E ec s o combined CBGA and cannabis-de i ed e pene nano o mula ions
on TRPV1 ac i a ion: Implica ions o enhanced pain managemen
Mazen M. El-Hammadi
a,*
, And ea L. Small-Howa d
b
, Me cedes Fe n´
andez-A ´
e alo
a
,
Helen Tu ne
c
, Lucía Ma ín-Bande as
a,d,*
a
Depa amen o de Fa macia y Tecnología Fa mac´
eu ica, Facul ad de Fa macia, Uni e sidad de Se illa, c/P o . Ga cía Gonz´
alez, n◦2, 41012 Se illa, Spain
b
GB Sciences, Inc. (OTCQB:GBLX), 9205 W. Russell Road, Sui e 240 Las Vegas, Ne ada 89148, Uni ed S a es
c
Labo a o y o Pha macology and Analy ics, School o Na u al Sciences and Ma hema ics, Chaminade Uni e si y o Honolulu, Honolulu, HI, Uni ed S a es
d
Ins i u o de Biomedicina de Se illa (IBiS), Hospi al Uni e si a io Vi gen del Rocio/CSIC/Uni e sidad de Se illa, Se ille, Spain
ARTICLE INFO
Keywo ds:
Cannabige olic acid
Cannabis-based e penes
PLGA polyme ic nanopa icles
Be a-my cene
Ne olidol
Be a-ca yophyllene
Nanomedicine
Ch onic pain
ABSTRACT
Cannabinoids and e penes, key bioac i e componen s o cannabis, a e inc easingly s udied o hei indi idual
and combined con ibu ions o he he apeu ic po en ial o cannabis-based ea men s, wi h ongoing esea ch
explo ing hei dis inc and in e ac i e e ec s. This s udy aimed o encapsula e cannabige olic acid (CBGA) in
poly(e hylene glycol)-poly(lac ic-co-glycolic acid) nanopa icles (PEG-PLGA NPs) and in es iga e he e ec s o
combining CBGA NPs wi h cannabis-de i ed e pene-loaded NPs (my cene [MC], ne olidol [NL], and ca -
yophyllene [CPh]) o po en ial applica ions in pain managemen . CBGA NPs (152 nm) and e pene-loaded NPs
(233–297 nm) we e p epa ed ia nanop ecipi a ion and emulsion-sol en e apo a ion, espec i ely, exhibi ing a
polydispe si y index <0.3 and nega i e ze a po en ials (−23 o −26 mV). Encapsula ion e iciency was 98.6 %
o CBGA and 13–33 % o e penes. CBGA elease ollowed a biphasic p o ile, wi h ~ 20 % eleased wi hin 4 h
and sus ained elease o e 72 h. In i o e alua ion used HEK293 cells exp essing he nocicep i e ansien e-
cep o po en ial anilloid-1 (TRPV1) channel, a key media o o pain pe cep ion. TRPV1 ac i a ion was assessed
ia calcium in lux kine ics (Fluo-4 indica o ). The EC50 alues we e 23.8 µg/mL (CBGA NPs), 8.0 µg/mL (MC
NPs), 6.7 µg/mL (NL NPs), and 13.3 µg/mL (CPh NPs). Combina o ial ea men s o CBGA NPs wi h e pene NPs
a hei espec i e EC50 concen a ions e ealed signi ican ly enhanced calcium in lux compa ed o indi idual
NPs, wi h he s onges in e ac ion obse ed o CBGA/NL and mode a e e ec s o CBGA/MC. Fluo escence
imaging u he co obo a ed hese indings. These esul s sugges ha combining CBGA NPs wi h e pene-
loaded NPs could po en ia e pain- elie e icacy, o e ing a p omising s a egy o ad anced he apeu ic
o mula ions.
1. In oduc ion
Cannabis sa i a has long been alued o i s di e se medicinal p op-
e ies. This e sa ile plan p oduces mo e han 500 bioac i e com-
pounds, including cannabinoids, e penes, la onoids, and o he
phy ochemicals, which collec i ely shape i s complex pha macological
p o ile. Mode n esea ch inc easingly highligh s i s he apeu ic po en-
ial o managing ch onic pain, in lamma ion, anxie y, and neu ological
diso de s (Russo and Ma cu, 2017). The plan ’s b oad ange o bioac i e
compounds o e s a mul i- ace ed app oach o ea men by a ge ing
a ious pa hways and mechanisms wi hin he body (B idgeman and
Abazia, 2017). Howe e , clinical u iliza ion o Cannabis sa i a aces
challenges, including s anda diza ion o o mula ions, consis ency in
dosing, and gaps in unde s anding long- e m e ec s. Fu he esea ch is
c i ical o ha ness i s ull po en ial o de eloping sa e, e ec i e, and
a ge ed he apies de i ed om his ancien plan .
Cannabinoids, a pha macologically ac i e class o compounds in
Cannabis sa i a, modula e calcium signaling h ough in e ac ions wi h
ansien ecep o po en ial (TRP) channels, such as TRPV1 (Jansen
e al., 2019; S a kus e al., 2019; De Pe ocellis e al., 2011; Mulle e al.,
2018; S o ozhuk and Zholos, 2018), and calcium elease-ac i a ed cal-
cium (CRAC) channels (Faouzi e al., 2022). The TRPV1 channel, a
* Co esponding au ho s a : Depa amen o de Fa macia y Tecnología Fa mac´
eu ica, Facul ad de Fa macia, Uni e sidad de Se illa, c/P o . Ga cía Gonz´
alez, n◦2,
41012 Se illa, Spain.
E-mail add esses: [email p o ec ed] (M.M. El-Hammadi), [email p o ec ed] (L. Ma ín-Bande as).
Con en s lis s a ailable a ScienceDi ec
In e na ional Jou nal o Pha maceu ics
jou nal homepage: www.else ie .com/loca e/ijpha m
h ps://doi.o g/10.1016/j.ijpha m.2025.125766
Recei ed 5 Ma ch 2025; Recei ed in e ised o m 22 May 2025; Accep ed 23 May 2025
In e na ional Jou nal o Pha maceu ics 679 (2025) 125766
A ailable online 24 May 2025
0378-5173/© 2025 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ).
majo pain modula ion a ge , is exp essed in nocicep i e neu ons and
o he cell ypes, whe e i ac s as a senso o noxious s imuli (e.g., hea ,
acidic pH) (S o ozhuk e al., 2019). Cannabinoid-TRPV1 in e ac ions
igge calcium in lux, a key mechanism in pain signal modula ion (De
Pe ocellis e al., 2013).
Cannabige olic acid (CBGA) (Fig. 1E), a non-psychoac i e “mo he
cannabinoid,” se es as he biosyn he ic p ecu so o majo cannabi-
noids such as e ahyd ocannabinolic acid (THCA), cannabidiolic acid
(CBDA), and cannabich omenic acid (CBCA) (Ande son e al., 2021).
P oduced in he cannabis plan ia enzyma ic condensa ion o ge anyl
py ophospha e and oli e olic acid, CBGA is he cen al in e media e in
cannabinoid biosyn hesis (ElSohly, 2017). Eme ging e idence un-
de sco es CBGA’s he apeu ic po en ial, pa icula ly o pain and in-
lamma o y managemen (Calapai e al., 2022; Ruhaak e al., 2011).
Mechanis ically, CBGA inhibi s s o e-ope a ed calcium en y (SOCE),
down egula es IL-2 p oduc ion in T cells, and a enua es kidney
in lamma ion (Faouzi e al., 2022; Suzuki e al., 2023).
Howe e , he he apeu ic po en ial o CBGA is limi ed by i s poo
solubili y, s abili y, and bioa ailabili y (Zi pel e al., 2015). Nano-
pa icle encapsula ion has eme ged as a p omising s a egy o add ess
hese limi a ions, by enhancing CBGA’s pha macokine ic p o ile while
imp o ing i s s abili y and enabling a ge ed deli e y (S ella e al., 2021;
Lazza o o Rebela o e al., 2023). In addi ion, CBGA has been iden i ied
as he mos po en cannabinoid o supp essing TRPM7 ac i i y, sug-
ges ing he apeu ic po en ial o TRPM7-media ed pa hologies
including cance , s oke, and kidney disease (Suzuki e al., 2024).
The plan ’s e pene p o ile signi ican ly in luences cannabinoid ac-
i i y h ough he “en ou age e ec ”—a syne gis ic ela ionship be-
ween cannabinoids and e penes ha po en ia es he apeu ic e icacy
(Anand e al., 2021). Speci ic e penes such as β-my cene (MC), β-ca -
yophyllene (CPh), and ne olidol (NL) (Fig. 1A-A-D) ha e been shown o
enhance analgesic, an i-in lamma o y, and anxioly ic e ec s when
combined wi h cannabinoids, su passing he e ec s o indi idual com-
pounds (Russo, 2011; Fe be e al., 2020; Ch is ensen e al., 2023). Fo
example, Jansen e al. demons a ed ha a Cannabis sa i a-de i ed
e pene mix u e po en ia ed in acellula calcium in lux in TRPV1-
exp essing HEK cells, wi h MC showing he s onges ac i i y and NL
exhibi ing mode a e e ec s. No ably, MC-induced calcium in lux was
TRPV1-dependen , as e idenced by comple e inhibi ion wi h he TRPV1
an agonis capsazepine. Molecula docking s udies sugges ed MC binds
TRPV1 ia hyd ophobic, non-co alen in e ac ions (Jansen e al., 2019).
Ou esea ch g oup ecen ly de eloped nano omula ions o MC, CPh,
and NL encapsula ed in poly(e hylene glycol)-poly(lac ic-co-glycolic
acid) nanopa icles (PEG-PLGA NPs) and e alua ed hei e icacy in
HEK TRPV1 cells. An in-dep h s udy was conduc ed on he ac i i y o
ee and encapsula ed e penes, assessing bo h indi idual and combi-
na ional e ec s. Th ough calcium in lux assays (Fluo-4 indica o , 1-hou
obse a ion), we demons a ed ha he e pene-loaded NPs and hei
combina ions signi ican ly enhanced luo escence in ensi y compa ed o
ee e penes, con i ming NP-media ed imp o emen in cellula up ake
and bioac i i y (Small-Howa d e al., 2020; El-Hammadi e al., 2021; El-
Hammadi e al., 2022). These PLGA-based nanosys ems p o ide dis inc
ad an ages o d ug deli e y, including FDA-app o ed biocompa ibili y,
unable biodeg adabili y, and sus ained elease kine ics (El-Hammadi
e al., 2015; El-Hammadi and A ias, 2022; Kuma e al., 2024).
The syne gis ic e ec s (en ou age e ec ) obse ed be ween canna-
binoids and e penes highligh he po en ial o op imized he apeu ic
o mula ions. By exploi ing he en ou age e ec , new ea men s can be
de eloped o enhance e icacy while educing ad e se e ec s (Fe be
e al., 2020), o e ing new oppo uni ies o managing complex condi-
ions including ch onic pain, epilepsy, and men al heal h diso de s
(Pe zke e al., 2022; Leinen e al., 2023; Hoch e al., 2024). Based on his
p emise, ou s udy e alua ed he he apeu ic po en ial o CBGA-loaded
PLGA-PEG NPs in combined wi h e pene-loaded NPs o pain man-
agemen using in i o expe imen s. Th ee key cannabis-based e penes
we e u ilized in his wo k, namely β–my cene, β–ca yophyllene, and
ne olidol (Fig. 1).
2. Ma e ials and me hods
2.1. Ma e ials
MC ( e . W276200), PEG-PLGA (PEG a e age Mn 2,000, PLGA
a e age Mn 11,500; lac ide:glycolide 50:50], Plu onic® F-68, Span® 60,
and ehalose we e pu chased om Sigma-Me ck (Ge many). CPh (≥80
% pu i y, ood g ade) and NL (a mix u e o cis- and ans-isome s, ≥97 %
pu i y) we e p o ided by GB Sciences (NV, USA). Poly inyl alcohol
(PVA) 72,000 g/mol (polyme iza ion g ade 1600) and HPLC-g ade
sol en s (e hyl ace a e, and dichlo ome hane) we e ob ained om
Pan eac Química (Spain). All o he chemicals we e analy ical-g ade and
sou ced om Me ck (Ge many). Ul apu e wa e was p epa ed using a
Milli-Q Ad an age A10 sys em (Millipo e, Spain).
2.2. P epa a ion o nanopa icles
2.2.1. P epa a ion CBGA-loaded nanopa icles
CBGA-loaded NPs we e p epa ed using a nanop ecipi a ion ech-
nique ollowing ou es ablished p o ocol (Du an-Loba o e al., 2014;
Be ocoso e al., 2017). B ie ly, CBGA (3.75 mg), PLGA (22.5 mg) and
Span® 60 (7.5 mg) we e dissol ed in 1.5 mL o ace one. The o ganic
phase was hen slowly in oduced d opwise in o 4.5 mL o an aqueous
Plu onic® F-68 solu ion (0.5 % w/ ) using a sy inge pump a a
con olled a e o 5 mL/h unde con inuous s i ing (500 pm). Ace one
was subsequen ly e apo a ed o e 1 h in a chemical hood a oom
Fig. 1. Chemical s uc u es o (A) β–my cene, (B) β–ca yophyllene, (C) ans-ne olidol, (D) cis-ne olidol, and (E) cannabige olic acid (CBGA).
M.M. El-Hammadi e al.
In e na ional Jou nal o Pha maceu ics 679 (2025) 125766
2
empe a u e. The concen a ions o Span® 60 and Plu onic® F-68 we e
selec ed based on p e iously op imized condi ions de eloped by ou
g oup o cannabinoid nanoencapsula ion, ensu ing consis en pa icle
size, s abili y, and encapsula ion e iciency ac oss ba ches (Du an-
Loba o e al., 2014; Be ocoso e al., 2017; Al a ez-Fuen es e al., 2012;
Ma in-Bande as e al., 2014). P ocess pa ame e s o nanop ecipi a ion,
including s i ing speed, sol en - o-nonsol en a io, and injec ion a e.
we e chosen based on p io op imiza ions o achie e minimal poly-
dispe si y index (PDI) and consis en pa icle size. To elimina e unen-
capsula ed compounds and excess su ac an , he esul ing suspension
was ans e ed o an Amicon® ube and spun a 4,000 ×g a 12 ◦C. Fo
lyophiliza ion, ehalose (1:5 w/w PLGA-PEG/ ehalose) was added o
he concen a ed NPs, ollowed by eeze-d ying (48 h, −80.0 ±0.5 ◦C
and 0.057 mba ; C yodos eeze-d ie , Tels a Indus ial S.L., Spain).
Pos -lyophiliza ion s abili y was assessed by DLS analysis o econ-
s i u ed NPs o size and ze a po en ial, wi h measu emen s pe o med
unde iden ical condi ions o p e-lyophiliza ion cha ac e iza ion.
2.2.2. Te pene-loaded nanopa icles
Te pene-loaded PEG-PLGA NPs we e p epa ed ia emulsion-sol en
e apo a ion (Small-Howa d e al., 2020; El-Hammadi e al., 2021).
B ie ly, 10 mg o e pene (MC, CPh, o NL) and 40 mg o PEG-PLGA we e
dissol ed in 1 mL o e hyl ace a e (o ganic phase), which was hen
added d opwise o 5 mL o a 0.5 % (w/ ) PVA aqueous solu ion unde
high-speed homogeniza ion (Poly on® PT 2500 E, Kinema ica AG,
Swi ze land) o 1 min. The 1:5 o ganic- o-aqueous phase a io p o-
mo ed op imal nanopa icle cha ac e is ics by ensu ing apid sol en
di usion and s able emulsion o ma ion (El-Hammadi e al., 2021).
Following sol en emo al ( o a y e apo a ion), NPs we e pu i ied ia
ul a il a ion (Amicon® ubes wi h Ul acel-100 kDa egene a ed cel-
lulose memb anes, 15 mL capaci y) a 4,000 ×g and 12 ◦C. The esul ing
NPs we e esuspended in a ehalose solu ion (1:5; PLGA-PEG/
ehalose), and lyophilized (−80 ◦C, <0.100 mba ; C yodos eeze-
d ie , Tels a Indus ial S.L., Spain) o ob ain a whi e, co on-like
powde .
2.3. Nanopa icle cha ac e iza ion
Pa icle size dis ibu ion and ze a po en ial we e de e mined by dy-
namic ligh sca e ing (DLS) and lase Dopple elec opho esis, espec-
i ely, using a Ze asize Nano ZS (Mal e n Ins umen s L d., UK). Fo
mo phological analysis, nanopa icle suspensions we e applied o
ca bon-coa ed coppe g ids, s ained wi h 2 % u anyl ace a e, and ai -
d ied p io o imaging by ansmission elec on mic oscopy (TEM;
Zeiss Lib a 120, Ca l Zeiss Mic oscopy, Ge many).
2.4. Measu emen o CBGA load capaci y
CBGA loading in o he PLGA-PEG NPs was de e mined using a
e e se phase-high pe o mance liquid ch oma og aphy (RP-HPLC)
me hod, as p e iously desc ibed o cannabinoids wi h some modi ica-
ions (Al a ez-Fuen es e al., 2012). The RP-HPLC analysis was pe -
o med using a Hi achi LaCh om Eli e® HPLC Sys em, equipped wi h a
qua e na y pump L-2130, a diode a ay de ec o L-2455, and an au o-
ma ic injec o L-2200. A C18 Wa e s A lan is T3 column (3 µm, 4.6 ×
100 mm) main ained a 25.0 ±0.1 ◦C (L-2350 column o en, Eli e
LaCh om1) was used in his analysis. The mobile phase was me hanol:
ace oni ile:wa e (52:30:18 / ) a pH 4.5. The mobile phase was
main ained a 1.8 mL/min low a e. The de ec ion wa eleng h was 227
nm and he injec ion olume was 20 µL. CBGA s anda d solu ions
(33.75–500 µg/mL in me hanol) yielded a sha p, symme ic peak
( e en ion ime: 3.7 ±0.1 min) wi hin an 8-min un ime. Fo analysis,
NPs we e dissol ed in ace oni ile, and da a we e p ocessed using
EZCh om Eli e So wa e.
Encapsula ion e iciency (EE%) and d ug loading (DL%) we e
calcula ed using Eqs. (1) and (2):
EE(%) = masso inco po a edCBGA(mg)
ini ialmasso CBGA(mg)×100 (1)
DL(%) = masso inco po a edCBGA(mg)
masso nanoca ie (mg)×100 (2)
2.5. Measu emen o e pene load capaci y
The e pene con en in nanopa icles was quan i ied by gas
ch oma og aphy-mass spec ome y (GC–MS) using a TRACE GC sys em
coupled wi h a DELTA V™ mass spec ome e (The mo Scien i ic, USA)
ollowing es ablished me hods (El-Hammadi e al., 2021; El-Hammadi
e al., 2022). Lyophilized NPs we e dissol ed in dichlo ome hane
(DCM) and analyzed using an SPB-1 capilla y column (30 m ×0.25 mm
×1.5 µm) wi h a 15-minu e un ime, showing e en ion imes o 6.92
min (MC), 5.73 min (CPh), 6.77 min (cis-NL), and 7.16 min ( ans-NL).
The mass spec ome e ope a ed in elec on impac (EI) mode (70 eV)
wi h ull scan acquisi ion (m/z 20–300) and an ion sou ce empe a u e
o 250 ◦C. Quan i ica ion employed selec ed ion moni o ing (SIM) o
cha ac e is ic agmen ions a m/z 69 (MC), 93 (CPh), and 133 (NL).
Da a we e p ocessed using Xcalibu 2.0.7 so wa e (The mo Scien i ic,
USA), and e pene con en was calcula ed as encapsula ion e iciency
(EE%) and d ug loading (DL%) acco ding o Eqs. (3) and (4).
EE(%) = masso e peneincopo a ed(mg)
ini ialmasso e pene(mg)×100 (3)
DL(%) = masso e peneincopo a ed(mg)
masso nanopa icles(mg)×100 (4)
2.6. In i o elease o CBGA om nanopa icles
The in i o elease p o ile o CBGA om nanopa icles was e alua ed
using a dialysis me hod unde sink condi ions. CBGA-loaded NPs (1 mL,
1.6 mg/mL CBGA equi alen ) we e placed in Visking dialysis ubing
(12–14 kDa MWCO, Medicell In e na ional, UK) and imme sed in 4 mL
o PBS (pH 7.4 ±0.1) con aining 0.5 % (w/ %) Tween 80. The sys em
was incuba ed a 37.0 ±0.5 ◦C in a he mos a ic shake (500 pm;
Ti amax 1000, Heidolph, Ge many), wi h ee (non-encapsula ed)
CBGA solu ion, ea ed unde he same condi ions as he nanopa icle
o mula ions, se ing as con ol. Aliquo s (350 µL) we e collec ed a
p ede e mined in e als (0.5–80 h) and immedia ely eplaced wi h esh
bu e o main ain cons an olume. CBGA concen a ion in he elease
medium was quan i ied by HPLC, wi h esul s exp essed as cumula i e
d ug elease pe cen age o e ime.
% Cumula i e d ug elease =amoun o d ug loaded in NPs [mg] − amoun o d ug emained in NPs [mg]
amoun o d ug loaded in NPs [mg]×100 (5)
M.M. El-Hammadi e al.
In e na ional Jou nal o Pha maceu ics 679 (2025) 125766
3
2.7. In i o expe imen s
2.7.1. Cell main enance
HEK293 cells s ably exp essing TRPV1 channels (kindly p o ided by
D . Helen Tu ne , Chaminade Uni e si y o Honolulu) we e cul u ed in
Minimum Essen ial Medium (MEM; Co ning, NY, USA) supplemen ed
wi h 10 % e al bo ine se um (FBS), 50 U/mL penicillin, 50 µg/mL
s ep omycin (Sigma-Ald ich), and 0.6 mg/mL gene icin (Gibco,
Swi ze land). Cells we e main ained a 37 ◦C wi h 5 % CO
2
humidi ied
a mosphe e.
2.7.2. Recons i u ion and p epa a ion o nanopa icles o in i o assays
Fo biological es ing, lyophilized NPs we e asep ically econs i u ed
in s e ile phospha e-bu e ed saline (PBS) o hei o iginal olume using
a lamina low hood. The esul ing nanopa icle suspensions we e hen
dilu ed in ei he assay bu e o comple e cul u e medium o achie e he
desi ed ea men concen a ions immedia ely p io o expe imen a ion.
2.7.3. De e mina ion o EC50 o CBGA- and e pene-loaded nanopa icles
The hal -maximal e ec i e concen a ion (EC50), de ined as he
concen a ion needed o achie e 50 % o he maximum e ec , was
de e mined o each NP o mula ion by e alua ing calcium in lux in
HEK-TRPV1 cells. Cells we e exposed o se ial dilu ions o CBGA-, MC-,
NL-, o CPh-loaded NPs o 60 min while moni o ing in acellula cal-
cium le els using luo escence. Pa allel cy o oxici y assessmen s we e
conduc ed unde iden ical exposu e condi ions o ensu e he selec ed
concen a ion anges main ained cell iabili y >80 %.
2.7.3.1. Calcium signaling assay. The calcium signaling assay can be
used o measu e he cy osolic concen a ion o calcium ions. In his
me hod, he non- luo escen Fluo-4 ace oxyme hyl es e (Fluo-4 AM;
The mo Fishe Scien i ic) is clea ed by in acellula es e ases o elease
he ee, luo escen calcium indica o Fluo-4. Upon binding o calcium
ions, Fluo-4 emi s a s ong g een luo escence when exci ed by ligh a a
wa eleng h o 488 nm, allowing o he de ec ion and isualiza ion o
calcium le els wi hin he cell.
2.7.3.1.1. Assay p ocedu e. HEK TRPV1 cells we e ha es ed using
ypsin, ollowed by deac i a ion wi h media and cen i uga ion a
1,000 pm o 5 min a oom empe a u e. The cells we e hen coun ed,
washed wice wi h 1 mM calcium assay bu e (composed o Na Ringe ’s
solu ion [140 mM NaCl, 2.8 mM KCl, 2 mM MgCl
2
, 11 mM glucose, 10
mM HEPES], 2 mM p obenecid, 1 mM CaCl
2
; pH 7.4), and collec ed by
cen i uga ion unde he same condi ions. The cells we e esuspended in
a 1
μ
M Fluo-4 AM solu ion (p epa ed by mixing 1
μ
L o 5 mM Fluo-4 AM
wi h 1
μ
L o 20 % Plu onic F-127 in DMSO and he addi ion o 5 mL o 1
mM calcium assay bu e , ollowed by incuba ion o 10 min a 37 ◦C in
he da k). The suspended cells we e hen u he incuba ed o 30 min a
37 ◦C in he da k. Following incuba ion, he cells we e washed wice
wi h he calcium assay bu e , esuspended in he bu e , and seeded in o
opaque-walled 96-well pla es a a densi y o 15 ×10
4
cells pe 180
μ
L/
well. Fluo escence was measu ed using a pla e eade (Syne gy HTX,
BioTek, USA) wi h exci a ion and emission wa eleng hs o 485 nm and
528 nm, espec i ely. A e es ablishing a baseline ( h ee measu e-
men s), 20
μ
L o he s imulan solu ion/suspension ( ee CBGA, CBGA-
loaded NPs, e pene-loaded NPs, o con ols) was added o each well,
and measu emen s con inued o 1 h a a a e o one ead e e y 26 sec.
Con ol esponses we e sub ac ed, and he esul ing da a we e a e aged
o gene a e calcium signal p o iles. The a ea unde he cu e (AUC) o
each concen a ion o CBGA/ e pene NPs was also calcula ed o u he
analysis.
2.7.3.1.2. P epa a ion o s imulan dispe sions. All NP dispe sions
we e p epa ed in 1 mM Ca Assay Bu e immedia ely p io o expe i-
men a ion. The NPs we e dilu ed di ec ly using he assay bu e , and
blank NPs we e also p epa ed a equi alen concen a ions. CBGA- and
e pene-loaded NPs we e p epa ed a concen a ions o 10, 40, 100, 200,
400, and 1000
μ
g/mL, yielding inal well concen a ions o 1, 4, 10, 20,
40, and 100
μ
g/mL a e 1:10 dilu ion in cell suspensions. Ionomycin (4
μ
M), a calcium ionopho e ha inc eases he in acellula concen a ion
o Ca
2+
, was used as a posi i e con ol. All nanopa icle suspensions
we e o exed (30 sec) and sonica ed (5 min, 37 ◦C) immedia ely be o e
use o ensu e monodispe sion.
2.7.3.2. In i o cy o oxici y. To de e mine he e ec i e and non- oxic
concen a ions o he NPs, he cy o oxici y o he CBGA and e pene-
loaded NPs was assessed using he MTT assay (3-(4,5-dime hyl hiazol-
2-yl)-3,5-diphenyl e azolium b omide) a e a sho 60 min incuba ion
pe iod. As demons a ed in ou p io wo k (El-Hammadi e al., 2022),
blank NPs showed no cy o oxici y (cell iabili y >95 % a all es ed
concen a ions) and we e consequen ly excluded om his assay. Cells
we e seeded in 96-well pla es a a densi y o 45 ×10
3
cells pe well and
cul u ed o 48 h a 37 ◦C in a 5 % CO
2
a mosphe e. The cells we e hen
ea ed wi h a ious concen a ions o CBGA- o e pene-loaded NPs (1,
4, 10, 20, 40, 100 µg/mL; co esponding o he concen a ion o
encapsula ed compound) in cul u e medium. A e 60 min o incuba ion
a 37.0 ±0.5 ◦C, he medium was emo ed and eplaced wi h 200
μ
L o
MTT solu ion (0.5 mg/mL in cul u e medium). Following a 4-hou in-
cuba ion a he same empe a u e, he medium was disca ded, and 200
μ
L o DMSO was added o each well o solubilize he o mazan c ys als.
Un ea ed cells and cells ea ed wi h 1 % T i on X-100 we e used as
con ols. The op ical densi y (OD) a 570 nm was measu ed using a
mic opla e eade (Syne gy HT, BioTek Ins umen s, Inc., Ve mon ,
USA). Rela i e cell iabili y (%) was compu ed using he ollowing
o mula:
Rela i e cell iabili y(%) = OD ea ed cells
OD con ol (un ea ed)cells ×100 (6)
Concen a ions esul ing in a iabili y dec ease below 80 % we e
conside ed cy o oxic adhe ing o ISO 10993-5:2009 s anda ds.
2.7.3.3. Calcula ion o EC50. The EC50 o CBGA and e pene-loaded
NPs we e de e mined based on he AUC alues, calcula ed om luo-
escence esponses measu ed o e 60 min in HEK cells incuba ed wi h
a ious concen a ions o he NPs unde in es iga ion. EC50 alues we e
de i ed om nonlinea eg ession o log- ans o med concen-
a ion– esponse da a (G aphPad P ism 8), wi h cu e i s cons ained o
baseline (0 %) and maximal esponse (100 % ionomycin con ol).
2.7.4. In es iga ing combined e ec s o CBGA NPs wi h e pene NPs
To e alua e he combined e ec s o CBGA NPs and e pene NPs on
calcium in lux in HEK TRPV1 cells, a calcium signaling assay was con-
duc ed, ollowed by luo escence imaging and analysis 30 min a e
ea men .
2.7.4.1. Calcium signaling assay. This calcium signaling assay was
employed o in es iga e he impac o combining CBGA NPs wi h
e pene NPs on he calcium in lux in HEK TRPV1 cells.
2.7.4.1.1. Assay p ocedu e. The p ocedu e ou lined in Sec ion
2.7.3.1.1 was ollowed, wi h he AUC calcula ed o each plo co e-
sponding o he speci ic ea men s.
2.7.4.1.2. P epa a ion o s imulan dispe sions. CBGA, MC, NL, and
CPh NPs, along wi h combina ions o CBGA NPs wi h ei he MC, NL, o
CPh NPs, we e dispe sed in 1 mM calcium assay bu e . Nanopa icle
dispe sions we e p epa ed a 10 imes hei EC50 concen a ion o
achie e he desi ed EC50 concen a ion upon addi ion o he cells. In
combina ion o mula ions, he inal concen a ions o bo h CBGA NPs
and e pene NPs we e adjus ed o ma ch hei espec i e EC50 alues.
Addi ionally, blank NPs we e p epa ed a co esponding concen a ions.
A ou
μ
M ionomycin solu ion was used as a posi i e con ol.
M.M. El-Hammadi e al.
In e na ional Jou nal o Pha maceu ics 679 (2025) 125766
4
2.7.4.2. Fluo escence imaging – Image acqui ing and analysis
2.7.4.2.1. Assay p ocedu e. The abili y o he nano o mula ions o
modula e calcium in lux in HEK TRPV1 cells was u he assessed by
measu ing in e cellula luo escence in ensi y using Fluo-4 AM unde a
luo escence mic oscope. In his se o expe imen s, HEK TRPV1 cells
we e seeded in 96-well cell cul u e pla es a densi y o 3 ×10
4
pe well in
200 µL o cell suspension. The pla es we e incuba ed o 48 h a 37 ◦C a
humidi ied a mosphe e wi h 5 % CO
2
. On he day o ea men , he cells
we e washed wice wi h 1 mM Ca Assay Bu e . Fluo-4 AM (1
μ
M) was
added o each well (100
μ
L pe well), and he pla es we e incuba ed o
30 min a 37 ◦C in he da k. A e incuba ion, he cells we e washed
wice wi h 1 mM Ca Assay Bu e , ollowed by he addi ion o 180
μ
L o
1 mM Ca Assay Bu e pe well p io o ea men .
2.7.4.2.2. P epa a ion o s imulan solu ions/dispe sions. S imulan
dispe sions we e p epa ed ollowing he same p ocedu e desc ibed in
Sec ion 2.7.3.1.2.
2.7.4.2.3. T ea men and imaging. A olume o 20
μ
L o he p e i-
ously p epa ed s imulan s o con ols was added o each well, b inging
he o al olume o 200
μ
L pe well (20
μ
L s imulan /con ol +180
μ
L
bu e ). The inal concen a ions o CBGA and e pene-loaded NPs, bo h
indi idually and in combina ion, we e adjus ed o ma ch he EC50
alues o each espec i e nanopa icle o mula ion. Fo example, in he
CBGA/MC combina ion, CBGA NPs and MC NPs we e applied a hei
espec i e EC50 concen a ions du ing incuba ion wi h he cells.
The in ensi y o in acellula g een luo escence was obse ed using
Nikon in e ed mic oscope Eclipse Ti (Japan). Li e-cell images we e
cap u ed 30 min pos - ea men wi h he ollowing exposu e se ings: 1-
second exposu e ime, 7.6 ×gain, and 470 nm emission wa eleng h,
using a FITC il e .
2.7.4.2.4. Image analysis. The acqui ed images we e analyzed using
ImageJ so wa e (Ve sion 1.52 ; NIH, USA). The luo escence in ensi y
was calcula ed using he ollowing equa ion:
cell luo esence =IT− (AT×IB
AB)
N(7)
whe e: I
T
, o al in ensi y o he image; A
T
: o al a ea o he image; I
B
,
backg ound in ensi y; A
B
, backg ound a ea; and N, numbe o cells.
Subsequen ly, he ela i e luo escence in ensi y was exp essed as a
pe cen age using he ollowing equa ion:
I%=IT ea −INegC
IIono −INegC
×100 (8)
whe e: I%, ela i e in ensi y pe cen age; I
T ea
, luo escence in ensi y o
ea ed cells; I
Iono
, luo escence in ensi y o cells ea ed wi h ionomycin;
and I
Neg C
, luo escence in ensi y o he nega i e con ol.
2.8. S a is ical analysis
All da a a e p esen ed as he mean ±s anda d de ia ion (SD). All
expe imen s we e conduc ed in a leas h ee independen assays. G oup
compa isons we e pe o med using one-way ANOVA wi h LSD pos hoc
es ing. Da a analysis was conduc ed using SPSS S a is ics e sion 26.
S a is ical signi icance was se a p <0.05 o all analyses.
3. Resul s
3.1. Nanopa icle cha ac e iza ion
The physicochemical cha ac e iza ion o nanopa icles e ealed
well-de ined o mula ions sui able o biological e alua ion (Table 1).
CBGA-loaded NPs displayed a mean hyd odynamic diame e o 152 nm,
while e pene-loaded NPs (MC, NL, CPh) showed sligh ly la ge sizes o
233 nm, 279 nm, and 297 nm, espec i ely. All o mula ions exhibi ed
excellen monodispe si y (PdI <0.3) and nega i e ze a po en ials
anging om −23 o −25 mV, indica ing s able colloidal suspensions.
Encapsula ion e iciency was ma kedly highe o CBGA (98.6 ±0.9 %)
compa ed o e penes (13.2 ±1.2 % o MC, 28.4 ±2.2 % o NL, and
32.9 ±2.3 % o CPh). Following lyophiliza ion and econs i u ion, NPs
main ained hei o iginal cha ac e is ics wi h less han 10 % a ia ion in
size, PdI, and ze a po en ial, con i ming he s abili y o he o mula ions
unde p ocessing condi ions. T ansmission elec on mic oscopy u he
e i ied he absence o pa icle agg ega ion pos -lyophiliza ion (Fig. 2).
3.2. CBGA elease kine ics
The in i o elease o CBGA om he PLGA-PEG NPs was assessed in
PBS (pH 7.4 ±0.1) con aining 0.5 % (w/ %) Tween® 80 a 37.0 ±
0.5 ◦C. The CBGA NPs exhibi ed biphasic elease kine ics, cha ac e ized
by an ini ial bu s elease phase (~20 % cumula i e elease wi hin 4 h)
ollowed by sus ained d ug elease o e 72 h (Fig. 3). In con as , ee
CBGA (con ol) apidly di used h ough he dialysis memb ane,
eaching equilib ium wi hin 2 h.
Finally, in i o elease p o ile was analyzed using Kine DS so wa e
Table 1
Cha ac e is ics o CBGA-, MC-, NL- and CPh-loaded nanopa icles ( alues a e he mean ±SD).
Fo mula P epa a ion me hod Mean diame e
(nm)
PdI Ze a po en ial
(mV)
EE% DL%
CBGA NPs Nanop ecipi a ion 152.0 ±8.3 0.262 ±0.018 −25.6 ±1.5 98.6 ±0.9 12.1 ±0.3
MC NPs Emulsion-sol en e apo a ion 233.4 ±6.4 0.238 ±0.007 −24.2 ±1.1 13.2 ±1.2 3.4 ±0.3
NL NPs Emulsion-sol en e apo a ion 279.3 ±10.9 0.242 ±0.021 −24.9 ±2.2 28.1 ±2.2 7.0 ±0.5
CPh NPs Emulsion-sol en e apo a ion 296.9 ±9.7 0.224 ±0.027 −23.4 ±0.9 32.9 ±2.3 7.8 ±0.5
Fig. 2. TEM images o CBGA-loaded PEG-PLGA NPs. Scale ba : 100 nm.
M.M. El-Hammadi e al.
In e na ional Jou nal o Pha maceu ics 679 (2025) 125766
5
o iden i y he dominan elease mechanisms. The bes linea co ela ion
was ob ained when pe cen age o eleased d ug was i ed o adi ional
me hods, such as he Ko smeye -Peppas (K-P) and Weibull models, bo h
widely used o desc ibe in i o d ug elease kine ics om PLGA-based
sys ems (Table 2).
The K–P model desc ibes d ug elease using a powe -law equa ion,
while he Weibull model is o en p e e ed o i s g ea e lexibili y in
cap u ing bo h Fickian and non-Fickian di usion beha io s. In he
p esen s udy, he bes i was achie ed wi h he Weibull model (R
2
=
0.984), highligh ing i s sui abili y o cha ac e izing he elease p o ile
o CBGA om PLGA-based nanopa icles. In con as , he K–P model
showed a lowe co ela ion (R
2
=0.861), e lec ing i s mo e limi ed
applicabili y in complex, biphasic elease sys ems. The supe io i o he
Weibull model suppo s he biphasic elease beha io obse ed expe i-
men ally, cha ac e ized by an ini ial bu s ollowed by sus ained elease.
3.3. De e mina ion o EC50 alues
The EC50 alues o CBGA, MC, NL, and CPh NPs we e de e mined
by e alua ing a ange o NP concen a ions and moni o ing he calcium
signaling/ luo escence esponse in TRPV1 HEK cells (Fig. 4). The
e ec i e concen a ion ange was es ablished by compa ing he AUC
alues o he luo escence esponse plo s (Fig. 5A). In addi ion, he MTT
cy o oxici y assay was pe o med o iden i y he non-cy o oxic concen-
a ion ange o he de eloped NPs (Fig. 5B). As a esul , he maximum
e ec i e concen a ions (i.e., concen a ions beyond which no s a is i-
cally signi ican inc ease in e ec was obse ed) we e de e mined o be
40 µg/mL o CBGA NPs, 20 µg/mL o MC NPs, 10 µg/mL o NL NPs,
and 20 µg/mL o CPh NPs.
Nex , he EC50 o each NP o mula ion was calcula ed om he
concen a ion– esponse cu es gene a ed by plo ing no malized AUC
alues o calcium lux agains log- ans o med NP concen a ions
(Fig. 6). The AUCs o he luo escence esponse o cells ea ed wi h NPs
we e no malized agains he AUC o he ionomycin luo escence
esponse. The esul ing EC50 alues a e p esen ed in Table 3.
3.4. In es iga ing he combined e ec s o CBGA NPs wi h e pene NPs
The calcium esponses o HEK TRPV1 cells o co- ea men wi h
CBGA NPs wi h MC, NL, and CPh NPs we e in es iga ed (Fig. 7). O e all,
all combina ions esul ed in a signi ican inc ease in calcium in lux
compa ed o he e ec s o he cannabinoid o indi idual e penes alone
(Fig. 7B).
Fig. 7C p esen s a compa ison be ween he calcula ed and expe i-
men al AUC alues o calcium esponse plo s o cells ea ed wi h
Fig. 3. Cumula i e elease p o ile o CBGA om PLGA-PEG NPs. The nano-
pa icles we e enclosed in a dialysis bag, and he elease was moni o ed in
phospha e-bu e ed saline (PBS, pH 7.4) con aining 0.5 % (w/ ) Tween 80 a
37 ◦C. F ee CBGA, subjec ed o he same condi ions, was used as a e e ence
o compa ison.
Table 2
Co ela ion coe icien s (R
2
), a e cons an s (K) and elease exponen (A) ob-
ained om i ing elease da a o Weibull and Ko smeye -Peppas models.
Model Ma hema icla equa ion R
2
A K
Weibull
y=100⎛
⎜
⎝1−e− ( −lag
k)A⎞
⎟
⎠
0.984 0.831 10.407
Ko smeye -
Peppas
ln(y) = a×ln(x) + b; Which is
equal o y=K×xa
0.861 0.998 3.052
Fig. 4. Rela i e luo escence uni s (RFU) measu ed using a calcium signaling assay. HEK TRPV1 cells (15 ×10
4
cells/well, 180
μ
l) we e p e- ea ed wi h 1
μ
M Fluo-4
AM and suspended in 1 mM Ca Assay Bu e p io o he addi ion o s imulan s (CBGA NPs o e pene-loaded NPs) a a ious concen a ions. Ionomycin (4
μ
M) was
used as a posi i e con ol. Fluo escence was measu ed o e a 1-hou pe iod, wi h eadings aken e e y 26 s a an exci a ion/emission wa eleng hs o 485/528 nm.
(A) Calcium esponses induced by ee and encapsula ed CBGA and (B) co esponding AUCs (p <0.05); (C) calcium esponses by CBGA NPs; (D) calcium esponses
induced by MC NPs; (E) calcium esponses induced by NL NPs; and (F) calcium esponses induced by CPh NPs. The expe imen was conduc ed in iplica e (n =3),
and he calcium esponse esul s a e p esen ed as he mean alues.
M.M. El-Hammadi e al.
In e na ional Jou nal o Pha maceu ics 679 (2025) 125766
6
combina ions o CBGA NPs and e pene NPs. The calcula ed alues we e
ob ained by summing he indi idual AUCs o CBGA NPs and e pene NPs
p esen ed in Fig. 6B. The combina ion o CBGA/NL NPs demons a ed a
signi ican ly highe expe imen al alue compa ed o he calcula ed
alue, whe eas no s a is ically signi ican di e ences we e obse ed o
he CBGA/MC and CBGA/NL combina ional o mula ions.
3.5. Fluo escence imaging – Image acquisi ion and analysis
Fig. 8 shows luo escence images o HEK TRPV1 cells acqui ed 30
min a e ea men wi h (Fig. 8A) assay bu e , (Fig. 8B) blank NPs,
(Fig. 8C) ionomycin, e pene-loaded NPs (Fig. 8D-F), CBGA NPs
(Fig. 8G), and combina ions o CBGA NPs wi h e pene-loaded NPs
(Fig. 8H-J). Fluo escence in ensi y om hese images was quan i ied and
exp essed as a ela i e pe cen age by compa ing he luo escence o cells
ea ed wi h NPs o ha o cells ea ed wi h he posi i e con ol, ion-
omycin (Equa ion (8)).
O e all, a he es ed concen a ions (EC50), all combina ions o
CBGA NPs wi h e pene-loaded NPs showed signi ican ly highe luo-
escence in ensi y compa ed o indi idual NPs (p <0.05; Fig. 8K).
Fu he mo e, he expe imen al ela i e luo escence in ensi ies (RFI) o
CBGA/ e pene combina ions we e gene ally highe han calcula ed
alues, wi h s a is ically signi ican di e ences obse ed o CBGA/NL
and CBGA/CPh combina ional nano o mula ions (p <0.05; Fig. 8L).
4. Discussion
This s udy highligh s he po en ial o nanopa icle-based deli e y
sys ems o enhance he bioac i i y o cannabinoids and e penes o
a ge ed he apeu ic applica ions. PEG–PLGA NPs encapsula ing CBGA
and h ee cannabis-de i ed e penes, including MC, NL, and CPh we e
success ully o mula ed and cha ac e ized, and hei e ec s we e sub-
sequen ly e alua ed in HEK cells o e exp essing he TRPV1 ecep o .
To ensu e ep oducibili y and consis ency in nanopa icle o mula-
ion, we employed s anda dized and alida ed p epa a ion p o ocols o
bo h CBGA- and e pene-loaded NPs, esul ing in minimal ba ch- o-
ba ch a iabili y (<10 %) in size and ze a po en ial. Consis en d ug
loading and encapsula ion e iciencies we e con i med using HPLC and
GC–MS, ensu ing accu a e dosing ac oss expe imen s. The lowe
encapsula ion e iciency obse ed o e penes, compa ed o CBGA, is
likely a ibu able o hei ola ile and liquid na u e, which makes hem
mo e suscep ible o di usion losses du ing o mula ion (El-Hammadi
e al., 2021); unlike he mo e s able, solid-s a e CBGA. In addi ion,
TRPV1 ac i a ion assays we e conduc ed using EC50 concen a ions
de i ed om independen ly alida ed dose– esponse cu es, enabling
Fig. 5. De e mina ion o non-cy o oxic and e ec i e concen a ions o CBGA- and e pene-loaded NPs. (A) In i o iabili y o HEK TRPV1 a e a 1-hou exposu e o
a ious concen a ions o nanopa icles. Un ea ed cells se ed as he con ol o calcula ing ela i e cell iabili y (%). (B) A eas unde he cu e (AUCs) o luo-
escence in ensi y o HEK TRPV1 cells incuba ed wi h di e en concen a ions o NPs, measu ed o e a 1-hou pe iod (co esponding o he plo s in Fig. 3). Da a a e
ep esen ed as means ±SD om iplica e cul u es (n =6).
Fig. 6. A ea unde he cu e (AUC) o luo escence esponses in HEK cells
when incuba ed wi h a ying concen a ions o CBGA- and e pene-loaded NPs
o 1 h. Resul s we e no malized o he luo escence esponses o cells ea ed
wi h ionomycin. Da a a e p esen ed as means ±SD om iplica e expe imen s.
Table 3
Hal -maximal e ec i e concen a ion (EC50) alues calcula ed o CBGA, MC,
NL, and CPh NPs. Da a a e ep esen ed as means ±SD om iplica e cul u es.
Fo mula EC50 Co esponding esponse
(%AUC no malized o ionomycin)µg/mL µM
CBGA NPs 23.8 ±2.4 65.9 ±6.5 45.8 ±2.0
MC NPs 8.0 ±0.6 61.5 ±4.1 14.1 ±1.6
NL NPs 6.7 ±0.9 29.9 ±4.2 21.9 ±5.4
CPh NPs 13.3 ±0.5 65.3 ±2.4 3.3 ±0.7
M.M. El-Hammadi e al.
In e na ional Jou nal o Pha maceu ics 679 (2025) 125766
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Fig. 7. Fluo escence changes measu ed using a calcium signaling assay. HEK TRPV1 cells (15 ×10
4
cells/well; 180
μ
L) we e p e- ea ed wi h 1
μ
M Fluo-4 AM and
suspended in 1 mM Ca Assay Bu e be o e he addi ion o s imulan s (CBGA NPs, e pene-loaded NPs, o hei combina ion) a a inal concen a ion co esponding o
he EC50 o each ype o nanopa icle. Ionomycin (4
μ
M) was used as a posi i e con ol. Fluo escence was moni o ed o e 1 h, wi h eadings aken e e y 40 s a an
exci a ion/emission wa eleng h o 485/528 nm. (A) Calcium esponse plo s; (B) co esponding AUCs, and (C) compa isons o calcula ed and expe imen al AUC
alues co esponding o combina ions o CBGA NPs and e pene NPs. The expe imen was conduc ed in iplica e (n =3). Calcium esponses and AUC esul s a e
p esen ed as mean alues and mean ±SD, espec i ely. S a is ically signi ican di e ences a e indica ed as ollows: * o combina ion o mula s. CBGA NPs (p <
0.05); † o combina ion o mula s. espec i e e pene NPs (p <0.05); # o expe imen al combina ion o mula s. calcula ed combina ion o mula (p <0.05).
M.M. El-Hammadi e al.
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8
biologically ele an and consis en compa isons. While la ge-scale
p oduc ion and long- e m s abili y equi e u he de elopmen , ou
cu en me hodology o e s a obus and ep oducible pla o m o
p eclinical applica ions.
The physicochemical p ope ies o he nano o mula ions (such as
size, su ace cha ge, and composi ion) play a key ole in modula ing
biological in e ac ions. All NPs exhibi ed uni o m pa icle sizes wi hin
he op imal sub-300 ange wi h a na ow size dis ibu ion (PdI <0.3),
which a e gene ally a o able o cellula up ake and in e ac ion wi h
memb ane-localized ecep o s like TRPV1. Al hough up ake is ypically
mo e e icien o pa icles unde 200 nm, se e al s udies ha e demon-
s a ed ha nanopa icles app oaching 300 nm can also be in e nalized
by cells h ough al e na i e endocy ic pa hways, depending on hei
su ace p ope ies and he cell ype in ol ed (L´
opez-Vio a e al., 2023;
G a on e al., 2008; Pe i ho y e al., 2021). Ne e heless, signi ican
di e ences we e obse ed be ween CBGA and e pene-loaded NPs in
Fig. 8. Fluo escence images o HEK TRPV1 cells, p e-incuba ed wi h Fluo-4, cap u ed 30 min a e he addi ion o : (A) bu e , (B) blank NPs, (C) ionomycin, (D) CPh
NPs, (E) MC NPs, (F) NL NPs, (G) CBGA NPs, (H) CBGA NPs/CPh NPs, (I) CBGA NPs/MC NPs, and (J) CBGA NPs/NL NPs. The concen a ions o CBGA- and e pene-
loaded NPs in bo h indi idual and combina ion o mula ions we e se a he EC50 o each espec i e nanopa icle. The images shown a e ep esen a i e o h ee
independen expe imen s. Scale ba : 50
μ
m. (K) co esponding Rela i e luo escence in ensi y (RFI) o cell images, and (L) compa isons o calcula ed and expe i-
men al RFI alues co esponding o combina ions o CBGA NPs and e pene NPs. The expe imen was conduc ed in iplica e (n =3). RFI alues a e p esen ed as
mean ±SD, espec i ely. S a is ically signi ican di e ences a e indica ed as ollows: * o combina ion o mula s. CBGA NPs (p <0.05); † o combina ion o mula
s. espec i e e pene NPs (p <0.05); # o expe imen al combina ion o mula s. calcula ed combina ion o mula (p <0.05).
M.M. El-Hammadi e al.
In e na ional Jou nal o Pha maceu ics 679 (2025) 125766
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