Development, Characterization and In Vitro Gastrointestinal Release of PLGA Nanoparticles Loaded with Full-Spectrum Cannabis Extracts

Vol.:(0123456789)
AAPS Pha mSciTech (2024) 25:120
h ps://doi.o g/10.1208/s12249-024-02836-4
RESEARCH ARTICLE
De elopmen , Cha ac e iza ion andIn Vi o Gas oin es inal Release
o PLGA Nanopa icles Loaded wi hFull‑Spec um Cannabis Ex ac s
Ai o Villa e1,2· Gas ónPabloBa e o3,4 · Ma kelSanNicolás1,2,5· Oie Aizpu ua‑Olaizola5· Mai aneOli a es1,2·
A esa zUsobiaga1,2
Recei ed: 30 Janua y 2024 / Accep ed: 8 May 2024
© The Au ho (s) 2024
Abs ac
Cannabinoids, such as ∆9- e ahyd ocannabinol (THC) and cannabidiol (CBD), a e e ec i e bioac i e compounds ha
imp o e he quali y o li e o pa ien s wi h ce ain ch onic condi ions. The copolyme poly(lac ic-co-glycolic acid) (PLGA)
has been used o encapsula e such compounds sepa a ely, p o iding pha maceu ical g ade edible p oduc s wi h unique ea-
u es. In his wo k, a a ie y o PLGA based nano o mula ions ha main ain he na u al cannabinoid p o ile ound in he plan
(known as ull-spec um) a e p oposed and e alua ed. Th ee di e en cannabis sou ces we e used, ep esen ing he h ee
mos ele an cannabis chemo ypes. PLGA nanocapsules loaded wi h di e en amoun s o cannabinoids we e p epa ed by
nanoemulsion, and we e hen unc ionalized wi h h ee o he mos common coa ing polyme s: pec in, algina e and chi osan.
In o de o e alua e he sui abili y o he p oposed o mula ions, all he syn hesized nanocapsules we e cha ac e ized, and
hei cannabinoid con en , size, ze a-po en ial, mo phology and in i o bioaccessibili y was de e mined. Rega dless o he
employed cannabis sou ce, i s load and he unc ionaliza ion, high cannabinoid con en PLGA nanocapsules wi h sui able
pa icle size and ze a-po en ial we e ob ained. S udy o nanocapsules’ mo phology and in i o elease assays in gas o-
in es inal media sugges ed ha high cannabis sou ce load may comp omise he s uc u e o nanocapsules and hei elease
p ope ies, and hence, he use o lowe con en o cannabis sou ce is ecommended.
Keywo ds cannabinoids· ull-spec um· PLGA· nanoencapsula ion
Abb e ia ions
CI Chemo ype I
CII Chemo ype II
CIII Chemo ype III;
CBC Cannabich omene
CBCA Cannabich omenic acid
CBD Cannabidiol
CBDA Cannabidiolic acid
CBDV Cannabidi a in
CBDVA Cannabidi a inic acid
CBG Cannabige ol
CBGA Cannabige olic acid
CBN Cannabinol
CBNA Cannabinolic acid
DAD Diode-a ay de ec o
EE % Encapsula ion-e iciency
HPLC High-pe o mance liquid ch oma og aphy
IS In e nal s anda d
KH Higuchi´s model cons an
2 Coe icien o de e mina ion
SGF Simula ed gas ic luid
SIF Simula ed in es inal luid
THC ∆9-Te ahyd ocannabinol
THCA ∆9-Te ahyd ocannabinolic acid
* Ai o Villa e
ai o [email p o ec ed]
Gas ón Pablo Ba e o
gas onpabloba [email p o ec ed]
1 Depa men o Analy ical Chemis y, Facul y o Science
andTechnology, Uni e si y o  heBasque Coun y (UPV/
EHU), 48940Leioa, Basque, Spain
2 Resea ch Cen e o Expe imen al Ma ine Biology
andBio echnology (PIE), Uni e si y o  heBasque Coun y
(UPV/EHU), 48620Plen zia, Basque, Spain
3 Facul ad de Ingenie ía, Uni e sidad Nacional del Cen o
de la P o incia de Buenos Ai es, A del Valle 5737,
CP7400Ola a ía, BuenosAi es, A gen ina
4 Cen o de In es igaciones en Física e Ingenie ía del
Cen o de la P o incia de Buenos Ai es, CIFICEN
(UNCPBA-CICPBA -CONICET), A . Del Valle 5737,
B7400JWIOla a ía, BuenosAi es, A gen ina
5 So e eign Fields S.L, La amendi Kalea 3, 20006Donos ia,
Basque, Spain
AAPS Pha mSciTech (2024) 25:120 120 Page 2 o 16
THCV ∆9-Te ahyd ocannabi a in
THCVA ∆9-Te ahyd ocannabi a inic acid
PDI Polydispe si y index
PLGA Poly(lac ic-co-glycolic acid)
PVA Poly inyl alcohol
In oduc ion
The he apeu ic s eng h o Cannabis Sa i a L. has been
well-known in a ious cul u es om 3000 BC o he p esen
days [1]. The medicinal p ope ies o he plan comes om
he a ious bioac i e compounds ha ha e hei own palm,
such as, e penes, la onoids, alkaloids and, mos impo -
an ly, cannabionids, which a e speci ic o he Cannabis
plan [1, 2]. Cannabinoids in e e e in he endocannabinoid
sys em, a o ing a a ie y o psychological and physiologi-
cal e ec s due o he di e en a ini ies be ween cannabi-
noids and endocannabinoid sys em’s ecep o s (mainly CB1
and CB2) [2, 3].
Among he a ious cannabinoids (mo e han 120) iden i-
ied in he li e a u e, ∆9- e ahyd ocannabinol (∆9-THC o
THC) and cannabidiol (CBD) s ill a e he mos known and
widely used ones, since hey a e he majo cannabinoids in
mos cannabis plan s and hey ha e shown o be e ec i e
o elie he symp oms o ch onic pain and o he diseases
[4]. THC is widely known o i s psychoac i i y, bu also
o i s analgesic, an i-in lamma o y, appe i e s imulan and
an ieme ic p ope ies [5]. CBD, has shown also analgesic
and an i-in lamma o y e ec s, oge he o anxioly ic, an i-
epilepsy and an ipsycho ic e ec s [6]. Because hey ha e
such di e se e ec s, he choice be ween THC o CBD is
made depending on he he apeu ic applica ion, al hough he
combina ion o bo h has p o en o be p ac ical in educing
he side e ec s o THC (i.e., oxic psychosis, dizziness, d y
mou h…), so he combina ion o bo h is o en sough [7].
Besides THC and CBD, mino cannabinoids such as canna-
bige ol (CBG), cannabich omene (CBC), cannabinol (CBN)
and e ahyd ocannabi a in (THCV), ha e demons a ed
he apeu ic e icacy o di e en applica ions, al hough he
main in e es owa ds hese cannabinoids is hei use as
enhance s o he e ec s o majo cannabinoids [8]. As wi h
he combina ion o THC and CBD, he simula enous use o
mul iple cannabinoids can imp o e he he apeu ic bene i s
o majo cannabinoid. Indeed, i is belie ed ha consuming
he ull spec um o cannabinoids p esen in he plan may
p omo e a syne gis ic e ec o “en ou age” e ec o majo
cannabinoids. Fo his eason cannabis-de i ed p oduc s ha
main ain he cannabinoid composi ion o he plan (usually
e e ed o as ull-spec um p oduc s) ha e gained in e es
in he ecen yea s [5, 8, 9]. Rela ed o his, he ex ended
knowledge in he plan , including gene ics and cul i a ion
condi ions, has led o he op ion o de elop a as a ie y o
Cannabis a ie ies, each o hem ha ing a unique spec um
o cannabinoids [10].
Nowadays, a wide a ie y o cannabis-de i ed p oduc s
a e a ailable anging om apo izable plan ex ac s o
c eams and lo ions. Ne e heless, when i comes o medici-
nal use, p oduc s designed o o al adminis a ion a e p e-
e ed because o hei ease o dosing and sel -medica ion,
hei educed oxici y and he abili y o p o iding long-las -
ing e ec s [7, 11, 12]. The mayo d awback aced by edible
p oduc s is he low o al bioa ailabili y o cannabinoids.
This is pa ly due o he lipophilic na u e o cannabinoids
(log P ~ 6–7), which makes hem o ha e a poo solubili y
in aqueous media (2–10μg/mL) and nega i ely a ec s hei
abso p ion in he in es ine [13, 14]. Addi ionally, cannabi-
noids ha e low s abili y in acidic and oxida i e en i on-
men s, especially in he p esence o ligh and hea [15, 16].
The e o e, cannabinoids can easily be deg aded h ough he
diges i e ac , pa icula ly in he s omach [17]. The ma ix
in which cannabinoids a e ound can play a c ucial ole in
imp o ing hei solubili y and physicochemical s abili y,
and hence, hei pha macokine ics and bioa ailabili y [18].
Encapsula ion o cannabinoids in polyme ic ehicles has
p o ed o be a sui able s a egy o apping cannabinoids in
ma ixes ha p o ide enhanced o al bioa ailabili y, while
o e ing he possibili y o design o mula ions wi h speci ic
elease p o iles [18–20]. In his ega d, polyme ic nano o -
mula ions ha e eme ged as a p omising ool o p o ide a a -
ge ed deli e y o cannabinoids, p omo e imp o ed o al bio-
a ailabili y and desi ed pha macokine ic p o iles [20–23].
Wi hin he wide ange o polyme ic ma e ials ha can be
used o elabo a e nanoca ie s, poly(lac ic-co-glycolic acid)
(PLGA) can be highligh ed due o i s: (i) biodeg adabili y
and biocompa ibili y, (ii) abili y o p o ide a sus ained, con-
olled and a ge ed elease, and, (iii) e sa ili y in e ms o
ou es o adminis a ion (e.g., o al, opical, ocula , ans-
de mal) among o he p ope ies [24–27]. Those p ope ies
ha e led o PLGA as one o he mos used polyme ic ma e-
ial o design pha maceu icals con aining a wide a ie y o
bioac i e compounds [25, 27]. Fo ins ance, PLGA-based
nano o mula ions ha e ecen ly been p oposed o encapsu-
la e a ious d ugs and bioac i e compounds, such as cou-
ma in C75, oxalipla in, Leishmania in an um an igens and
que ce in. The use o PLGA nanoca ie s has imp o ed
solubili y and cellula up ake o such compounds, and in
gene al, inc eased he e icacy o hei pha amacological
ac ion [28–31].
In he ield o cannabinoids, F aguas-Sánchez and
cowo ke s, de eloped CBD-loaded PLGA injec able
nanopa icles o he ea men o di e en ypes o can-
ce (o a ian and b eas ) wi h e y p omising esul s [32,
33]. Likewise, he esea ch g oup o Ma in-Bande as
has de eloped a se ies o PLGA nano o mula ions loaded
wi h CB13 (a syn he ic cannabinoid de i a i e) designed
AAPS Pha mSciTech (2024) 25:120 Page 3 o 16 120
o o al adminis a ion. These o mula ions ha e shown o
o e an imp o ed in es inal abso p ion and biodis ibu-
ion o cannabinoids, sus ained long- e m (10–14days)
elease o cannabinoids, and minimal cy o oxici y, mak-
ing hem p omising o mula ions o ea ing ch onic
pain, among o he po en ial uses [34–37].
None heless, i has been demons a ed ha he addi ion
o an ou e laye (o coa ing) by he unc ionaliza ion o
PLGA nanocapsules, using di e en polyme s can e en
u he imp o e hei cha ac e is ics in e ms o abso p-
ion, biodis ibu ion and he apeu ic e icacy [30, 34–39].
Fo ins ance, chi osan is a na u al ca ionic polysaccha-
ide ha has been widely used o unc ionalize PLGA
nanopa icles. The inclusion o a chi osan coa ing can
signi ican ly imp o e in es inal pe meabili y and abso p-
ion o PLGA nanopa icles, as i is able o open igh
junc ions in he in es inal epi helium [40–44]. Despi e
he p o en e icacy o chi osan, o he well-known na u-
al polysaccha ides, such as algina e and pec in, can also
be used o imp o e he ea u es o PLGA nanopa icles
[45]. Pec in and algina e a e anionic polyme s, unlike chi-
osan, a e mo e s able a low pH alues and a e he e o e
used o in es inal and colonal d ug deli e y [44]. Pec in
is commonly used o enhance shel -li e o encapsual ed
bioac i e compounds, p e en s i i a ion o he in es inal
mucosa, and can imp o e in es inal e en ion, in es inal
mucoadhesi i y and anspo p ope ies o capsules [44,
46–48]. Simila ly, algina e is a common coa ing ha can
imp o e encapsula ion e iciency, in eg i y o capsules
h ough he gas oin es inal ack, and hei mucoadhe-
si i y, so i has been used p e iously o imp o e elease
and abso p ion p ope ies o PLGA nano o mula ions [38,
49–51].
In his con ex , and highligh ing he ac ha he
cannabinoid-PLGA o mula ions de eloped o da e only
con ain THC, CBD o CB13 [23, 52], a a ie y o PLGA
based nano o mula ions ha main ain he na u al cannabi-
noid p o ile ound in he plan , o ull-spec um o mula-
ions, a e p oposed and e alua ed in he p esen wo k. Fo
his main goal, h ee cannabis s ains, con aining di e en
cannabinoid p o iles, we e used, and non- unc ionalized
PLGA capsules oge he wi h pec in, algina e o chi osan
coa ed capsules we e p epa ed wi h each o hem. In o de
o e alua e he o e all ea u es o he p oposed o mula-
ions, o ule ou possible inadequa e o mula ions and o
ha e a p elimina y idea o hei possible beha io in case
o inges ion, a basic physicochemical cha ac e iza ion
was ca ied. Thus, he cannabinoid con en , pa icle-size,
mo phology and ze a-po en ial o he p oposed o mu-
la ios we e e alua ed, and he po en ial elease p o ile
and o al bioaccessibili y o en apped cannabinoids we e
s udied by simula ed in i o diges ion.
Ma e ials andMe hods
Ma e ials andS anda ds
The PLGA (powde , 50:50) employed o he syn hesis
o nanocapsules was ob ained om MedChemExp ess
(Monmou h Junc ion, NJ, USA). Poly inyl alcohol (PVA,
13.000—23.000g/mol, 98% hyd olyzed), alginic acid
sodium sal om b own algae (low iscosi y), chi osan
(medium molecula weigh ), suc ose ( o molecula biol-
ogy, ≥ 99.5%), pepsin om po cine gas ic mucosa (pow-
de , ≥ 250 uni s/mg solid, lo esul : 444 U/mg), panc ea in
om po cine panc eas (powde , sui able o cell cul u e,
4 × USP speci ica ions), bile sal s ( o mic obiology) and
sodium bica bona e (ACS eagen , ≥ 99.7%) we e pu -
chased om Sigma-Ald ich Chemie GmbH (Schnelldo ,
Ge many). Pec in was p oduced by Guinama (La Pobla
de Vallbona, Spain) and phospha idylcholine (Phospho-
lipon® 90 G, ≥ 94%) by Phospholipid GmbH (Cologne,
Ge many). Hyd ochlo ic acid (HCl, 36%) was pu chased
om Me ck KGaA (D ams ad , Ge many). E hanol
(99.5%) was ob ained om Pan eac Química S.L.U. (Ba -
celona, Spain) and e hyl ace a e (≥ 99.8%, Ch omAR® o
HPLC) and ace oni ile (≥ 99.8%, Ch omAR® o HPLC)
om Mac on Fine Chemicals (Gliwice, Poland). Ac i a ed
cha coal (powde , eagen g ade), me hanol (UHPLC-MS
g ade) and wa e (UHPLC-MS g ade) we e pu chased
om Sha lab (Sen mena , Spain), whe eas Milli-Q qual-
i y wa e (< 0.05 µS∙cm−1) was p oduced using a Millipo e
185 om Millipo e (Bu ling on, MA, USA).
The solu ions o indi idual s anda ds o cannabinoids
∆9- e ahyd ocannabinolic acid-A (THCA, 1000µg/mL in
me hanol), ∆9- e ahyd ocannabinol (THC, 1000µg/mL
in me hanol) ∆9- e ahyd ocannabi a in (THCV, 1000µg/
mL in ace oni ile), ∆9- e ahyd ocannabi a inic acid
(THCVA, 1000µg/mL in ace oni ile), cannabich omene
(CBC, 1000µg/mL in me hanol), cannabich omenic acid
(CBCA, 1000µg/mL in ace oni ile), cannabidiol (CBD,
1000µg/mL in me hanol), cannabidiolic acid (CBDA,
1000µg/mL in ace oni ile), cannabige ol (CBG, 1000µg/
mL in me hanol), cannabige olic acid (CBGA, 1000µg/
mL in ace oni ile), cannabinol (CBN, 1000µg/mL in
me hanol) and cannabinolic acid (CBNA, 1000µg/mL
in me hanol), cannabidi a in (CBDV, 1000µg/mL in
me hanol), cannabidi a inic acid (CBDVA, 1000µg/mL
in ace oni ile) we e pu chased by D . Eh ens o e GmbH
(Augsbu g, Ge many). The deu e a ed analogue ∆9- e -
ahyd ocannabinol (THC, 1000µg/mL in me hanol) was
supplied by Me ck KGaA (D ams ad , Ge many) and
phenan ene, used as in e nal s anda d (IS), was pu chased
om Sigma-Ald ich Chimie (Sain -Quen in-Falla ie ).
A mixed esh s ock solu ion con aining 100μg/mL o
AAPS Pha mSciTech (2024) 25:120 120 Page 4 o 16
all a ge compounds was p epa ed mon hly in me ha-
nol, whe eas in e media e dilu ions we e p epa ed daily
acco ding o he expe imen a ion.
P epa a ion o Pu i ied Cannabis Ex ac s
Cannabis lowe s om h ee di e en cannabis s ains we e
made in o en iched cannabis ex ac s. The s ains we e cho-
sen o be ep esen a i e o he h ee mos ele an canna-
bis chemo ypes: (i) Chemo ype I (CI) – a THC ich s ain
(~ 12% THC), (ii) Chemo ype II (CII) – a THC/CBD le eled
s ain (~ 5% THC and ~ 7% CBD) and (iii) Chemo ype III
(CIII) – a CBD ich s ain (~ 11% CBD). Flowe s (a ound
15g) o each s ain we e g inded and placed in o sepa a e
closed Py ex con aine s, and we e main ained a 120°C o
1h o assu e comple e deca boxyla ion o acidic cannabi-
noids while keeping loses o a minimum [53]. A e ha ,
he Py ex con aine s we e s o ed a –40°C, and once hey
we e cooled down, cold e hanol (–40°C) was added un il he
deca boxyla ed lowe s we e ully co e ed. Cold ex ac ion
o neu al cannabinoids was pe o med by main aining he
e hanol solu ions in an ul asonic ba h o 1min and, hen
5min a –40°C. Once ex ac ion was comple ed, e hanol
ex ac s we e acuum il e ed using 10—12µm cellulose
il e s, and he esul ing ex ac s we e s o ed a –80°C o e -
nigh o pu i y hem by win e iza ion [54]. The waxes, and
lipids ha p ecipi a ed du ing his pe iod, we e emo ed
by a as acuum il a ion using 0.45µm cellulose ni a e
il e s. The emaining chlo ophyll was emo ed by adding
80mg o ac i a ed cha coal pe ini ial cannabis lowe g am
(1.25g o each ex ac app oxima ely), o exing o 10min
and il e ing using 0.45µm polyp opylene sy inge disk il-
e s. Finally, he excess o e hanol was emo ed by a o a y
e apo a o (35°C, 100mba ) un il iscous ex ac s we e
ob ained (a ound 2g pe s ain). The h ee ex ac s we e
s o ed a –20°C un il u he use.
Cannabinoid Con en inPu i ied Cannabis Ex ac s
The concen a ion o main cannabinoids o each cannabis
s ain’s ex ac was de e mined by means o high pe o -
mance liquid ch oma og aphy coupled o a diode a ay
de ec o (HPLC–DAD) in iplica e ollowing a p e iously
alida ed me hod [55]. B ie ly, 0.1g o cannabis ex ac
we e sonica ed wi h 5mL o me hanol in an ul asonic ba h
o 15min. Subsequen ly, he solu ion was cen i uged o
5min a 10000g in a 5804 R Eppendo cen i uge (Ham-
bu g, Ge many), and he supe na an solu ion was dilu ed
wi h me hanol con aining IS ( esul ing in a concen a ion o
5µg/mL IS in he inal solu ion). The inal solu ions we e
sy inge- il e ed wi h 0.22µm polyp opylene disks and ana-
lyzed by means o HPLC–DAD using he me hod desc ibed
in he ollowing sec ions.
P epa a ion o PLGA Nanopa icles
PLGA nanopa icles we e p epa ed based on an emulsion
sol en e apo a ion echnique p e iously desc ibed o
encapsula ion o pu e CBD [33] wi h some modi ica ions
due o he na u e o he cannabis ex ac s. Fi s , 12 PLGA
solu ions, each o hem con aining 500mg o PLGA dis-
sol ed in 10mL o e hyl ace a e, we e p epa ed. The PLGA-
e hyl ace a e solu ions we e loaded wi h cannabinoids by
adding he di e en cannabis ex ac s. Owing o he lack o
p e ious e e ences ela ed o cannabis ex ac con aining
PLGA o mula ions, ou cannabis ex ac o PLGA powde
mass a ios ( e e ed as load a io om now on) we e es ed
o each o he cannabis s ains (see TableI). Depending on
his, 50, 150, 300 o 500mg o each s ain’s ex ac we e
added o each o he 12 ini ial PLGA solu ions ( esul ing in
4 loading a ios pe s ain). Once he ex ac s we e dissol ed,
he PLGA-e hyl ace a e-cannabis solu ions we e added d op-
wise in o 50mL o PVA 0.5% aqueous solu ions, which
Table I Scheme and
Codi ica ion o Syn he ized
PLGA Nanocapsules
S ain Load a io
(mgex ac :mgPLGA)
Coa ing
Uncoa ed Algina e Pec in Chi osan
Chemo ype I
(CI)
1:10 CI10∅CI10A CI10P CI10C
3:10 CI30∅CI30A CI30P CI30C
6:10 CI60∅CI60A CI60P CI60C
10:10 CI100∅CI100A CI100P CI100C
Chemo ype I
(CII)
1:10 CII10∅CII10A CII10P CII10C
3:10 CII30∅CII30A CII30P CII30C
6:10 CII60∅CII60A CII60P CII60C
10:10 CII100∅CII100A CII100P CII100C
Chemo ype I
(CIII)
1:10 CIII10∅CIII10A CIII10P CIII10C
3:10 CIII30∅CIII30A CIII30P CIII30C
6:10 CIII60∅CIII60A CIII60P CIII60C
10:10 CIII100∅CIII100A CIII100P CIII100C
AAPS Pha mSciTech (2024) 25:120 Page 5 o 16 120
we e s i ed a 24000 pm by an Ul a- u ax (IKA, S au en,
Ge many) in o de o emulsi y nanopa icles. This p ocess
was ca ied ou o all o mula ions excep o he highes
loading a io (i.e., 10:10) since an indi isible agglome a e
ha blocked he Ul a- u ax was o med du ing he p ocess.
A e emulsi ica ion, excess o e hyl ace a e was emo ed
by o a y e apo a ion (40°C, 100mba ) while excess o
PVA and non-ecapsula ed cannabinoids we e emo ed by
cen i uga ion and decan a ion o h ee imes by adding
u he Milli-Q wa e . The ob ained we PLGA-cannabis
nanopa icles o each s ain and load we e sepa a ed in ou
aliquo s o u he unc ionaliza ion. One o he aliquo s pe
load and s ain was s o ed a 4°C (uncoa ed capsules) and
he o he h ee we e unc ionalized using di e en coa ing
laye s: algina e, pec in and chi osan (see TableI). The unc-
ionaliza ion o hose PLGA-cannabis nanopa icles was ca -
ied ou by he addi ion o 10mL o 0.2% aqueous solu ions
o he co esponding coa ing agen (i.e., sodium algina e,
pec in, and chi osan) on o each o he aliquo s and con inu-
ously agi a ed a oom empe a u e o 2h. Then, Milli-Q
wa e was added and he excess o coa ing polyme , along
wi h po en ial aces o non-encapsula ed cannabinoids
we e emo ed h ough cen i uga ion and decan a ion. This
p ocess was epea ed h ee imes by adding u he Milli-
Q wa e . Once cleaned, 10mL o c yop o ec ing solu ion
(i.e., suc ose 1% aqueous solu ion [56]), was added o each
o he 4 ac ions (non- unc ionalized and unc ionalized)
o each s ain and load; and we e d ied subsequen ly in a
Cool acuum Lyomic on eeze-d ye (Ba celona, Spain) a
–60°C and 0.037mba o 48h. The suc ose was sepa a ed
and he ob ained d y capsules we e weighed and s o ed a
–20°C un il u he use.
Cannabinoid Con en inPLGA Nanopa icles
The concen a ion o 14 cannabinoids in he syn he ized
PLGA-cannabinoid nanopa icles was de e mined in ipli-
ca e by means o HPLC–DAD ollowing a me hod desc ibed
in he li e a u e used o de e mine he con en o a syn he ic
cannabinoid in PLGA nanopa icles [37]. 1mL o ace oni-
ile was added on o accu a ely weighed 4mg o lyophilized
PLGA nanopa icles, and he mix u e was o exed (5min)
o dissol e PLGA nanopa icles and p omo e libe a ion o
en apped compounds in he o ganic phase [37, 57–59]. The
ob ained ace oni ile solu ions we e dilu ed in me hanol con-
aining IS ( esul ing in a concen a ion o 5µg/mL in he
inal solu ions) and il e ed using 0.22µm polyp opylene
disks. The cannabinoid con en in he inal solu ions was
de e mined by means o HPLC–DAD analyisis.
5 µL o he inal solu ions we e analyized using an In ini y
1260 LC Sys em (HPLC) coupled o an In ini y 1260 Diode
A ay De ec o WR (DAD), bo h om Agilen Technologies
(San a Cla a, CA, USA). Sepa a ion o cannabinoids was
achie ed using a Kine ex C18 column (150 × 3mm, 2.6µm)
wi h a Secu i y Gua d Ul a C18 p ecolumn (2 × 3mm), bo h
om Phenomenex (To ance, CA, USA). The sepa a ion o
cannabinoids was done using a g adien me hod a a cons an
low o 0.7mL/min consis ed o mobile phases A (Wa e ,
0.1% ace ic acid) and B (Me hanol, 0.1% ace ic acid). The
g adien me hod s a ed a 30% A and main ained 3min;
hen, i was dec eased i s o 20% in 6min and hen o 5%
in 3min, which was main ained o 3min. A was inc eased
o 30% in 5min and main ained o o he 4min o each ini-
ial condi ions be o e he nex ch oma og aphic un, which
las 24min in o al. Cannabinoids we e de ec ed using DAD
and we e quan i ied a 230nm using an ex e nal calib a ion
cu e p epa ed in he ange o 0.1µg/mL and 50µg/mL o
all a ge compounds. All he calib a ion solu ions, as well
as he measu ed ex ac s, con ained IS in a concen a ion o
5µg/mL in o de o co ec he DAD signal and minimize
ins umen al measu emen s a iabili y.
Encapsula ion‑e iciency
The encapsula ion-e iciencies (EE %) o each o mula ion
and cannabinoid we e de e mined by compa ing encapsu-
la ed mass (mgcap) o each cannabinoid wi h he mass o
cannabinoid ha was loaded along wi h he cannabis ex ac
(mgex ) (Eq.1):
whe e, mcap is he ob ained mass in g o nanopa icles, Ccap
is he concen a ion in mg/g o each cannabinoid in each
o mula ion, mex is he employed mass in mg o cannabis
ex ac and Cex is he concen a ion o each cannabinoid in
mg/g in each o he cannabis ex ac s.
Physical Cha ac e iza ion o PLGA Nanopa icles:
Pa icle Size, Ze a‑Po en ial andSEM Imaging
The pa icle size and ze a-po en ial o PLGA nanocapsules
we e measu ed using a Ze asize Nano se ies om Mal-
e n Ins umen s (Mal e n, UK). Fo his pu pose, 4mg
o d ied PLGA nanocapsules we e suspended in 4mL o
Milli-Q wa e unde con inuous magne ic s i ing o p omo e
homogenei y o he suspension. Aliquo s o he homogenous
dispe sions we e used o p epa e dilu ions o 0.05mg/mL
(pa icle size) and 0.5mg/mL (ze a-po en ial). The pa icle
size and ze a-po en ial o h ee aliquo s o he inal dilu ions
we e de e mined espec i ely by dynamic ligh sca e ing
and elec opho e ic ligh sca e ing (a an angle o 90°).
The mo phology o he syn he ized nanocapsules was
checked by means o Scanning Elec on Mic oscopy (SEM).
The d ied PLGA nanocapsules we e coa ed wi h 15nm o
(1)
EE
%= mgcap
mgex
=
m
cap ⋅
C
cap
mex
⋅
Cex

AAPS Pha mSciTech (2024) 25:120 120 Page 6 o 16
gold on a K550X spu e coa e om Emi ech (Mon igny-
le-B e onneux, F ance) and measu ed using a FEG SEM
S4800 om Hi achi (Tokyo, Japan) wi h an accele a ion
ol age o 5kV.
In Vi o Gas o‑In es inal Diges ion Simula ion
Release o he main cannabinoids was de e mined by a s a ic
in i o diges ion simula ion, ollowing he ecommenda-
ions o INFOGEST [60] and Minekus e al. [61]. Simu-
la ed gas ic luid (SGF) con ained 2000 U/mL o pepsin and
0.17mM o phospha idylcholine in Milli-Q wa e and he
pH was adjus ed o 3 using a 2M HCl solu ion. Simula ed
in es inal luid (SIF) con ained 3.2g/L o panc ea ine and
8.16g/L o bile sal s in Milli-Q wa e . The con en o main
cannabinoids (THC and CBD) in d y PLGA nanocapsules
was onside ed o s anda dize he assays and p o ide an eas-
ie compa ison be ween o mula ions. The mass employed
in each assay was he one equi ed o p o ide 10mg o THC
(CI nanopa icles), 10mg o CBD (CIII nanocapsules) o
10mg o THC + CBD (CII capsules).
Fo each o mula ion he calcula ed PLGA nanopa -
icles mass was weighed and 10mL o SGF we e added
and main ained a 37 ˚C unde con inuous s i ing o 2h,
simula ing he gas ic phase. A e ha , 10mL o SIF we e
added ( esul ing in 1.6g/L o panc ea ine and 4.08g/L o
bile sal s), pH was adjus ed o 7 using a sa u a ed sodium
bica bona e aqueous solu ion, and he mix u e was main-
ained a 37 ˚C unde con inuous s i ing o 4h, simula ing
he in es inal phase. The elease p o ile o cannabinoids was
de e mined by aking aliquo s o 200 µL a di e en imings
(12 aliquo s du ing he 6h o he expe imen ). Each ime
he aliquo s we e aken, aliquo s o 200 µL o esh SGF o
SGF:SIF 1:1 mix u e we e added o he assay-solu ion in
o de o main ain he same a io wi hin mic ocapsules and
simula ed luids in he whole simula ion. All he assays we e
un in iplica e. All he aliquo s we e dilu ed wi h me hanol
con aining IS ( esul ing in a concen a ion o 5µg/mL IS in
he inal solu ion) and cen i uged using a 5424 R Eppen-
do Cen i uge (Hambu g, Ge many) o 5min a 10000g.
The supe na an s we e collec ed and il e ed wi h 0.22µm
polyp opylene sy inge disks p io o HPLC–DAD analysis.
The esul s we e exp essed as he eleased cumula i e
ac ion om he o al cannabinoid con en h ough diges-
ion ime. In es inal bioa ailabili y was calcula ed by es ing
he ac ion eleased in he gas ic phase o he inal eleased
ac ion.
Resul s
Cannabinoid Con en andEncapsula ion‑e iciency
The con en o 14 cannabinoids in he syn he ized PLGA
nano o mula ions is compiled in TableS1. Since he applied
polyme ic coa ing did no esul in signi ican di e ences
(p- alue > 0.05) in he cannabinoid con en o capsules made
wi h he same cannabis s ain and ex ac loading, he mean
con en o he uncoa ed and h ee coa ed o mula ions pe
s ain and ex ac loading a e shown in Fig.1. The cannabi-
noid con en in he ob ained PLGA nanocapsules a e in line
wi h he cannabinoid p o ile o he employed ex ac and he
loaded ex ac amoun in each case (cannabinoid con en o
he ex ac s a e shown in TableS2).
CI capsules, con ained high amoun s o THC, be ween 8
and 35% o he capsules’ mass, and con ained CBD, CBC,
CBG, CBN and THCV ( oge he wi h hei acidic analogs)
in concen a ions be ween 0.01% and 0.7%. This made CI
capsules o ha e he highes con en o cannabinoids and o
ha e he wides spec um o neu al cannabinoids, compa ed
o he capsules ob ained om CII and CIII s ains. CII cap-
sules, con ained be ween 3.8% and 14% o THC and be ween
4.0% and 15% o CBD, and he con en o CBC, CBG and
CBN ( oge he wi h hei acidic analogs) we e encoun e ed
in concen a ions be ween 0.01% and 1.16%. CIII capsules,
on he o he hand, con ained high amoun s o CBD, be ween
Fig. 1 Con en o THC,
CBD,CBG,CBC and sum
o o he mino cannabinoids
(CBN, THCV, THCA, CBDA,
CBGA and CBCA) in PLGA
nanocapsules (w/w %) o each
cannabis s ain and load a io
(mgex ac :mgPLGA) (n = 12,
exp essed a a 95% con idence
le el)
AAPS Pha mSciTech (2024) 25:120 Page 7 o 16 120
6.9% and 26% o he capsules’ mass and con ained CBC and
CBG in concen a ions be ween 0.01% and 1.3%. Thus, he
o al cannabinoid con en in CII and CIII is sligh ly lowe ,
and al hough he cannabinoid spec um is igh e , he con-
en in mino cannabinoids such as CBC and CBG, is highe
han in CI capsules. Conside ing ha he a e age o al dose
is abou 30mg o THC, 60mg o THC + CBD (30mg o
each) and abou 100mg o CBD [7, 62, 63], be ween 360
and 80mg o CI capsules, 790 and 200mg o CII capsules
and 1400 and 400mg o CIII capsules would be enough o
ensu e hose doses.
In addi ion o he expec ed di e ences ela ed o he
na u e o he cannabis s ain and he ex a c load, some
simila i ies can be highligh ed. The con en o acidic can-
nabinoids in all he o mula ions is minimum, especially he
con en o THCA (e en in high THC con en capsules) due
o i s lowe deca boxyla ion empe a u e [53]. This obse -
a ion indica es ha he deca boxyla ion p ocess ca ied in
he p epa a ion o he h ee cannabis ex ac s was app opi-
a e. Simila ly, low con en o CBN ( he main deg ada ion
p oduc o THC’s oxida ion) was obse ed in all capsules,
which is a ac o g ea in e es in he case o CI and CII cap-
sules (bo h con aining high amoun s o THC). Based on his
esul , we can conclude ha he employed cannabis lowe s
we e esh ( he highe CBN con en is, he olde a cannabis
p oduc is) and ha THC was no oxidized nei he in he
p epa a ion o he cannabis ex ac s no in he encapsula ion
p ocess [64, 65]. This could be achie ed by a oiding high
empe a u es (a e deca boxyla ion), p olonged exposu e o
ligh and exposu e o oxidan species.
THCVA, CBDV and CBDVA we e unde he me hod lim-
i s o de ec ion o he measu ed dilu ions (0.01%). Al hough
he concen a ion o hese mino cannabinoids could ha e
been de e mined by measu ing a highe concen a ion
ex ac , his op ion was disca ded since he equi ed dilu-
ion o measu e hese cannabinoids, based on he low con en
in he pu i ied ex ac s (see TableS2), would sa u a e he
column and he HPLC sys em.
Conce ning he encapsula ion e iciencies (EE) o majo
cannabinoids, a sligh dec ease was obse ed as he cannabis
ex ac loading was inc eased in all o mula ions (TableII).
The highes EEs we e ob ained in nanocapsules loaded wi h
10mg o cannabis ex ac pe 100mg o PLGA, whe e mean
EEs be ween 85 and 99% we e ob ained. When he cannabis
ex ac load was inc eased o 3:10, he mean EE dec eased o
alues be ween 65 and 84%, and when inc eased o 6:10, he
EE % dec eased o alues be ween 56 and 73%. Likewise,
Ma in-Bande as e al. and F aguas-Sanchez e .al epo ed
lowe EE % o CB13 o CBD in hei PLGA based encap-
sula ions when cannabinoid load was inc eased [33, 37],
which is a ached o capsules’ maximum loading capaci y
o o loss o a ailable d ug due o i s mig a ion in o he
emulsi ie aqueous phase. In con as o his phenomenon,
no signi ican di e ences we e obse ed in he EE % o di -
e en s ains and cannabinoids.
Physical P ope ies o PLGA Nanocapsules: Pa icle
Size, Ze a‑po en ial andSEM Imaging
Pa icle size and ze a-po en ial o PLGA nanopa icles we e
de e mined using a ze a-size , measu emen plo s o bo h
pa ame e s a e shown in Figu esS1 and S2 espec i ely.
The polidispe si y index (PDI) alues o mos o mula-
ions we e a ound 0.3 (TableS3), indica ing ha hey had
an accep able dispe si y [66]. Fo his eason, and acco d-
ing o ISO 22412:2017, Z-a e age was elec ed o epo he
mean pa icle size o each o mula ion [67]. The pa icle
size anged be ween 150 and 700nm, being he applied
coa ing in each o ma ion he possible esponsible o such
a iabili y (Fig.2A). In ac , pa icles wi h he same coa -
ing showed s a is ically compa able pa icle sizes, being
he pec in coa ed capsules he la ges ones (380—700nm)
compa ed o ei he uncoa ed (160—440nm), algina e coa ed
(200—480nm) o chi osan coa ed (150—450nm) capsules
(depending on he case). On he con a y, no clea di e -
ences we e obse ed among s ains and ex ac load a ios.
Uncoa ed, algina e coa ed and chi osan coa ed nanocapsules
showed compa able pa icle sizes among hem in mos cases.
The ob ained pa icle sizes all in o he same size ange han
hose ob ained in THC, CBD o CB13 loaded PLGA nano-
pa icles in p e ious wo ks, which anged be ween 250 and
450nm in non- unc ionalized pa icles and be ween 600 and
900nm in unc ionalized pa icles [52].
The success in he su ace-modi ica ion (o coa ing) p o-
cess and i s e ec on he su ace-cha ge o nanopa icles
was assessed using he ze a-po en ial alues (Fig.1b). As
expec ed, uncoa ed, pec in coa ed and algina e coa ed cap-
sules showed nega i e ze a-po en ials due o hei anionic
na u e, whe eas chi osan coa ed capsules showed posi i e
ze a-po en ials due o hei ca ionic na u e. The addi ion
o pec in and algina e, inc eased signi ican ly he nega i e
cha ge o uncoa ed PLGA capsules in CI 1:10 and 3:10
Table II Encapsula ion-
e iciencies (EE) o THC and
CBD o Each S ain and
Load Ra io (n = 12, 2s a 95%
Con idence Le el)
S ain CI CII CIII
Load a io 1:10 3:10 6:10 1:10 3:10 6:10 1:10 3:10 6:10
THC (EE %) 98 ± 9 84 ± 13 73 ± 4 99 ± 5 76 ± 7 66 ± 8
CBD (EE %) 87 ± 5 65 ± 6 56 ± 6 85 ± 3 73 ± 1 57 ± 2
AAPS Pha mSciTech (2024) 25:120 120 Page 8 o 16
loaded capsules, CII 3:10 loaded capsules and all CIII
capsules. In e es ingly, in all cases (excep o CIII 1:10
loaded capsules) he ze a-po en ial o pec in and algina e
we e compa able be ween hem (p > 0.05), as well as com-
pa able among nanocapsules de i ed om di e en ex ac
load a ios and cannabis s ains. In he case o uncoa ed
capsules, despi e no clea endency was obse ed, signi i-
can di e ences we e obse ed among o mula ions, since
CI 1:10 loaded, and CIII capsules showed less nega i e
ze a-po en ials compa ed o he es . Fo his eason, su -
ace modi ica ion using pec in and algina e, seems a good
s a egy o inc ease nega i e ze a-po en ial and o s anda d-
ize he su ace-cha ge o o mula ions, ega dless o he
s ain and ex ac loading a io. Indeed, pec in and algina e
coa ed capsules showed ze a-po en ial alues mo e nega i e
han − 30mV in all o mula ions, which echnically ensu es
colloidal suspension s abili y and p e en ion o pa icle
agg ega ion and loccula ion [68]. Rega ding he possible
in e ac ion o capsules wi h he in es inal mucosa, anionic
polyme s such as algina e and chi osan ha e p o en o
enhance he e en ion o capsules in he in es ine by adhesion
mechanisms o he in es inal mucosa, leading o inc eased
in es inal abso p ion o encapsula ed compunds [69–71].
Al hough he in es inal memb ane is nega i ely cha ged, he
ca boxylic g oups o pec in and algina e can o m hyd ogen
bonds wi h oligosaccha ide chains o in es inal mucins [72].
These in e ac ions along wi h chain en anglemen and an
de Waal's in e ac ions, a e s onge ha elec os a ic epul-
sion, and esul in high mucoadhesion o hese biopolyme s
[73, 74]. In e es ingly, a highe nega i e cha ge densi y in
hese polyme s is ela ed o he mucoadhesi e s eng h and
hence, he ob en ion o such nega i e ze a-po en ials sug-
ges s ha mucoadhesion and abso p ion o cannabinoids
may be highe in pec in- and algina e-coa ed o mula ions
compa ed o uncoa ed capsules [75, 76].
On he o he hand, mo e di e ences we e obse ed in he
posi i e ze a-po en ials o he capsules coa ed wi h chi osan.
CI and CII 1:10 loaded capsules showed signi ican ly highe
ze a-po en ials compa ed o CIII 1:10 loaded capsules, and
CI 6:10 loaded capsules showed signi ican ly highe alues
compa ed o CII and CIII 1:10 loaded capsules; whe eas
3:10 loaded capsules showed compa able ze a-po en ials
ega dless o he s ain. Thus, CIII capsules showed sligh ly
lowe posi i e ze a-po en ials compa ed o CI and CII cap-
sules. Howe e , he cannabis ex ac loading employed was
he mos c i ical ac o ha appea ed o nega i ely incluence
he ze a-po en ial alue o chi osan-coa ed capsules. Inc eas-
ing he loading a io o cannabis ex ac led o signi ican
Fig. 2 Mean pa icle size
(A) and ze a-po en ial (B)
o PLGA nanopa icles o
each s ain, ex ac load a io
(mgex ac :mgPLGA) and coa ing
(n = 3, exp essed a 95% con i-
dence le el)
AAPS Pha mSciTech (2024) 25:120 Page 9 o 16 120
dec eases in he ze a-po en ial alues o he chi osan-
loaded capsules in all s ains. O e all, ze a-po en ial alues
abo e + 30mV we e only achie ed wi h 1:10 loaded CI and
CII capsules, sugges ing ha he emaining chi osan-coa ed
o mula ions would no esul in s able aqueous dispe sions,
and mucoadhesi e o mucope meable p ope ies migh also
be comp omised [68]. The imp o ed in es inal adhesion
and pe meabili y o chi osan (and o he ca ionic polyme s)
coa ed capsules is he esul o he elec os a ic a ac ion
be ween he posi i e cha ge o chi osan and he nega i e
cha ge o in es inal memb anes [77, 78]. A less posi i e su -
ace cha ge o he capsules could comp omise elec os a ic
a ac ion be ween capsules and in es inal memb anes, and
he e o e, o mula ions wi h highe cannabis ex ac load
(and less posi i e ze a-po en ials) may exhibi less e icien
abso p ion and a lowe bioa ailabil y o cannabinoids [75,
79].
In addi ion o pa icle-size and ze a-po en ial, he mo -
phology o PLGA nanocapsules was e alua ed by SEM
analysis. Conside ing he mino in luence o he na u e o
he employed cannabis s ain in pa icle-size and ze a-po en-
ial, i was assumed ha mo phology o capsules would no
a y signi ican ly be ween capsules de i ed om di e en
cannabis chemo ypes. Hence, SEM images o all he o -
mula ions o one o he chemo ypes (chemo ype II) we e
aken and assumed o be ep esen a i e o equi alen o -
mula ions (i.e. same ex ac load a io and coa ing) o he
o he chemo ypes (Figu eS3). SEM images e ealed ce ain
he e ogenei y in he mo pohology o nanocapsules in mos
o mula ions. Al hough sphe ical o quasi-sphe ical shapes
we e p edominan , elonged and i egula shapes we e also
obse ed. The polyme ic coa ing used did no p oduce con-
clusi e di e encies in he shape o capsules. The ex ac
loading a io did no lead o clea di e ences in mo phology
nei he , bu appea ed o be ela ed o he agglome a ion o
nanocapsules. Rega dless o he polyme ic coa ing, a highe
deg ee o agglome a ion is obse ed wi h he inc ease o he
loading a io. This obse a ion is in line wi h he ac ha
10:10 loaded caspules could no be p ocessed due o he
o ma ion o big agglome a es. This phenomenom led o
he o ma ion o some nanopa icle-ne wo ks in 6:10 loaded
capsules, as i was also ob se ed in he li e a u e [51].
Howe e , hese agglome a ions in d y nanocapsules seem
o be educed o o disappea when capsules a e dispe sed in
wa e owing o he obse ed pa icle sizes and ze a-po en ial
(and he measu emen plo s o bo h pa ame e s). In ega d o
he su ace, capsules wi h 1:10 ex ac load a io seemed o
ha e a smoo he su ace compa ed o capsules wi h highe
loading a ios, bu o e all capsules p esen ed ough and
i egula su aces.
Conside ing he obse ed endencies and compa ing he
o e all capsule mo phologies wi h he sphe ical, egula and
smoo h su aces ob ained by F aguas-Sanchez e . al, whose
pa icle syn hesis p o ocol was ollowed, i seems ha he
use o cannabis ex ac s ins ead o pu e CBD, may in luence
nega i ely he mo phology o capsules [33]. The obse ed
angula and i egula mo phology in some capsules could
nega i ely a ec hei anspo and memb ane pe mea ion
ea u es, and he lack o homogenei y may comp omise he
homogenous elease o encapsula ed compounds [80–82].
Al hough mo phology o some nanocapsules could be
imp o ed, mos o mula ions showed sa is ac o y pa icle
sizes (con i med by SEM imageing) and ze a-po eni als, and
so, elease and abso p ion o PLGA nanocapsules may s ill
be sui able o a p ope deli ey o encapsula ed cannabinoids
[83, 84]. Howe e , he use o lowe cannabis ex ac loading
a ios is ecommended o assu e he in eg i y o PLGA nano-
capsules s uc u e and he e ec i i y o he unc ionaliza ion
i chi osan is used. Simila ly, i unc ionaliza ion ia anionic
polyme s is in ended, he use o algina e is ecommend o e
pec in, as pec in coa ing seemed o lead o a highe pa icle
size in some cases.
In Vi o Gas o‑in es inal Diges ion Simula ion:
Release P o ile andBioaccessibili y
In i o gas o-in es inal diges ion simula ions we e ca ied
in o de o ge a p elimina y idea o he elease-p o ile and
in es inal bioaccessibili y o cannabinoids in he p oposed
PLGA nano o mula ions. Mo eo e , hese assays allow
de e mining whe he he employed cannabis s ain, ex ac
load a io and coa ing has any e ec on he elease o can-
nabinoids o PLGA nanocapsules in gas o-in es inal media
o no .
The elease-p o ile o each o mula ion was de e -
mined based on he cumula i e elease o main cannabi-
noids (THC, THC and CBD, and CBD, in CI, CII and CIII
s ains espec i ely) du ing he in i o gas o-in es inal
diges ion simula ion. The aise o cumula i e elease
du ing simula ed diges ion was g aphically ep esen ed
o each o mula ion (Figu eS4). All o mula ions ol-
lowed a sus ained elease p o ile o cannabinoids in bo h
gas ic and in es inal phases. In some cases hough, he
sus ained elease in he in es inal phase was p eceded by
a na ow bu s elease a he beginning o he in es inal
phase. Despi e he simila i ies in he elease p o iles,
he e a e di e ences in elease a e o main cannabinoids
depending on he o mula ion. Wi h he aim o ha ing a
deepe insigh in o he in i o elease p o iles o he o -
mula ions s udied in his wo k elease kine ic models we e
buil and compa ed. Acco ding o he li e a u e, elease
mechanisms o d ugs om PLGA nanocapsules a e based
on ze o-o de , i s -o de , Higuchi´s and Ko smeye –Pep-
pas´ Eqs.[85–88]. In his wo k, ze o-o de , i s -o de and
Higuchi´s kine ic models we e es ed, which a e common
and basic kine ic models ha wo k o mos o mula ions,
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Publishe 's No e Sp inge Na u e emains neu al wi h ega d o
ju isdic ional claims in published maps and ins i u ional a ilia ions.