molecules
A icle
Cy o oxic Plan Ex ac s owa ds Insec Cells:
Bioac i i y and Nanoencapsula ion S udies o
Applica ion as Biopes icides
Ana I. F. Lopes 1,2, Ma iana Mon ei o 1,2, Ana R. L. A aújo 1, Ana Ri a O. Rod igues 2,
Elisabe e M. S. Cas anhei a 2, Da id M. Pe ei a 3, Ped o Olim 3, A. Gil Fo es 1
and M. Samei o T. Gonçal es 1,*
1Cen e o Chemis y, Depa men o Chemis y, Campus o Gual a , Uni e si y o Minho,
4710-057 B aga, Po ugal; [email p o ec ed] (A.I.F.L.); [email p o ec ed] (M.M.);
[email p o ec ed] (A.R.L.A.); [email p o ec ed] (A.G.F.)
2Cen e o Physics, Depa men o Physics, Campus o Gual a , Uni e si y o Minho,
4710-057 B aga, Po ugal; [email p o ec ed] (A.R.O.R.); [email p o ec ed] (E.M.S.C.)
3REQUIMTE/LAQV, Labo a o y o Pha macognosy, Depa men o Chemis y, Facul y o Pha macy,
Uni e si y o Po o, R. Jo ge Vi e bo Fe ei a, 228, 4050-313 Po o, Po ugal; dpe ei a@ .up.p (D.M.P.);
up201706671@ .up.p (P.O.)
*Co espondence: [email p o ec ed]; Tel.: +351253604372
Recei ed: 12 No embe 2020; Accep ed: 8 Decembe 2020; Published: 11 Decembe 2020


Abs ac :
The po en ial o plan ex ac s as bioinsec icides has been desc ibed as a p omising
ield o ag icul u al de elopmen . In his wo k, he ex ac s o Punica g ana um (pomeg ana e),
Phy olacca ame icana (Ame ican pokeweed), Glando a p os a a (sh ubby g omwell), Ulex eu opaeus
(go ce), Tage es pa ula (F ench ma igold), Camellia japonica ed (camellia), Ru a g a eolens ( ue o
he b-o -g ace) we e ob ained, pu i ied, and hei ac i i y agains Spodop e a ugipe da (S 9) insec cells
was in es iga ed. F om he pool o o e wen y ex ac s ob ained, comp ising di e en pola i ies
and ege able ma e ials, less pola samples we e shown o be mo e oxic owa ds he insec cell
line S 9. Among hese, a dichlo ome hane ex ac o R. g a eolens was capable o causing a loss o
iabili y o o e 50%, exceeding he e ec o he comme cial insec icide chlo py i os. This ex ac
elici ed ch oma in condensa ion and he agmen a ion in ea ed cells. Nanoencapsula ion assays
o he cy o oxic plan ex ac s in soybean liposomes and chi osan nanos uc u es we e ca ied ou .
The nanosys ems exhibi ed sizes lowe o a ound 200 nm, low polydispe si y, and gene ally high
encapsula ion e iciencies. Release assays showed ha chi osan nanoemulsions p o ide a as and o al
ex ac elease, while liposome-based sys ems a e sui able o a mo e delayed elease. These esul s
ep esen a p oo -o -concep o he u u e de elopmen o bioinsec icide nano o mula ions based on
he cy o oxic plan ex ac s.
Keywo ds: biopes icides; bioinsec icides; Ru a g a eolens; chlo py i os; nanoencapsula ion
1. In oduc ion
Many syn he ic chemicals ha e been used in plan p o ec ion agains ag oindus ial and medicinal
pes s o he las 60 yea s [
1
–
3
]. This has caused a s ong nega i e impac a di e en le els, and he e
a e se ious conce ns abou hei side e ec s, such as he p esence o esidues on ood and d inking
wa e , oxici y o humans as well as o o he non- a ge o ganisms, and high pe sis ence, leading o
an eme gence o pes esis ance. These ac s ha e demanded o a d as ic dec ease in he use o
chemical pes icides and opening up space o new and sa e al e na i e s a egies [
1
]. The use o
na u al compounds o con ol weeds has long been accep ed as an en i onmen iendly s a egy,
Molecules 2020,25, 5855; doi:10.3390/molecules25245855 www.mdpi.com/jou nal/molecules
Molecules 2020,25, 5855 2 o 14
bu a me s ha e mos ly elied on oxic syn he ic ag ochemicals. New ools in pes managemen a e
based on biopes icides, which include semiochemicals and plan -inco po a ed p o ec an s, bo anical
and mic obial de i ed chemicals, and also new syn he ic analogues [
1
]. The majo p oblems in o ganic
ag icul u e aced by a me s a e weed managemen , c op diseases and insec con olling ools. Due o
he di e si y o chemicals p esen , na u al p oduc s ha e become impo an sou ces o mos biological
ac i i y op imiza ion, also playing also an impo an ole in d ug disco e y [
4
]. In ecen yea s,
esea ch has been ocused on he pes icidal e iciency o many phy ocompounds, om essen ial oils
o mo e complex mix u es ex ac ed om di e en plan s [
5
,
6
]. Compa ed o syn he ic pes icides,
phy ochemical biopes icides p esen lowe oxici y, a e less pe sis en , and biodeg adable [
1
,
7
].
Phy ochemical biopes icides, such as nico ine, o enone, py e h ins and sabadilla alkaloids, a e some
examples o he i s se ies o bo anical pes icides used. Howe e , due o ei he hei s abili y o
oxici y and ad e se e ec s on human heal h, hei use has been es ic ed, and despi e an inc easing
applica ion, hese emain less han 5% o he o al pes icide use [8].
Phy ochemical biopes icides comp ise a se ies o seconda y me aboli es ha ac in a syne gis ic
way, which s ongly educes he chances o insec pes s o de elop esis ance. Li e a u e has co e ed
in de ail many plan species and hei use as biopes icides.
Punica g ana um L. 1753, Phy olacca ame icana L. 1753, Glando a p os a a Loisel, 1844, Ulex eu opaeus
L. 1753, Tage es pa ula L. 1753, Camellia japonica L. 1753 and Ru a g a eolens L. 1753 displayed a huge ange
o biological ac i i ies [
3
,
9
–
19
], including an ibac e ial (e.g., P. g ana um) [
9
], an i i al (e.g., P. g ana um,
C. japonica) [
10
,
11
], an i ungal (e.g., P. g ana um,T. pa ula,C. japonica) [
12
], nema icide (T. pa ula) [
13
],
he bicide (U. eu opaeus) and insec icide (e.g., T. pa ula and R. g a eolens) [14–19].
The chemical composi ion and bioac i i y o R. g a eolens lea es we e s udied and es ed agains
he maize wee il. The insec icidal and epellen p ope ies o R. g a eolens we e analyzed and e alua ed
in con ac and apo o ms, wi h a o al o 45 compounds de ec ed by ch oma og aphic echniques,
mos o which we e 2-ke ones. The esea ch ca ied ou o da e suppo s he e idence ha R. g a eolens
lea es con ain bioac i e seconda y plan me aboli es, which jus i y i s use agains S.zeamais in s o age
pes managemen p og ams [
20
]. Lee e al. isola ed essen ial oils om he lowe s and lea es o
R. g a eolens, which we e e alua ed using umigan and con ac oxici y bioassays agains s o e ood
pes s, suppo ing he eco-po en ial o using R. g a eolens essen ial oil and i s majo cons i uen s in he
managemen o s o ed pes s [21,22].
Despi e he epo ed ac i i y agains pes s, se e al plan compounds exhibi ed some limi a ions in
hei applica ion, due o hei easy deg ada ion and high ola ili y. Nanoencapsula ion echniques a ise
as sui able s a egies o allow o he p ese a ion and con olled elease o plan componen s [
23
,
24
].
Speci ically, lipid-based nanosys ems ha e been widely used as ehicles o bioac i es in cosme ic and
pha maceu ical indus ies [
25
,
26
], as well as o he encapsula ion o plan ex ac s wi h insec icidal
ac i i y [
27
,
28
], being a p omising ou e o a sa e applica ion o pes icides [
29
]. Plan ex ac s ha e also
been encapsula ed in cyclodex ins [
24
] and polyme ic nanopa icles, he la e including algina e [
30
],
gum a abic/mal odex in [
31
], gela in [
32
], poly inylalcohol (PVA) [
33
], poly(dl-lac ide-co-glycolide)
(PLGA) [34] and chi osan nanos uc u es [35,36].
In his wo k, ex ac s om se en plan species, P. g ana um (pomeg ana e), P. ame icana
(Ame ican pokeweed), G. p os a a (sh ubby g omwell), U. eu opaeus (go ce), T. pa ula (F ench ma igold),
C. japonica (camellia) and R. g a eolens ( ue o he b-o -g ace), we e isola ed using wa e /e hanol and
dichlo ome hane as sol en s. The ob ained ex ac s we e e alua ed as po en ial insec icides, h ough he
assessmen o hei biological ac i i y agains S 9 insec cell lines, compa ed wi h a comme cial syn he ic
pes icide. Conside ing he insec icidal ac i i y exhibi ed by he R. g a eolens dichlo ome hane ex ac ,
encapsula ion assays in chi osan nanoemulsions and lipid nanosys ems we e ca ied ou , o he u u e
de elopmen o g een insec icide nano o mula ions.
Molecules 2020,25, 5855 3 o 14
2. Resul s and Discussion
2.1. Plan Ex ac s
A se ies o se en plan species we e selec ed, aking in o accoun hei p oduc ion in he coun y,
endemic cha ac e is ics, he in o ma ion o li e a u e, as well as hei unexploi ed po en ial in he ield
o bioinsec icides. A e he ha es , he pa s o plan s/ ui s ha we e in ended o be s udied we e
sepa a ed, namely ui peel in he case o P. g ana um (pomeg ana e); be ies in he case o P. ame icana
(Ame icanpokeweed); lowe sandlea esin hecaseo G.p os a a(sh ubbyg omwell),U.eu opaeus(go ce),
R. g a eolens ( ue o he b-o -g ace), and T. pa ula (F ench ma igold)—in he o me , lowe s (sepa a ed om
he ecep acle) and lea es we e ex ac ed sepa a ely—and lea es in he case o C. japonica.
The p epa a ion o he plan ma e ial included d ying unde sunligh (20–25
◦
C, 45 days) (peel o
P. G ana um), he lyophiliza ion (be ies o P. ame icana, lea es and lowe s o T. pa ula), o d ying
in an o en (40–45
◦
C, 3 days), ollowed in bo h cases by g ounding wi h a sh edde o ob ain he
co esponding powde , which was passed h ough a sie e un il a pa icle size
≤900 µm
was ob ained.
Fu he mo e, hese ma e ials we e subjec ed o Soxhle ex ac ion wi h sol en sys ems o di e en
pola i ies, namely in a pola sys em, using a wa e /e hanol solu ion (1:1), and in a less pola one,
using dichlo ome hane, o 9 o 4 h, espec i ely. The sol en s we e emo ed by e apo a ion unde
educed p essu e (dichlo ome hane ex ac s) ollowed by lyophiliza ion (aqueous e hanolic ex ac s).
In o de o emo e he chlo ophyll con en , dichlo ome hane ex ac s we e passed h ough a Ch omabond
C18 column, using me hanol as eluen , ollowed by sol en e apo a ion. The 22 ex ac s hus ob ained
(11 in wa e /e hanol and 11 in dichlo ome hane, Table 1) we e submi ed o assays o cell iabili y using
he insec cell line Spodop e a ugipe da (S 9), o assess hei capaci y as po en ial bioinsec icides.
Table 1.
Ex ac s o a ious species ob ained using wo di e en sol en sys ems and s udied agains S 9.
En y Specie Pa o he Plan Sol en Code
1Phy olacca ame icana Lea es DCM A1
2Phy olacca ame icana Be ies DCM A2
3Phy olacca ame icana Be ies Wa e /E OH (1:1) D2
4Phy olacca ame icana Lea es Wa e /E OH (1:1) D3
5Tage es pa ula ( ed lowe s) Lea es DCM (F.1-3) A3
6Tage es pa ula (yellow lowe s) Lea es DCM (F.1-4) A4
7Tage es pa ula (o ange lowe s) Lea es DCM (F.1-3) A5
8Tage es pa ula ( ed lowe s) Flowe s DCM A6
9Tage es pa ula ( ed lowe s) Lea es Wa e /E OH (1:1) B6
10 Tage es pa ula Flowe s ( ed) Wa e /E OH (1:1) C1
11 Tage es pa ula (yellow lowe s) Lea es Wa e /E OH (1:1) C2
12 Tage es pa ula (o ange lowe s) Lea es Wa e /E OH (1:1) C3
13 Ru a g a eolens Lea es DCM B1
14 Ru a g a eolens Lea es Wa e /E OH (1:1) D4
15 Ulex eu opaeus
Lea es and lowe s
DCM B2
16 Ulex eu opaeus
Lea es and lowe s
Wa e /E OH (1:1) C5
17 Glando a p os a a
Lea es and lowe s
DCM B3
18 Glando a p os a a
Lea es and lowe s
Wa e /E OH (1:1) D1
19 Punica g ana um F ui peel DCM B4
20 Punica g ana um F ui peel Wa e /E OH (1:1) C4
21 Camellia japonica Lea es DCM B5
22 Camellia japonica Lea es Wa e /E OH (1:1) C6
2.2. E alua ion o he Ac i i y Agains S 9 Cells
Gi en he high numbe o samples unde s udy (22), we we e in e es ed in knowing which samples
we e he mos oxic owa ds he S 9 cell line. To his end, all ex ac s we e e alua ed a he same
concen a ion (100
µ
g/mL) o hei impac in he iabili y o hese cells a e 24 h o incuba ion. Fo a
clea e unde s anding, a hea map o all esul s can be ound in Figu e 1(le ). Conside ing he esul s,
Molecules 2020,25, 5855 4 o 14
he e was a clea clus e o a g oup o samples ha we e ac i e ( ed) and hose ha we e mos ly inac i e
(blue). The mos ac i e samples we e, in ac , hose a ising om he use o non-pola sol en s, as i
can be seen in clea e de ail in Figu e 1( igh ), when plo ing he esul s om iabili y assays agains
he pola i y o he ex ac s. This esul can be explained by he ac ha he use o nonpola sol en s
allows he ex ac ion o classes o na u al p oduc s ha a e usually no ex ac able wi h highly pola
sol en s, such as wa e . As so, while pola sol en s will ex ac molecules such as suga s, polyphenols
and hei glycosyla ed de i a i es, he use o non-pola sol en s widens he chemical di e si y o he
a ge molecules o include classes such as alkaloids, e penes, couma ins, among o he s.
Molecules 2020, 25, x FOR PEER REVIEW 4 o 15
same concen a ion (100 μg/mL) o hei impac in he iabili y o hese cells a e 24 h o incuba ion.
Fo a clea e unde s anding, a hea map o all esul s can be ound in Figu e 1 (le ). Conside ing he
esul s, he e was a clea clus e o a g oup o samples ha we e ac i e ( ed) and hose ha we e
mos ly inac i e (blue). The mos ac i e samples we e, in ac , hose a ising om he use o non-pola
sol en s, as i can be seen in clea e de ail in Figu e 1 ( igh ), when plo ing he esul s om iabili y
assays agains he pola i y o he ex ac s. This esul can be explained by he ac ha he use o
nonpola sol en s allows he ex ac ion o classes o na u al p oduc s ha a e usually no ex ac able
wi h highly pola sol en s, such as wa e . As so, while pola sol en s will ex ac molecules such as
suga s, polyphenols and hei glycosyla ed de i a i es, he use o non-pola sol en s widens he
chemical di e si y o he a ge molecules o include classes such as alkaloids, e penes, couma ins,
among o he s.
Figu e 1. (Le ): Resul s o he iabili y (%) o insec cells a e ea men wi h he indica ed samples.
(Righ ): Compa ison o he iabili y o cells ea ed wi h non-pola (A1–B5) s. pola (B6–C4) ex ac s.
Ha ing educed he numbe o samples unde s udy, we we e in e es ed in knowing which
species we e igge ing highe le els o oxici y. As seen in Figu e 2, mos samples educed cell
iabili y o he 50–75% ange; howe e , he R. g a eolens dichlo ome hane ex ac (B1, Table 1) was
able o educe he iabili y below 50%. Fo benchma king pu poses, we addi ionally es ed he
iabili y o S 9 cells a e exposu e o he comme cial syn he ic insec icide chlo py i os. As shown in
Figu e 2, his molecule elici ed ca. 50% o iabili y loss, hus being less po en han B1. Fo his eason,
he la e was selec ed o subsequen s udies.
Ex ac
Viabili y (%)
Pola
Apola
0
50
100
Figu e 1.
(
Le
): Resul s o he iabili y (%) o insec cells a e ea men wi h he indica ed samples.
(
Righ
): Compa ison o he iabili y o cells ea ed wi h non-pola (
A1
–
B5
) s. pola (
B6
–
C4
) ex ac s.
Ha ing educed he numbe o samples unde s udy, we we e in e es ed in knowing which species
we e igge ing highe le els o oxici y. As seen in Figu e 2, mos samples educed cell iabili y o he
50–75% ange; howe e , heR. g a eolens dichlo ome haneex ac (
B1
,Table 1) wasable o educe he iabili y
below 50%. Fo benchma king pu poses, we addi ionally es ed he iabili y o S 9 cells a e exposu e o
he comme cial syn he ic insec icide chlo py i os. As shown in Figu e 2, his molecule elici ed ca. 50% o
iabili y loss, hus being less po en han
B1
. Fo his eason, he la e was selec ed o subsequen s udies.
Molecules 2020, 25, x FOR PEER REVIEW 5 o 15
Figu e 2. Viabili y o S 9 cells a e exposu e o he indica ed samples a 100 μg/mL, o 24 h. CHPY =
chlo py i os. *** p < 0.001. Iden i y o samples (A1–A6 and B1–B5) as in Table 1.
The UHPLC ESI-Q-TOF-MME analysis o R. g a eolens ex ac e ealed he p esence o en
compounds, some o which show a molecula mass compa ible wi h quinolin, benzopy an, and
ac idone de i a i es (Table 2). A ch oma og am o he ex ac is shown in he Suppo ing
In o ma ion, Figu e S1.
Table 2. Mass spec ome y da a and pu a i e iden i ica ion o he R. g a eolens ex ac . The peak
men ioned is he ch oma og am peak numbe shown in he Suppo ing In o ma ion, Figu e S2.
Peak RT M+ Compound Re e ence
10 19.477 279
G a eoline
[37]
11 20.455 259
Skimmianin
[37]
11 20.455 261
4,6,7-T ime hoxy- u o[2,3-b]quinoline
[38]
Figu e 2.
Viabili y o S 9 cells a e exposu e o he indica ed samples a 100
µ
g/mL, o 24 h.
CHPY =chlo py i os. *** p<0.001. Iden i y o samples (A1–A6 and B1–B5) as in Table 1.
Molecules 2020,25, 5855 5 o 14
The UHPLC ESI-Q-TOF-MME analysis o R. g a eolens ex ac e ealed he p esence o en
compounds, some o which showa molecula mass compa iblewi h quinolin, benzopy an, andac idone
de i a i es (Table 2). A ch oma og am o he ex ac is shown in he Suppo ing In o ma ion, Figu e S1.
Table 2.
Mass spec ome y da a and pu a i e iden i ica ion o he R. g a eolens ex ac . The peak
men ioned is he ch oma og am peak numbe shown in he Suppo ing In o ma ion, Figu e S2.
Peak RT M+Compound Re e ence
10 19.477 279
Molecules 2020, 25, x FOR PEER REVIEW 5 o 15
Figu e 2. Viabili y o S 9 cells a e exposu e o he indica ed samples a 100 μg/mL, o 24 h. CHPY =
chlo py i os. *** p < 0.001. Iden i y o samples (A1–A6 and B1–B5) as in Table 1.
The UHPLC ESI-Q-TOF-MME analysis o R. g a eolens ex ac e ealed he p esence o en
compounds, some o which show a molecula mass compa ible wi h quinolin, benzopy an, and
ac idone de i a i es (Table 2). A ch oma og am o he ex ac is shown in he Suppo ing
In o ma ion, Figu e S1.
Table 2. Mass spec ome y da a and pu a i e iden i ica ion o he R. g a eolens ex ac . The peak
men ioned is he ch oma og am peak numbe shown in he Suppo ing In o ma ion, Figu e S2.
Peak RT M+ Compound Re e ence
10 19.477 279
G a eoline
[37]
11 20.455 259
Skimmianin
[37]
11 20.455 261
4,6,7-T ime hoxy- u o[2,3-b]quinoline
[38]
G a eoline
[37]
11 20.455 259
Molecules 2020, 25, x FOR PEER REVIEW 5 o 15
Figu e 2. Viabili y o S 9 cells a e exposu e o he indica ed samples a 100 μg/mL, o 24 h. CHPY =
chlo py i os. *** p < 0.001. Iden i y o samples (A1–A6 and B1–B5) as in Table 1.
The UHPLC ESI-Q-TOF-MME analysis o R. g a eolens ex ac e ealed he p esence o en
compounds, some o which show a molecula mass compa ible wi h quinolin, benzopy an, and
ac idone de i a i es (Table 2). A ch oma og am o he ex ac is shown in he Suppo ing
In o ma ion, Figu e S1.
Table 2. Mass spec ome y da a and pu a i e iden i ica ion o he R. g a eolens ex ac . The peak
men ioned is he ch oma og am peak numbe shown in he Suppo ing In o ma ion, Figu e S2.
Peak RT M+ Compound Re e ence
10 19.477 279
G a eoline
[37]
11 20.455 259
Skimmianin
[37]
11 20.455 261
4,6,7-T ime hoxy- u o[2,3-b]quinoline
[38]
Skimmianin
[37]
11 20.455 261
Molecules 2020, 25, x FOR PEER REVIEW 5 o 15
Figu e 2. Viabili y o S 9 cells a e exposu e o he indica ed samples a 100 μg/mL, o 24 h. CHPY =
chlo py i os. *** p < 0.001. Iden i y o samples (A1–A6 and B1–B5) as in Table 1.
The UHPLC ESI-Q-TOF-MME analysis o R. g a eolens ex ac e ealed he p esence o en
compounds, some o which show a molecula mass compa ible wi h quinolin, benzopy an, and
ac idone de i a i es (Table 2). A ch oma og am o he ex ac is shown in he Suppo ing
In o ma ion, Figu e S1.
Table 2. Mass spec ome y da a and pu a i e iden i ica ion o he R. g a eolens ex ac . The peak
men ioned is he ch oma og am peak numbe shown in he Suppo ing In o ma ion, Figu e S2.
Peak RT M+ Compound Re e ence
10 19.477 279
G a eoline
[37]
11 20.455 259
Skimmianin
[37]
11 20.455 261
4,6,7-T ime hoxy- u o[2,3-b]quinoline
[38]
4,6,7-T ime hoxy- u o[2,3-b]quinoline
[38]
16 23.932 216
Molecules 2020, 25, x FOR PEER REVIEW 6 o 15
16 23.932 216
8-Me hoxypso alen 5-Me hoxypso alen
[39]
17 24.082 229
P eleine
[37]
18 33.006 314
Chalepin
[37]
21 38.681 356
Ru ama in
[37]
20 37.305 271
2-N-Nonyl-4-quinolone
[38]
21 38.681 357
G a ac idone iol
[37]
2.3. Mo phological Assessmen
In ligh o he p onounced impac o B1 in S 9 cell iabili y, we we e in e es ed in unde s anding
he unde lying e ec o he ex ac . To his end, he con ol and B1- ea ed cells ha e been imaged in
o de o assess pu a i e di e ences in o e all cell mo phology and ch oma in. As shown in Figu e 3,
B1 elici s a signi ican deg ee o ch oma in condensa ion (blue a ows) and e en agmen a ion
(yellow a ows), al hough o e all cell in eg i y is main ained. This esul sugges s ha B1 may be
elici ing he loss o cell iabili y by igge ing an o ganized p ocess o cell dea h, in which
mo phological ai s a e compa ible o hose desc ibed he e.
Molecules 2020, 25, x FOR PEER REVIEW 6 o 15
16 23.932 216
8-Me hoxypso alen 5-Me hoxypso alen
[39]
17 24.082 229
P eleine
[37]
18 33.006 314
Chalepin
[37]
21 38.681 356
Ru ama in
[37]
20 37.305 271
2-N-Nonyl-4-quinolone
[38]
21 38.681 357
G a ac idone iol
[37]
2.3. Mo phological Assessmen
In ligh o he p onounced impac o B1 in S 9 cell iabili y, we we e in e es ed in unde s anding
he unde lying e ec o he ex ac . To his end, he con ol and B1- ea ed cells ha e been imaged in
o de o assess pu a i e di e ences in o e all cell mo phology and ch oma in. As shown in Figu e 3,
B1 elici s a signi ican deg ee o ch oma in condensa ion (blue a ows) and e en agmen a ion
(yellow a ows), al hough o e all cell in eg i y is main ained. This esul sugges s ha B1 may be
elici ing he loss o cell iabili y by igge ing an o ganized p ocess o cell dea h, in which
mo phological ai s a e compa ible o hose desc ibed he e.
8-Me hoxypso alen 5-Me hoxypso alen
[39]
17 24.082 229
Molecules 2020, 25, x FOR PEER REVIEW 6 o 15
16 23.932 216
8-Me hoxypso alen 5-Me hoxypso alen
[39]
17 24.082 229
P eleine
[37]
18 33.006 314
Chalepin
[37]
21 38.681 356
Ru ama in
[37]
20 37.305 271
2-N-Nonyl-4-quinolone
[38]
21 38.681 357
G a ac idone iol
[37]
2.3. Mo phological Assessmen
In ligh o he p onounced impac o B1 in S 9 cell iabili y, we we e in e es ed in unde s anding
he unde lying e ec o he ex ac . To his end, he con ol and B1- ea ed cells ha e been imaged in
o de o assess pu a i e di e ences in o e all cell mo phology and ch oma in. As shown in Figu e 3,
B1 elici s a signi ican deg ee o ch oma in condensa ion (blue a ows) and e en agmen a ion
(yellow a ows), al hough o e all cell in eg i y is main ained. This esul sugges s ha B1 may be
elici ing he loss o cell iabili y by igge ing an o ganized p ocess o cell dea h, in which
mo phological ai s a e compa ible o hose desc ibed he e.
P eleine
[37]
18 33.006 314
Molecules 2020, 25, x FOR PEER REVIEW 6 o 15
16 23.932 216
8-Me hoxypso alen 5-Me hoxypso alen
[39]
17 24.082 229
P eleine
[37]
18 33.006 314
Chalepin
[37]
21 38.681 356
Ru ama in
[37]
20 37.305 271
2-N-Nonyl-4-quinolone
[38]
21 38.681 357
G a ac idone iol
[37]
2.3. Mo phological Assessmen
In ligh o he p onounced impac o B1 in S 9 cell iabili y, we we e in e es ed in unde s anding
he unde lying e ec o he ex ac . To his end, he con ol and B1- ea ed cells ha e been imaged in
o de o assess pu a i e di e ences in o e all cell mo phology and ch oma in. As shown in Figu e 3,
B1 elici s a signi ican deg ee o ch oma in condensa ion (blue a ows) and e en agmen a ion
(yellow a ows), al hough o e all cell in eg i y is main ained. This esul sugges s ha B1 may be
elici ing he loss o cell iabili y by igge ing an o ganized p ocess o cell dea h, in which
mo phological ai s a e compa ible o hose desc ibed he e.
Chalepin
[37]
21 38.681 356
Molecules 2020, 25, x FOR PEER REVIEW 6 o 15
16 23.932 216
8-Me hoxypso alen 5-Me hoxypso alen
[39]
17 24.082 229
P eleine
[37]
18 33.006 314
Chalepin
[37]
21 38.681 356
Ru ama in
[37]
20 37.305 271
2-N-Nonyl-4-quinolone
[38]
21 38.681 357
G a ac idone iol
[37]
2.3. Mo phological Assessmen
In ligh o he p onounced impac o B1 in S 9 cell iabili y, we we e in e es ed in unde s anding
he unde lying e ec o he ex ac . To his end, he con ol and B1- ea ed cells ha e been imaged in
o de o assess pu a i e di e ences in o e all cell mo phology and ch oma in. As shown in Figu e 3,
B1 elici s a signi ican deg ee o ch oma in condensa ion (blue a ows) and e en agmen a ion
(yellow a ows), al hough o e all cell in eg i y is main ained. This esul sugges s ha B1 may be
elici ing he loss o cell iabili y by igge ing an o ganized p ocess o cell dea h, in which
mo phological ai s a e compa ible o hose desc ibed he e.
Ru ama in
[37]
20 37.305 271
Molecules 2020, 25, x FOR PEER REVIEW 6 o 15
16 23.932 216
8-Me hoxypso alen 5-Me hoxypso alen
[39]
17 24.082 229
P eleine
[37]
18 33.006 314
Chalepin
[37]
21 38.681 356
Ru ama in
[37]
20 37.305 271
2-N-Nonyl-4-quinolone
[38]
21 38.681 357
G a ac idone iol
[37]
2.3. Mo phological Assessmen
In ligh o he p onounced impac o B1 in S 9 cell iabili y, we we e in e es ed in unde s anding
he unde lying e ec o he ex ac . To his end, he con ol and B1- ea ed cells ha e been imaged in
o de o assess pu a i e di e ences in o e all cell mo phology and ch oma in. As shown in Figu e 3,
B1 elici s a signi ican deg ee o ch oma in condensa ion (blue a ows) and e en agmen a ion
(yellow a ows), al hough o e all cell in eg i y is main ained. This esul sugges s ha B1 may be
elici ing he loss o cell iabili y by igge ing an o ganized p ocess o cell dea h, in which
mo phological ai s a e compa ible o hose desc ibed he e.
2-N-Nonyl-4-quinolone
[38]
21 38.681 357
Molecules 2020, 25, x FOR PEER REVIEW 6 o 15
16 23.932 216
8-Me hoxypso alen 5-Me hoxypso alen
[39]
17 24.082 229
P eleine
[37]
18 33.006 314
Chalepin
[37]
21 38.681 356
Ru ama in
[37]
20 37.305 271
2-N-Nonyl-4-quinolone
[38]
21 38.681 357
G a ac idone iol
[37]
2.3. Mo phological Assessmen
In ligh o he p onounced impac o B1 in S 9 cell iabili y, we we e in e es ed in unde s anding
he unde lying e ec o he ex ac . To his end, he con ol and B1- ea ed cells ha e been imaged in
o de o assess pu a i e di e ences in o e all cell mo phology and ch oma in. As shown in Figu e 3,
B1 elici s a signi ican deg ee o ch oma in condensa ion (blue a ows) and e en agmen a ion
(yellow a ows), al hough o e all cell in eg i y is main ained. This esul sugges s ha B1 may be
elici ing he loss o cell iabili y by igge ing an o ganized p ocess o cell dea h, in which
mo phological ai s a e compa ible o hose desc ibed he e.
G a ac idone iol
[37]

Molecules 2020,25, 5855 6 o 14
2.3. Mo phological Assessmen
In ligh o he p onounced impac o
B1
in S 9 cell iabili y, we we e in e es ed in unde s anding he
unde lying e ec o he ex ac . To his end, he con ol and
B1
- ea ed cells ha e been imaged in o de
o assess pu a i e di e ences in o e all cell mo phology and ch oma in. As shown in Figu e 3,
B1
elici s
a signi ican deg ee o ch oma in condensa ion (blue a ows) and e en agmen a ion (yellow a ows),
al hough o e all cell in eg i y is main ained. This esul sugges s ha
B1
may be elici ing he loss
o cell iabili y by igge ing an o ganized p ocess o cell dea h, in which mo phological ai s a e
compa ible o hose desc ibed he e.
Molecules 2020, 25, x FOR PEER REVIEW 7 o 15
Figu e 3. S 9 cells exposed o B1 (100 μg/mL) o 24 h (S Plan Fluo ELWD 40 × DIC N1 objec i e). Cell
mo phology was e alua ed using 4′,6-diamidino-2-phenylindole (DAPI, ch oma in) and phalloidin (ac in).
Yellow a ow: ch oma in agmen a ion; Blue a ow: ch oma in condensa ion. (C)—con ol. Colo blind-
iendly image in Suppo ing In o ma ion Figu e S2.
2.4. Nanoencapsula ion S udies
The size and size dis ibu ion o he chosen nanoencapsula ion sys ems was de e mined by
dynamic ligh sca e ing (DLS), Table 3. The liposomes p epa ed by he e hanolic injec ion me hod
ha e a smalle size han he ones p epa ed by hin ilm hyd a ion bu exhibi a sligh ly highe
polydispe si y. The chi osan nanos uc u es (p epa ed by ionic gela ion) a e he encapsula ion
sys ems wi h he lowes hyd odynamic diame e . The sizes we e main ained cons an o wo weeks,
wi h no signi ican change in polydispe si y, p o ing he s abili y o he p epa ed nanosys ems.
Table 3. Size (hyd odynamic diame e ) and he polydispe si y o soybean liposomes and chi osan
nanos uc u es de e mined by dynamic ligh sca e ing (DLS) (SD: s anda d de ia ion om i e
independen measu emen s).
Sys em Size ± SD (nm) PDI ± SD
Liposomes (e hanolic injec ion) 121 ± 28 0.256 ± 0.08
Liposomes ( hin ilm hyd a ion) 203 ± 17 0.185 ± 0.05
Chi osan nanos uc u es 118 ± 23 0.203 ± 0.02
Liposomes ha e been desc ibed as ideal bioac i e deli e y sys ems, due o hei biocompa ibili y
and hei abili y o ca y bo h hyd ophilic (in he aqueous lumen) and hyd ophobic (in he lipid
bilaye ) payloads [40]. The esul s in Table 3 a e in acco dance wi h he ones epo ed in he li e a u e
o leci hin-based liposomal sys ems p epa ed by e hanolic injec ion [41,42] and hin ilm hyd a ion
[43,44], he la e being sligh ly la ge . Chi osan nanos uc u es ha e also been desc ibed as
ad an ageous o he encapsula ion o pes icides and na u al ex ac s [45–48]. The hyd odynamic
diame e and polydispe si y ob ained o chi osan nanocapsules (Table 3) a e also in acco dance wi h
he epo ed esul s o a simila p epa a ion me hod [45].
The encapsula ion e iciencies o he ex ac wi h he bes insec icidal ac i i y B1 in soybean
liposomes and chi osan nanoemulsions a e p esen ed in Table 4.
Figu e 3.
S 9 cells exposed o
B1
(100
µ
g/mL) o 24 h (S Plan Fluo ELWD 40
×
DIC N1 objec i e).
Cell mo phology was e alua ed using 4
0
,6-diamidino-2-phenylindole (DAPI, ch oma in) and phalloidin
(ac in). Yellow a ow: ch oma in agmen a ion; Blue a ow: ch oma in condensa ion. (
C
)—con ol.
Colo blind- iendly image in Suppo ing In o ma ion Figu e S2.
2.4. Nanoencapsula ion S udies
The size and size dis ibu ion o he chosen nanoencapsula ion sys ems was de e mined by
dynamic ligh sca e ing (DLS), Table 3. The liposomes p epa ed by he e hanolic injec ion me hod ha e
a smalle size han he ones p epa ed by hin ilm hyd a ion bu exhibi a sligh ly highe polydispe si y.
The chi osan nanos uc u es (p epa ed by ionic gela ion) a e he encapsula ion sys ems wi h he lowes
hyd odynamic diame e . The sizes we e main ained cons an o wo weeks, wi h no signi ican change
in polydispe si y, p o ing he s abili y o he p epa ed nanosys ems.
Table 3.
Size (hyd odynamic diame e ) and he polydispe si y o soybean liposomes and chi osan
nanos uc u es de e mined by dynamic ligh sca e ing (DLS) (SD: s anda d de ia ion om i e
independen measu emen s).
Sys em Size ±SD (nm) PDI ±SD
Liposomes (e hanolic injec ion) 121 ±28 0.256 ±0.08
Liposomes ( hin ilm hyd a ion) 203 ±17 0.185 ±0.05
Chi osan nanos uc u es 118 ±23 0.203 ±0.02
Molecules 2020,25, 5855 7 o 14
Liposomes ha e been desc ibed as ideal bioac i e deli e y sys ems, due o hei biocompa ibili y
and hei abili y o ca y bo h hyd ophilic (in he aqueous lumen) and hyd ophobic (in he lipid bilaye )
payloads [
40
]. The esul s in Table 3a e in acco dance wi h he ones epo ed in he li e a u e o
leci hin-based liposomal sys ems p epa ed by e hanolic injec ion [
41
,
42
] and hin ilm hyd a ion [
43
,
44
],
he la e being sligh ly la ge . Chi osan nanos uc u es ha e also been desc ibed as ad an ageous
o he encapsula ion o pes icides and na u al ex ac s [
45
–
48
]. The hyd odynamic diame e and
polydispe si y ob ained o chi osan nanocapsules (Table 3) a e also in acco dance wi h he epo ed
esul s o a simila p epa a ion me hod [45].
The encapsula ion e iciencies o he ex ac wi h he bes insec icidal ac i i y
B1
in soybean
liposomes and chi osan nanoemulsions a e p esen ed in Table 4.
Table 4.
Encapsula ion e iciencies (EE%
±
SD) o he ex ac wi h he bes insec icidal ac i i y in
liposomes and chi osan nanoemulsions (SD: s anda d de ia ion o h ee independen measu emen s).
Sys em Sample B1
Liposomes (e hanolic injec ion) 93.3 ±6
Liposomes ( hin ilm hyd a ion) 73.0 ±9
Chi osan nanoemulsions 94.0 ±2
The encapsula ion e iciencies we e gene ally high (abo e 70%), especially in liposomes p epa ed
by he e hanolic injec ion me hod and in chi osan nanos uc u es.
The elease o he encapsula ed ex ac was ollowed o 24 h a oom empe a u e (25
◦
C) owa ds
phospha e bu e , pH =7.3 (Figu e 4). The esul s e idence ha chi osan nanos uc u es a e sui able
o a as e and comple e elease, while liposomes allow a delayed elease. The di e ence in he elease
p o iles ob ained om liposomes p epa ed by he wo echniques ( hin ilm hyd a ion and e hanolic
injec ion) may be ela ed wi h he hyd ophobic cha ac e o his ex ac , as e hanolic injec ion is mo e
ad an ageous o his ype o bioac i es [41].
Molecules 2020, 25, x FOR PEER REVIEW 8 o 15
Table 4. Encapsula ion e iciencies (EE% ± SD) o he ex ac wi h he bes insec icidal ac i i y in
liposomes and chi osan nanoemulsions (SD: s anda d de ia ion o h ee independen measu emen s).
Sys em Sample B1
Liposomes (e hanolic injec ion) 93.3 ± 6
Liposomes ( hin ilm hyd a ion) 73.0 ±9
Chi osan nanoemulsions 94.0 ± 2
The encapsula ion e iciencies we e gene ally high (abo e 70%), especially in liposomes
p epa ed by he e hanolic injec ion me hod and in chi osan nanos uc u es.
The elease o he encapsula ed ex ac was ollowed o 24 h a oom empe a u e (25 °C)
owa ds phospha e bu e , pH = 7.3 (Figu e 4). The esul s e idence ha chi osan nanos uc u es a e
sui able o a as e and comple e elease, while liposomes allow a delayed elease. The di e ence in
he elease p o iles ob ained om liposomes p epa ed by he wo echniques ( hin ilm hyd a ion and
e hanolic injec ion) may be ela ed wi h he hyd ophobic cha ac e o his ex ac , as e hanolic
injec ion is mo e ad an ageous o his ype o bioac i es [41].
The expe imen al elease p o iles we e analyzed by a modi ied Ko smeye –Peppas model [49] and
he esul s a e summa ized in Table 5 ( he i ings a e shown in Suppo ing In o ma ion Figu e S3).
Figu e 4. Release p o iles o sample B1 om he liposomes and chi osan nanos uc u es.
Table 5. Release pa ame e s o he Ko smeye –Peppas model i ed o he elease p o iles (R is he
coe icien o de e mina ion).
Nanosys em Ks n l

Liposomes ( hin ilm hyd a ion) 0.07 0.50 0.9 0.98
Liposomes (e hanolic injec ion)
Liposomes (e hanolic injec ion) *
0.01 1.39 0 0.98
0.0045 1.74 0 0.99
Chi osan nanos uc u es
Chi osan nanos uc u es *
0.04 1.07 2.13 0.98
0.0314 1.21 2.11 0.99
Conside ing ha he nanoencapsula ion sys ems a e sphe ical, he n alue in he ange 0.43 < n
< 0.85 sugges s ha he elease p o iles om liposomes p epa ed by hin ilm hyd a ion can be mainly
asc ibed o di usion and swelling, i.e., bo h con ibu ions ha e compa able a es [49]. The samples
ma ked wi h * in Table 5 we e subjec ed o a di e en analysis, o which he elease is ini ially
assumed o be associa ed wi h he supe case-ii anspo mechanism, whe e e osion, elaxa ion and
di usion a e esponsible o ex ac elease. Despi e he easonable i ing o he model o he
Figu e 4. Release p o iles o sample B1 om he liposomes and chi osan nanos uc u es.
The expe imen al elease p o iles we e analyzed by a modi ied Ko smeye –Peppas model [
49
]
and he esul s a e summa ized in Table 5( he i ings a e shown in Suppo ing In o ma ion Figu e S3).
Molecules 2020,25, 5855 8 o 14
Table 5.
Release pa ame e s o he Ko smeye –Peppas model i ed o he elease p o iles (Ris he
coe icien o de e mina ion).
Nanosys em Ksn l R2
Liposomes ( hin ilm hyd a ion)
0.07 0.50 0.9 0.98
Liposomes (e hanolic injec ion)
Liposomes (e hanolic injec ion) *
0.01 1.39 0 0.98
0.0045 1.74 0 0.99
Chi osan nanos uc u es
Chi osan nanos uc u es *
0.04 1.07 2.13 0.98
0.0314 1.21 2.11 0.99
Conside ing ha he nanoencapsula ion sys ems a e sphe ical, he n alue in he ange
0.43 <n<0.85
sugges s ha he elease p o iles om liposomes p epa ed by hin ilm hyd a ion can be mainly asc ibed
o di usion and swelling, i.e., bo h con ibu ions ha e compa able a es [
49
]. The samples ma ked wi h *
in Table 5we e subjec ed o a di e en analysis, o which he elease is ini ially assumed o be associa ed
wi h he supe case-ii anspo mechanism, whe e e osion, elaxa ion and di usion a e esponsible o
ex ac elease. Despi e he easonable i ing o he model o he expe imen al da a ob ained in liposomes
p epa ed by injec ion and chi osan nanos uc u es (as implied by he coe icien s o de e mina ion), i does
no p ope ly explain he da a om a quali a i e poin o iew. The e o e, he elease p o iles we e also
i ed o he Gompe z empi ical unc ion (Table 6), de ined as
X =Xmaxe−aeblog10
whe e
X
and
Xmax
a e he dissol ed ac ion pe cen ages a ime and i s maximum,
a
is a shape
pa ame e and
b
is he dissolu ion a e pe uni o ime [
50
]. The i ings o his model a e also
gene ally good (see Suppo ing In o ma ion Figu e S4). Al hough he model does no p o ide
mechanis ic in o ma ion, he la ge b alues o he samples in chi osan indica e as e elease kine ics,
while liposomes ob ained by hyd a ion seem o be mo e e ec i e in ensu ing a slowe elease.
Table 6. Release pa ame e s ob ained om he i ing o he Gompe z model o he elease p o iles.
Nanosys em Xmax a B R2
Liposomes ( hin ilm hyd a ion)
49.38 3.61 1.55 0.99
Liposomes (e hanolic injec ion) 100 680.48 5.99 0.99
Chi osan nanos uc u es 100 163.23 5.34 0.97
The esul s ob ained he ein indica e ha lipid-based and chi osan nanoencapsula ion sys ems allow
high encapsula ion e iciencies o he ex ac s wi h insec icidal ac i i y and p o ide di e en elease
a es. This ep esen s a p oo -o -concep o u u e de elopmen s o bioinsec icide nano o mula ions
using he mos ac i e ex ac s.
3. Expe imen al Sec ion
3.1. Chemicals and Reagen s
E hanol, me hanol, dichlo ome hane, isop opanol, DMSO, ace ic acid and e ahyd o u an we e
pu chased om Me ck KGaA (Da ms ad , Ge many). Ch omabond C18 column was pu chased om
Sigma-Ald ich (S . Louis, MO, USA). T ypan blue and 3-(4,5-dime hyl hiazolyl-2)-2,5-diphenyl e azolium
b omide (MTT) we e ob ained om Sigma-Ald ich (S . Louis, MO, USA). Dulbecco’s modi ied eagle
medium (DMEM), Hank’s balanced sal solu ion (HBSS), e al bo ine se um (FBS), penicillin–s ep omycin
solu ion (penicillin 5000 uni s/mL and s ep omycin 5000
µ
g/mL) and 0.25% ypsin-EDTA we e ob ained
om GIBCO, In i ogen™(G and Island, NY, USA).
Molecules 2020,25, 5855 9 o 14
3.2. Plan Ma e ial
The se en species o plan s we e ha es ed in he No h o Po ugal, namely in Melgaço (42.1144
◦
N,
8.2580
◦
W) (P. g ana um,P. ame icana,G. p os a a,U. eu opaeus,T. pa ula,C. japonica ed) and Guima
ã
es
(41.4425
◦
N, 8.2918
◦
W) (R. g a eolens), a he lowe ing s age in he case o G. p os a a,U. eu opaeus and
T. pa ula in Ma ch 2019 (G. p os a a,U. eu opaeus) o Oc obe 2019 o he emaining plan s. The plan
ma e ial included ui peel (P. g ana um), be ies (P. ame icana), lowe s and lea es (G. p os a a,
U. eu opaeus, and T. pa ula), as well as only lea es (C. japonica and R. g a eolens). In he case o T. pa ula,
he lea es and yellow, o ange and ed lowe s (sepa a ed om he ecep acle) we e used in sepa a e
expe imen s. A leas h ee specimens o each species we e collec ed.
The pomeg ana e peel (P. g ana um) was d ied unde sunligh a 20–25
◦
C du ing 45 days.
The be ies o P. ame icana, as well as he lea es and lowe s o T. pa ula, we e ozen a
−
20
◦
C un il
lyophiliza ion in an Alpha 1-4 ld Plus–Ch is eeze d ye (Ma in Ch is Ge ie ocknungsanlagen
GmbH, Os e ode am Ha z, Ge many). The lea es and lowe s o he emaining species we e d ied in
an o en a 40–45 ◦C du ing 3 days.
A e d ying o lyophiliza ion, he ege able ma e was g ound wi h a sh edde , and he esul ing
powde passed h ough a sie e un il a pa icle size
≤
900
µ
m was ob ained. The ma e ial was s o ed
unde acuum o u he use.
3.3. Plan Ma e ials Ex ac ion
The d ied o lyophilized plan ma e ials o P. g ana um,P. ame icana,G. p os a a,U. eu opaeus,
T. pa ula,C.japonica,R. g a eolens (8 g) we e subjec ed o Soxhle ex ac ion wi h wa e /e hanol (1:1)
(50 mL) o dichlo ome hane (50 mL) o 9 o 4 h, espec i ely.
The o ganic sol en s we e emo ed by e apo a ion unde educed p essu e a 40
◦
C (Ro a apo
®
R-210, BÜCHI Labo echnik AG, Flawil, Swi ze land), ollowed by lyophiliza ion (Alpha 1-4 ld
Plus–Ch is eeze d ye , Ma in Ch is Ge ie ocknungsanlagen GmbH, Os e ode am Ha z, Ge many)
in he case o ex ac ions wi h wa e /e hanol (1:1). The esul ing lyophilized ex ac s we e s o ed
unde acuum o u he use. The e apo a ed ex ac s o dichlo ome hane we e passed h ough a
Ch omabond C18 column, using me hanol as eluen , o he emo al o chlo ophylls. A e he sol en
e apo a ion unde acuum a 35 ◦C, he esul ing ex ac s we e s o ed a −20 ◦C o u he use.
3.4. Cell Cul u e
S 9 (Spodop e a ugipe da)cells we e omATCC/LGC S anda ds(Spain)main ained as asuspension
cul u e and cul i a ed in G ace’s medium wi h 10% FBS and an ibio ics a 28
◦
C. Cells we e used
in he expe imen s while in he exponen ial phase o g ow h. HaCaT (human ke a inocy es) cells
we e cul u ed in DMEM supplemen ed wi h 10% FBS and 1% penicillin/s ep omycin a 37
◦
C, in a
humidi ied a mosphe e o 5% CO2.
3.5. Assessmen o Viabili y
Fo he assessmen o iabili y, a esazu in-based me hod was used, as p e iously desc ibed [
51
].
S 9 cells we e pla ed a a densi y o 3
×
10
4
cells/well, incuba ed o 24 h and hen exposed o he
molecules unde s udy o 24 h. A e his pe iod, a comme cial solu ion o esazu in was added (1:10)
and he kine ic eac ion o luo escence inc ease was moni o ed. An incuba ion ime o 30 min was used.
3.6. Mo phological Assessmen
Fo mo phological s udies, he cells we e cul u ed in 96-well pla es, as desc ibed abo e o iabili y
expe imen s, in he p esence o he samples unde s udy. A e incuba ion, he cells we e washed wi h
HBSS and ixed in 10% o malin solu ion o 30 min, a oom empe a u e. DAPI (0.25
µ
g/mL) and
CF543 (5 U/mL) we e added and he cells we e s ained o 25 min a oom empe a u e and washed
wi h HBSS, as desc ibed be o e [52,53].