca boxyl g oups o FA. Densi y unc ional
heo y calcula ions showed ha he p e-ac i a ion o FA by DCC is equi ed o g a ing o he
FA o he EDA-BNNPs
PT
. I was also demons a ed ha a e FA immobiliza ion, he elec onic
cha ac e is ics o he p e idine ing emain unchanged, indica ing ha he a ge ing p ope ies o he
FA/EDA-BNNPsPT nanohyb ids a e p ese ed.
Keywo ds: BN nanopa icles; chemical apo deposi ion; plasma su ace polyme iza ion; olic acid
conjuga es; d ug deli e y nanoca ie s; densi y unc ional heo y
1. In oduc ion
The cu en he apies a e no su icien o p o ide e ec i e ea men o di e en o ms o cance s.
Ta ge ed d ug deli e y (TDD) has become a widely used cance esea ch s a egy o sol e he main
p oblems ela ed o ad e se e ec s o chemo he apeu ic agen s on heal hy cells [
1
]. An impo an
addi ional ad an age o he TDD app oach is he abili y o c ea e he apeu ic sys ems wi h p olonged
ac ion [
2
]. Due o mode n nanoindus y achie emen s, a ious ypes o nanopa icles (NPs) we e
syn hesized and u ilized as p omising nanoca ie s o cance he apy. G a ing pa icula bioma ke s
Nanoma e ials 2019,9, 1658; doi:10.3390/nano9121658 www.mdpi.com/jou nal/nanoma e ials
Nanoma e ials 2019,9, 1658 2 o 14
ha a e o e exp essed speci ically on umo cells enables he a ge ed deli e y o he apeu ical agen s,
he eby minimizing oxic side e ec s o he whole body. Howe e , o de elop he apeu ically e ec i e
and biologically sa e ehicles o TDD i is necessa y o selec a a ge ing molecule, a he apeu ic agen ,
and a p omising chemically s able and biologically sa e suppo and hen ind a way o combine hem
in o a single sys em [3,4].
Plasma su ace unc ionaliza ion and polyme iza ion a e powe ul ools o NP su ace ac i a ion,
since he modi ica ion p ocess can be well con olled and eco- iendly [
5
]. Plasma ea men allows
he deposi ion o ul a hin plasma polyme using o ganic monome s ha do no polyme ize unde
con en ional chemical condi ions. In e ms o gas p essu e, all plasma p ocesses can be di ided
in o wo main g oups: low-p essu e (LP) plasma modi ica ion and non- he mal a mosphe ic-plasma
(AP) ea men [
6
]. Table 1compa es he s abili y o unc ional g oups o med a LP and AP
plasma ea men s.
Table 1.
S abili y o unc ional g oups o med a low-p essu e (LP) and a mosphe ic-plasma (AP)
plasma ea men s. XPS: X- ay pho oelec on spec oscopy.
No. Subs a e Gas G oups Plasma N/C and O/C
Ra io (XPS)
S abili y, H
(Thickness loss, %) Re
1 Si Cyclop opylamine
(CPA) NH2LP 0.24 (N/C) 20%, 48 h [7]
2 Si n-Hep ylamine NH2LP 0.12 (N/C) 15%, 24 h [8]
3 Ti Allylamine NH2LP 0.20 (N/C) 50%, 24 h [9,10]
4 Ti Ai /H2O2/TEOS
(Te ae hoxysilane) OH LP 0.58 (O/C) 100%, 12 h
(in boiling oluene) [11]
5 Polyme Allylamine Ac ylic
Acid NH2,
COOH LP 0.20 (N/C)
0.64 (O/C) - [12]
6 Polyme O2OH,
COOH AP 0.09 (O/C) 55%, 24 h [13]
7 Polyme Ai OH,
COOH AP 0.43 (O/C) - [14]
8 Polyme Ai OH,
COOH AP 0.68 (O/C) - [15]
9 Si CO2/C2H4/A OH,
COOH LP 0.45 (O/C) 39%, 24 h (high
s abili y o COOH
g oups) [16]
10 Si Maleic Anhyd ide
Vinyl ime hoxysilane
OH,
COOH AP 0.20 (O/C) 75%, 20 h [17]
Oxygen-plasma ea men is equen lyused o emo e ca bon con amina ion om heNPsu aces.
The con e sion o hyd oca bons in o ola ile compounds, such as CO, CO
2
, and H
2
O, allows one o
emo e hem by pumping and o c ea e he app op ia e condi ions o enhanced adhesion be ween he
deposi ed polyme ilm and he NP su ace [
18
]. In o de o p oduce plasma polyme ilm con aining
high concen a ions o amino unc ional g oups, plasma polyme iza ion o cyclop opylamine (CPA)
was pe o med. The monome selec ion was ca ied ou based on a ailable li e a u e da a, which ha e
shown high s abili y and eac i i y o he CPA ilms. Recen ly, LP plasma polyme iza ion o CPA
o unc ionalize ZnO, Al
2
O
3
, and Z O
2
NPs has been success ully es ed [
19
]. TEM images clea ly
demons a ed ha a hin (5 nm) plasma polyme ilm was o med on he su ace o indi idual NPs,
while he emaining NPs emained uncoa ed.
Bo h LP and AP plasmas we e used o ac i a e he polye hylene su aces [
20
]. The in luence o
plasma p ocess pa ame e s, such as ope a ing equencies (40 kHz and 13.56 MHz a LP) and dielec ic
ba ie discha ge (50 Hz a AP), on he polyme su ace ac i a ion we e compa ed. A signi ican
inc ease in su ace ene gy and imp o ed we abili y was obse ed a e LP plasma p ocessing a
40 kHz, whe eas AP plasma ea men was less e ec i e owa d su ace ac i a ion a he same powe
couplings. Compa ing he wo me hods, i can be no ed ha he absence o a acuum sys em is he
main ad an age o he non- he mal AP plasma ea men , whe eas LP plasma p ocessing usually leads
o he o ma ion o mo e s able ilms con aining a highe amoun o unc ional g oups on hei su ace.
Due o high chemical s abili y [
21
], di e en ino ganic NPs, such as mesopo ous silica [
22
], i on
oxide [
23
], hexagonal bo on ni ide (h-BN) [
24
], and ca bon nano ubes [
25
], ha e been de eloped
Nanoma e ials 2019,9, 1658 3 o 14
and es ed in NP-based cance he apy. Among hem, h-BN nanopa icles (BNNPs) a e o pa icula
in e es due o hei excellen biocompa ibili y [
26
], high d ug loading capaci y [
27
], and he apeu ic
e icacy owa d umo cells wi h mul iple d ug esis ance [28].
Sil e NPs we e p ecipi a ed on he su ace o BNNPs o p o ide coupling o olic acid (FA) wi h
BNNPs [
29
]. The sho comings o his s a egy include di icul ies in ob aining a uni o m Ag NP size
and hei uni o m dis ibu ion on he ca ie su ace. As a esul , he FA-coa ed su ace a ea was
ela i ely small. He ein, we desc ibe a new LP plasma-based app oach o ac i a e he su ace o BNNPs
o acili a e he a achmen o ola es, which a e ecognized as bioma ke s o a ge ed chemo he apeu ic
d ug deli e y o cance cells [
30
,
31
]. BNNPs we e ob ained by a chemical apo deposi ion (CVD)
p ocess (S ep (i)). Ca boxy-con aining plasma polyme was deposi ed on he su ace o BNNPs
(he ea e e e ed o as BNNPs
PT
) by means o a adio equency plasma eac o using e hylene
and ca bon dioxide monome s (S ep (ii)). FA molecules we e hen conjuga ed o BNNPs in h ee
consecu i e s ages (labeled as S eps (iii)–( )): (iii) FA p eac i a ion by N,N
0
-dicyclohexylca bodiimide
(DCC); (i ) NH
2
- unc ionaliza ion o polyme -coa ed BNNPs wi h e hylenediamine, EDA (he ea e
designa ed as EDA-BNNPs
PT
); and ( ) inal conjuga ion o DCC-ac i a ed FA o modi ied BNNPs
(he ea e abb e ia ed as FA/EDA-BNNPs
PT
). Densi y unc ional heo y (DFT) calcula ions we e
ca ied ou o unco e he FA/EDA-BNNPs
PT
chemical bonding mechanism. Fo ha , a de ailed
ene ge ic analysis o he a omic s uc u e and s abili y o he FA/EDA-BNNPs
PT
sys em was pe o med.
2. Ma e ials and Me hods
2.1. Ma e ials
The ollowing eagen s we e used: FA and EDA (PanReac AppliChem, Da ms ad , Ge many); DCC
and dime hyl sul oxide, DMSO (P ime Chemical G oup, Moscow, Russia); bo on powde (AVIABOR,
Dze zhinsk, Russia); ammonia solu ion, dichlo ome hane, o mic acid, and ace oni ile (Cupa na eac i e).
2.2. Syn hesis o BNNPs
BNNPs we e syn hesized in a bo on oxide CVD p ocess using a e ical induc ion-hea ing u nace,
ope a ing a 41.4 kHz [28]. The u nace consis ed o a qua z cylind ical chambe , g aphi e suscep o ,
BN-based ce amic eac o , gas supply, and exhaus gas sys ems. The BN c ucible wi h a p ecu so was
placed a he bo om o eac o abo e he a gon inle . P ecu so powde mix u e o B (>99%), MgO
(analy ical g ade), annealed in ai a 450
◦
C o 1 h, and FeO (pu e) aken in a mola a io o 3.5:0.1:1
was used as a sou ce o bo on oxide apo . Syn hesis was ca ied ou unde he empe a u e g adien
along he heigh o eac o om 1430
◦
C in he p ecu so zone o 700–750
◦
C in he BNNPs ou le zone.
The ammonia and a gon gas lows we e con olled a 100 and 500 cm
3
/min, espec i ely. A e he
syn hesis o 8 h, a hick whi e-colo ed deposi was obse ed in he collec ing c ucible and on he inne
su ace o he eac o .
2.3. Plasma Su ace Polyme iza ion o BNNPs
The deposi ion o ca boxy-con aining plasma polyme was ca ied ou using a UVN-2M acuum
sys em e acua ed o a p essu e below 5
×
10
−3
Pa. The capaci i ely coupled adio- equency (RF)
plasma was gene a ed by a Ci o1310-ACNA-N37A-FF (Come ) RF powe supply uni coupled wi h
a RFPG-128 plasma gene a o (Beams&Plasmas). High- equency powe (500 W, 13.56 MHz) was
supplied in a pulsed mode (du y cycle 5%, pulse du a ion 2 ms). The deposi ion ime was 10 min.
A (99.998%), CO
2
(99.995%), and C
2
H
4
(99.95%) gases we e used as p ecu so s. The gas low a es
we e se a 0.4, 2.5, and 3.5 sccm o C
2
H
4
, CO
2
, and A , espec i ely. Gas low was con olled using a
Mul i Gas Con olle 647C (MKS). Wo king and esidual gas p essu es we e measu ed by a VMB-14
uni (Tokamak Company) and a D395-90-000 BOC Edwa ds con olle . BNNP suspension in isop opyl
alcohol (4 mg/mL) was sonica ed o 10 min. Then, 30 mL o he BNNP suspension was applied o
he glass su ace and d ied. The dis ance be ween he RF-elec ode and he subs a e was 8 cm. A e
Nanoma e ials 2019,9, 1658 4 o 14
deposi ion o he ca boxy-con aining polyme , he BNNPs we e washed o he glass subs a e o
s uc u al cha ac e iza ion. To analyze he elemen al composi ion and hickness o plasma polyme ,
he plasma modi ied BNNPs we e cha ac e ized by Fou ie - ans o m in a ed (FTIR) spec oscopy
and X- ay pho oelec on spec oscopy (XPS).
2.4. NH2-Func ionaliza ion o Ca boxy-Modi ied BNNPs
NH
2
- unc ionaliza ion o he BNNPs
PT
was ca ied ou acco ding o he me hod desc ibed
elsewhe e [
32
]. B ie ly, 40 mg o BNNPs
PT
was added in o 10 mL o DMSO a e which he mix u e was
dispe sed o 15 min. Then, 5 mg o DCC was added o he solu ion and s i ed a oom empe a u e
o 10 min. Finally, 10
µ
L o amino-con aining compound (EDA) was added o he eac ion mix u e and
s i ed a oom empe a u e o 2 h. The pa icles we e insed h ee imes in dis illed wa e and d ied.
The NH2- unc ionalized BNNPs (EDA-BNNPsPT) we e cha ac e ized by XPS and FTIR spec oscopy.
2.5. Conjuga ion o FA o EDA-BNNPsPT
Ten millig ams o FA we e dissol ed in 10 mL o DMSO and hen 2 mg o DCC was added.
The mix u e was added o a p e-dispe sed solu ion o EDA-BNNPs
PT
in 10 mL o me hylene chlo ide
(CH
2
Cl
2
). The eac ion mix u e was s i ed a oom empe a u e o 24 h, hen he FA/EDA-BNNPs
PT
we e insed in dis illed wa e and d ied. The FA/EDA-BNNPs
PT
powde samples we e cha ac e ized by
XPS, FTIR spec oscopy, and Fou ie - ans o m ion cyclo on esonance mass spec ome y (FT ICR MS).
2.6. Ma e ial Cha ac e iza ion
The as-syn hesized BNNPs and hei conjuga es we e cha ac e ized using a scanning elec on
mic oscope JSM-7600F (JEOL) equipped wi h he ene gy-dispe si e X- ay (EDX) de ec o and a
ansmission elec on mic oscope JEM 2100 (JEOL). Chemical and phase composi ions we e s udied by
means o EDX spec oscopy using an 80 mm
2
X-Max EDX de ec o (Ox o d Ins umen s) and FTIR
spec oscopy wi h a Ve ex 70 acuum spec ome e (B uke ) in he ange 400
−
4000 cm
−1
. XPS spec a
o he BNNPs, BNNPs
PT
, EDA-BNNPs
PT
, and FA/EDA-BNNPs
PT
samples we e eco ded on an Axis
Sup a ins umen (K a os Analy ical L d., Manches e , UK) equipped wi h a monoch oma ic Al K
α
X- ay sou ce (h
υ
=1486.6 eV). The pass ene gy and X- ay beam cu en we e se o 40 eV and 15 mA,
espec i ely. The acqui ed spec a we e i ed using CasaXPS so wa e as desc ibed
elsewhe e [16,17].
The s uc u e o FA/EDA-BNNPs
PT
was addi ionally s udied by FT ICR MS. To de e mine he molecula
o mula and ion ype, ChemCalc so wa e was used [
33
]. The FA/EDA-BNNPs
PT
nanohyb ids we e
added in o he mix u e o wa e
−
ace oni ile (50/50) wi h 0.1% o mic acid and hen cen i uged.
The supe na an s we e analyzed by FT ICR MS using an Apex Ul a ins umen (B uke ) wi h
elec osp ay ioniza ion a ionizing po en ial on a capilla y o 4 kV. Ze a po en ials we e measu ed
using a Ze asize Nano ZS sys em (Mal e n Ins umen s).
2.7. A omis ic Simula ions
The a omic s uc u e and s abili y o he FA conjuga ed o BNNP we e analyzed using densi y
unc ional heo y (DFT) [
34
,
35
]. The gene alized g adien app oxima ion (GGA) using he no malized
T oullie –Ma ins pseudopo en ials [
36
] in he SIESTA so wa e package was applied [
37
–
39
]. As a
basis o a omic localized o bi als, nume ical pseudoa omic wa e unc ions we e used. To neglec
in e molecula in e ac ions, he sys em was modelled as a supe cell wi h he su icien ly la ge space
be ween slabs (
≥
15 Å). The geome y o s uc u es was op imized un il esidual o ces became less
han 0.03 eV/Å. The eal-space mesh cu o was se o a leas 175 Ry. The Monkho s –Pack [
40
] special
k-poin scheme was used wi h k-g id cu o equaled o 6 and 24 Å o geome y elaxa ion and elec onic
s uc u e calcula ions, espec i ely.
Nanoma e ials 2019,9, 1658 5 o 14
3. Resul s and Discussion
3.1. P epa a ion and SEM Cha ac e iza ion o FA-BNNPs Conjuga es
Mic os uc u es o as-syn hesized and plasma polyme ized BNNPs a e depic ed in Figu e 1.
As- ab ica ed BNNPs ha e an almos sphe ical shape and size o 150
−
250 nm. The BNNP su ace is
o med by nume ous h-BN nanoshee s, as shown in Figu e 1a (inse ). A e he plasma ea men ,
he BNNPs
PT
we e co e ed by a hin laye o plasma polyme ha signi ican ly changes he BNNP
mo phology. The diame e o BNNPs
PT
sligh ly inc eased o 170–270 nm, he eby sugges ing ha he
hickness o he polyme laye is abou 10 nm.
Figu e 1.
SEM (
a
,
b
) and TEM (inse in (
a
)) images o BN nanopa icles (BNNPs) and hei conjuga es.
Figu e 2illus a es i e main s ages o he FA/EDA-BNNP
PT
conjuga e ab ica ion: (i) BNNP
syn hesis, (ii) hei plasma polyme iza ion, (iii) FA ac i a ion, (i ) ab ica ion o EDA-BNNPs
PT
,
and ( ) conjuga ion
o he p e-ac i a ed FA o he EDA-BNNPs
PT
, as well as he chemical s uc u es o
he modi ied BNNPs. The ca boxyl g oups deposi ed on he su ace o BNNPs by plasma ea men
(ii) a e ac i a ed by DCC and pa icipa e in he condensa ion eac ion wi h EDA o o m a hin
amino-con aining laye on he BNNP su ace (i ). Then, he ca boxyl-g oups o FA we e p eac i a ed
by DCC (iii) and he FA was g a ed o he su ace o BNNPs
PT
h ough chemical in e ac ion be ween
amino g oups o EDA-BNNPsPT and ca boxyl g oups o DCC-ac i a ed FA ( ).
Figu e 2.
Scheme o BNNP ab ica ion and subsequen modi ica ion: BNNP syn hesis using he eac ion
o bo on oxide apo wi h ammonia (
i
), plasma polyme iza ion (
ii
), ola e acid (FA) ac i a ion using
DCC (iii), ab ica ion o EDA-BNNPsPT (i ), and FA/EDA-BNNPsPT ( ) complexes.

Nanoma e ials 2019,9, 1658 6 o 14
3.2. FTIR Spec oscopy
The FTIR spec a o he BNNPs
PT
, EDA-BNNPs
PT
, and FA/EDA-BNNPs
PT
nanohyb ids a e
p esen ed in Figu e 3. The BNNPs sample [
27
] (no shown) e eals a s ong asymme ic band a
app oxima ely 1354 cm
−1
esul ing om he B
−
N s e ching ib a ion, and a less in ense band a
772 cm
−1
associa ed wi h he B
−
N
−
B bending ib a ion [
41
]. The FTIR spec um o CO
2
/C
2
H
4
plasma
polyme (BNNPs
PT
spec um in Figu e 3) shows se e al b oad peaks loca ed in he anges o 3590–3340
(-OH g oups), 3030–2780 (hyd oca bon), and 1820–1650 cm
−1
(ca boxyl/es e g oups). In addi ion,
ew peaks obse ed a 1096, 965, and 900 cm−1we e assigned o C-C-(O)-C (es e s), C-OH s e ching,
and C=C- bending ib a ions, espec i ely. A e NH
2
- unc ionaliza ion o BNNPs
PT
wi h EDA, new
sha p peaks a 3316, 1627, and 1266 cm
−1
appea ed, which we e assigned o he amino g oups, C=O
amide, and C-N s e ch molecula mo ion, espec i ely. A e he g a ing o FA, he in ensi y o peak
a 1096 cm
−1
(C-C-(O)-C g oups) no iceably inc eased and addi ional peaks a 2961 cm
−1
(C-H
3
) and
1020 cm
−1
(C-OH s e ching) we e obse ed. The p onounced peak a 1096 cm
−1
can be explained
by he con ibu ion o C-C bending om olic acid [
42
–
44
]. To ob ain an ac i a ed FA de i a i e
(p oduc B), ca bodiimide was added o olic acid (s age 3 in Figu e 2). Since he FA con ains an amino
g oup, polyme iza ion o FA may occu due o he in e ac ion o he ac i a ed ca boxyl g oup wi h he
ee amino g oup esul ing in he o ma ion o a pep ide bond. Thus, no a single ola e molecule is
a ached, bu a whole chain, which leads o an inc ease in he ola e peak in ensi ies, and, in pa icula ,
he C-C s e ching band.
Figu e 3. FTIR spec a o unc ionalized BNNPs.
3.3. XPS Spec oscopy
Elemen al composi ion and chemical bonds o he BNNPs
PT
, EDA-BNNPs
PT
,
and FA/EDA-BNNPs
PT
nanohyb ids we e s udied by XPS. High- esolu ion XPS B 1s, C 1s,
and N 1s spec a a e illus a ed in Figu e 4. The XPS B 1s spec a o he EDA-BNNPs
PT
and
FA/EDA-BNNPs
PT
samples we e decon olu ed in o wo peaks loca ed a 190.7 and 192.9 eV,
co esponding o B-N and B
2
O
3
, espec i ely. Mo eo e , he XPS B 1s spec um o he BNNPs
PT
sample
Nanoma e ials 2019,9, 1658 7 o 14
had an addi ional peak loca ed a 191.5 eV, co esponding o he O-B-N bonds. The high- esolu ion
XPS C 1s spec um o he BNNPs
PT
sample was decon olu ed in o ou peaks posi ioned a 285.0,
286.4, 288.1,
and 288.9 eV
, which could be a ibu ed o CH
x
, C-O/C
−
N, C=O/N-C=O-, and C(O)O,
espec i ely. The C(O)O componen was no obse ed a e u he NH
2
- unc ionaliza ion o BNNPs
PT
wi h EDA and inal conjuga ion o FA o he EDA-BNNPs
PT
su ace. The XPS N 1s spec um o he
BNNPs
PT
was esol ed in o wo main componen s a ibu ed o B-N (398.3 eV) and O-B-N/C-N
(398.9 eV). In he XPS N 1s spec um o he EDA-BNNPs
PT
sample, addi ional ea u es can be seen a
399.6 and 401.8 eV due o he con ibu ion om amide N-C=O bonds and p o ona ed ee amino
g oups appea ing om EDA. In he XPS N 1s spec um o he FA-BNNPs sample, he N-C=O peak
shi s o lowe binding ene gy (BE) a 399.8 eV and gained i s in ensi y. On he con a y, he in ensi y
o he NH
3+
peak was obse ed o signi ican ly dec ease. This indica es he con ibu ion om
C=N-moie ies ypical o he ola e s uc u e. Thus, he XPS esul s clea ly demons a e he chemical
bond o he FA molecules wi h he EDA-BNNPsPT su ace h ough amide linkage.
Figu e 4.
High- esolu ion XPS B 1s (
A
–
C
), C 1s (
D
–
F
), and N 1s (
G
–
I
) spec a o BNNPs
PT
(
A,D,G
),
EDA-BNNPsPT (B,E,H), and FA/EDA-BNNPsPT (C,F,I) samples. BE: binding ene gy.
The g a ing o EDA and FA o he su ace o BNNPs
PT
was quan i a i ely analyzed by calcula ing
he N/B a io. The N/B alue in he BNNPs
PT
de e mined om hei XPS spec a is equal o 0.77,
as shown in Table 2. The N/B a io inc eased o 0.9 a e BNNP su ace ic ionaliza ion and u he o
1.01 a e subsequen FA c oss-linkage. This indica es g a ing e hylenediamine (EDA-BNNPs
PT
) and
olic acid (FA/EDA-BNNPs
PT
) molecules. The addi ional inc ease in ni ogen con en a e EDA and
FA modi ica ions was calcula ed acco ding o he ollowing equa ions.
∆NEDA−BNNPsPT ="NEDA−BNNPsPT
BEDA−BNNPsPT −NBNNPsPT
BBNNPsPT #×[B]EDA−BNNPsPT (1)
Nanoma e ials 2019,9, 1658 8 o 14
∆NFA/EDA−BNNPsPT ="NFA/EDA−BNNPsPT
BFA/EDA−BNNPsPT −NEDA−BNNPsPT
BEDA−BNNPsPT #×[B]FA/EDA−BNNPsPT (2)
The ob ained
∆
N alues a e shown in Table 2. The o al a omic concen a ion o C, O, and N
a oms in he FA molecule g a ed o he BNNPs (he ea e deno ed as C
FA
) was es ima ed assuming
ha he e was no hyd ogen in he BNNPs (acco ding o XPS da a) and he FA has he chemical
o mula C
19
H
19
N
7
O
6
. Taking in o accoun he numbe o N a oms in one FA molecule (C
19
H
19
N
7
O
6
),
he CFA =3.8 a .%
alue was ob ained by di iding he
∆
N by 7 and mul iplying his alue by he
o al numbe o C (19), O (6), and N (7) a oms in one FA molecule. No e ha addi ional C 1s and
O 1s signals om bo h BNNP su ace con amina ions and BNNP-suppo ed ca bon ape can a ec
he esul s o quan i a i e XPS analysis. Thus, he ob ained C
FA
alues we e no malized o he o al
concen a ion o B and N a oms in BNNPs assuming equal con en s o B and N in BN (deno ed as
C
BN
) using he ollowing equa ion: C
FA
/C
BN
=3.8/7.5 =0.51, whe e he C
BN
alue was de e mined as
CBN =2[B] =7.5
. The ob ained esul indica es ha FA molecules occupy app oxima ely 51% o he
BN su ace. Thus, ou new LP plasma-based app oach p o ides a la ge su ace a ea co e ed wi h FA
molecules compa ed wi h BNNPs deco a ed wi h Ag NPs [29].
Table 2. Resul s o XPS analysis.
Sample Concen a ion, a % N/B∆N
B C N O
BNNPsPT 33.3 16.8 25.6 24.3 0.77
EDA-BNNPsPT 21.5 34.2 16.8 27.5 0.78 0.27
FA/EDA-BNNPsPT 3.7 71.0 3.8 21.5 1.01 0.86
3.4. Ze a Po en ial
As-syn hesized BNNPs ha e a nega i e su ace cha ge (
−
30 mV) allowing he o ma ion o s able
suspensions, as shown in Figu e 5. A e plasma polyme iza ion, he nega i e su ace cha ge o
he BNNPs
PT
con aining nega i ely cha ged ca boxy-g oups was obse ed o inc ease o
−
37 mV.
Posi i ely cha ged amino g oups o EDA inc eased he ze a po en ial alue o he EDA-BNNPs
PT
conjuga es up o
−
27 mV. A u he shi o he su ace cha ge owa ds a mo e nega i e alue (
−
32.6 eV)
indica es ha he FA was success ully g a ed o he EDA-BNNPsPT conjuga es.
Figu e 5. Ze a po en ials o p is ine and su ace- unc ionalized BNNPs.
Nanoma e ials 2019,9, 1658 9 o 14
3.5. Fou ie -T ans o m Ion Cyclo on Resonance Mass Spec ome y
The g a ing o FA o he EDA-BNNPs
PT
nanopa icles was addi ionally s udied by a FT ICR
MS me hod. Since BN nanoca ie s a e oo hea y o FT ICR MS analysis, he FA/EDA-BNNPs
PT
sample was i s ea ed wi h o mic acid o clea e he pep ide bonds in he conjuga es, a e which
he decomposed p oduc s we e analyzed by FT ICR MS, as shown in Figu e 6. Expe imen ally
ob ained mass o cha ge (m/z) alues ( igh column in Table 3) we e used o de e mine he main
molecula ion ypes using a chemical Web se ice [
33
]. Besides peaks om he side p oduc o
N,N
0
-dicyclohexylu ea, he cha ac e is ic FA peaks obse ed a 442.147 and 313.389 m/z and he peak
a 148.151 m/z assigned o he glu amic acid pa o FA clea ly indica e ha he FA was success ully
conjuga ed o he BNNP su ace.
Figu e 6.
Fou ie - ans o m ion cyclo on esonance mass spec ome y (FT ICR MS) spec um o
o mic acid ea ed FA/EDA-BNNPsPT sample.
Table 3. Main ions de ec ed by FT ICR MS.
Compound Fo mula Molecula Ion Type m/z Theo e ical m/z Expe imen al
N,N0-dicyclohexylu ea C13H24N2O [M2+H]+449.385 449.385
Folic acid C19H19N7O6[M +H]+442.138 442.147
Pa o FA C14H12N6O3[M +H]+313.297 313.389
N,N0-dicyclohexylu ea C13H24N2O [M +H]+225.196 225.196
Glu amic acid C5H9NO4[M +H]+148.146 148.151
3.6. Simula ion o FA/EDA-BNNPsPT Nanohyb ids
To unde s and how he FA g a s o he EDA-BNNPs
PT
conjuga es, de ailed heo e ical analysis
was pe o med using a omis ic simula ions by conside ing in e ac ion o he FA con aining a ious
ca boxyl g oups wi h he BN su ace. The expe imen al da a indica e ha he BNNP has a size o mo e
han 10
2
nm, which allows us o accep he assump ion ha i s su ace is la . Since he elec on ans e
be ween he h-BN laye s is almos absen , only a ew a omic planes in he h-BN shee we e conside ed.
This allows us o apply pe iodic bounda y condi ions and o conside a ela i ely small supe cell in he
calcula ions. The binding ene gies (BEs) we e calcula ed du ing ac i a ion o FA wi h DCC, as well as
a each s ep o BNNPsPT modi ica ion wi h DCC, EDA, and DCC-ac i a ed FA.
Du ing plasma su ace polyme iza ion, he –CH
2
-COOH g oups we e deposi ed on he BNNP
su ace. The high esis i i y o he B–N
π
sys em o adso p ion leads o ema kable chemical s abili y
o he pe ec h-BN su ace, he e o e ca bon g oups will mos ly bind wi h su ace opological de ec s,
such as B o N acancies. The BNNPs
PT
con aining COOH g oups we e NH
2
- unc ionalized wi h EDA