Ci a ion: Ghidini, M.; Sil a, S.G.;
E angelis a, J.; do Vale, M.L.C.;
Fa ooqi, A.A.; Pinhei o, M.
Nanomedicine o he Deli e y o
RNA in Cance . Cance s 2022,14,
2677. h ps://doi.o g/10.3390/
cance s14112677
Academic Edi o : Cla e Hoskins
Recei ed: 26 Ap il 2022
Accep ed: 25 May 2022
Published: 28 May 2022
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cance s
Re iew
Nanomedicine o he Deli e y o RNA in Cance
Michele Ghidini 1, Sand a G. Sil a 2, Jessica E angelis a 3, Ma ia Luísa C. do Vale 2,
Ammad Ahmad Fa ooqi 4and Ma ina Pinhei o 5,6,*
1Medical Oncology Uni , Fondazione IRCCS Ca’ G anda Ospedale Maggio e Policlinico, 20122 Milan, I aly;
[email p o ec ed]
2LAQV/REQUIMTE, Depa men o Chemis y and Biochemis y, Facul y o Sciences, Uni e si y o Po o,
4169-007 Po o, Po ugal; [email p o ec ed] (S.G.S.); [email p o ec ed] (M.L.C.d.V.)
3Tho acic Su ge y, Fondazione Policlinico Uni e si a io A. Gemelli IRCCS, Uni e si àCa olica del Sac o
Cuo e, La go F. Vi o 1, 00168 Rome, I aly; [email p o ec ed]
4Ins i u e o Biomedical and Gene ic Enginee ing (IBGE), Islamabad 44000, Pakis an;
[email p o ec ed]
5REQUIMTE, Uni e si y o Po o, 4169-007 Po o, Po ugal
6ICVS, Li e and Heal h Sciences Resea ch Ins i u e, School o Medicine, Uni e si y o Minho,
4710-057 B aga, Po ugal
*Co espondence: mpinhei [email p o ec ed]
Simple Summa y:
Cance is a mul i ace ed, li e- h ea ening, and genomically complex disease. The
wo ldwide p e alence o cance is so high ha one in h ee people will de elop cance du ing hei
li e ime. Al hough he use o RNA he apy is p omising o igh cance , i s e icien and sa e deli e y
is s ill one o he signi ican challenges hampe ing i s he apeu ic applica ion. Thus, he aim o
he p esen e iew was o highligh he mos ecen de elopmen s in he ield o nanomedicine
RNA-associa ed he apies o igh cance .
Abs ac :
The complexi y, and he di e si y o he di e en ypes o cance s allied o he endency
o o m me as asis make ea men e iciency so icky and o en impossible due o he ad anced
s age o he disease in he diagnosis. In ecen yea s, due o emendous scien i ic b eak h oughs, we
ha e wi nessed exponen ial g ow h in he elucida ion o mechanisms ha unde lie ca cinogenesis
and me as asis. The de elopmen o mo e selec i e he apies made i possible o imp o e cance
ea men . Al hough in e disciplina y esea ch leads o encou aging esul s, scien is s s ill ha e a long
explo a ion jou ney. RNA echnology ep esen s a p omise as a he apeu ic in e en ion o a ge ed
gene silencing in cance , and he e a e al eady some RNA-based o mula ions in clinical ials. How-
e e , he use o RNA as a he apeu ic ool p esen s se e e limi a ions, mainly ela ed o i s low s abili y
and poo cellula up ake. Thus, he use o nanomedicine employing nanopa icles o encapsula e
RNA may ep esen a sui able pla o m o add ess he majo challenges hampe ing i s he apeu ic
applica ion. In his e iew, we ha e e isi ed he po en ial o RNA and RNA-associa ed he apies o
igh cance , also p o iding, as suppo , a gene al o e iew o nanopla o ms o RNA deli e y.
Keywo ds: cance ; d ug deli e y sys ems; RNA; nanopa icles
1. In oduc ion
Wi h inc easing awa eness o he in e disciplina i y needed o a comp ehensi e
cha ac e iza ion o he unde lying mechanisms o cance esea ch, we ha e wi nessed
g oundb eaking disco e ies in a ious ace s o molecula oncology. Compelling expe i-
men al e idence ob ained om high- h oughpu echnologies has o e ed a window in o
p e iously in ac able p oblems in ou comp ehension o cance gene ics/epigene ics,
de egula ed cell signaling pa hways, noncoding genome, and umo he e ogenei ies and
p o ided new insigh s in o he apeu ic op ions. Rapidly e ol ing unde s anding has
shown ha by gaining deepe insigh s in o nano-bio in e ac ions and pe sonaliza ion o
Cance s 2022,14, 2677. h ps://doi.o g/10.3390/cance s14112677 h ps://www.mdpi.com/jou nal/cance s
Cance s 2022,14, 2677 2 o 19
nanomedicines, and h ough he applica ions o nano echnology o eme ging and exis ing
he apeu ic modali ies, we ha e s a ed o ealize he ue po en ial o nanomedicines in
cance [1–4].
Recen s udies ha e p o ided e idence o an upsu ge in cy okines a e he admin-
is a ion o posi i ely cha ged nanopa icles. The e is su icien p oo o he co ela ion
o Complimen ac i a ion wi h nanopa icle adminis a ion. Impo an ly, nanopa icles
wi h a posi i e su ace cha ge igge ed ac i a ion o he classical complimen pa hways,
whe eas nega i ely cha ged pa icles “swi ched on” he al e na i e (lec in) pa hways [
5
,
6
].
I is becoming sequen ially mo e unde s andable ha a e in ense expe imen al
and clinical e alua ion o p o ein biologics and small molecules, gene he apy and RNA
medicines ep esen p omising models o d ug inno a ion. RNA accines agains di e en
cance s showcase an e icien echnology, as hey a e easie and as e o de elop and
manu ac u e compa ed o con en ional accines. Impo an ly, RNA accines a e comple ely
syn he ic and do no equi e cell cul u es.
In his e iew, we ha e a emp ed o highligh he mos ecen de elopmen s in he
ield o nanomedicine.
2. RNA o Cance The apy
RNA he apy ac s on messenge RNA (mRNA) by using oligonucleo ides ha can
in e e e wi h di e en me abolic p ocesses o a polynucleo ide, such as splicing, he ma u e
p ocess s a ing om p e-mRNA, anspo , ansla ion, and deg ada ion [
1
] Di e en ly
om s anda d chemo he apy, RNA he apy ha bo s high speci ici y and may be used
o a ge mul iple c i ical oncogenic d i e s, educe d ug esis ance o umo cells, and
a es g ow h o ad anced-s age umo s [
2
]. RNA he apeu ics may ac h ough se e al
mechanisms. They can inhibi he p oli e a ion and induce apop osis o umo cells, p e en
he me as asiza ion p ocess, dis up he gene’s exp ession, inhibi angiogenesis, econs uc
he umo en i onmen , ep og am, and dec ease d ug esis ance o umo cells [
2
] RNA
he apy may be di ided in o h ee majo classes: an isense oligonucleo ides (ASO), RNA
in e e ence (RNAi) he apies, and messenge (mRNA) he apy (Figu e 1and Table 1).
ASO a e single-s anded sequences o 15–25 nucleo ides ha bind speci ically o a ge
mRNA by complemen a y base pai ing. Because o ASO’s weak hyd ophilici y, a common
modi ica ion is hioliza ion o inc ease di usion in issues and abso p ion.
Cance s 2022, 14, x FOR PEER REVIEW 2 o 19
unde s anding has shown ha by gaining deepe insigh s in o nano-bio in e ac ions and
pe sonaliza ion o nanomedicines, and h ough he applica ions o nano echnology o
eme ging and exis ing he apeu ic modali ies, we ha e s a ed o ealize he ue po en ial
o nanomedicines in cance [1–4].
Recen s udies ha e p o ided e idence o an upsu ge in cy okines a e he
adminis a ion o posi i ely cha ged nanopa icles. The e is su icien p oo o he
co ela ion o Complimen ac i a ion wi h nanopa icle adminis a ion. Impo an ly,
nanopa icles wi h a posi i e su ace cha ge igge ed ac i a ion o he classical
complimen pa hways, whe eas nega i ely cha ged pa icles “swi ched on” he
al e na i e (lec in) pa hways [5,6].
I is becoming sequen ially mo e unde s andable ha a e in ense expe imen al and
clinical e alua ion o p o ein biologics and small molecules, gene he apy and RNA
medicines ep esen p omising models o d ug inno a ion. RNA accines agains
di e en cance s showcase an e icien echnology, as hey a e easie and as e o de elop
and manu ac u e compa ed o con en ional accines. Impo an ly, RNA accines a e
comple ely syn he ic and do no equi e cell cul u es.
In his e iew, we ha e a emp ed o highligh he mos ecen de elopmen s in he
ield o nanomedicine.
2. RNA o Cance The apy
RNA he apy ac s on messenge RNA (mRNA) by using oligonucleo ides ha can
in e e e wi h di e en me abolic p ocesses o a polynucleo ide, such as splicing, he
ma u e p ocess s a ing om p e-mRNA, anspo , ansla ion, and deg ada ion [1]
Di e en ly om s anda d chemo he apy, RNA he apy ha bo s high speci ici y and may
be used o a ge mul iple c i ical oncogenic d i e s, educe d ug esis ance o umo cells,
and a es g ow h o ad anced-s age umo s [2]. RNA he apeu ics may ac h ough
se e al mechanisms. They can inhibi he p oli e a ion and induce apop osis o umo
cells, p e en he me as asiza ion p ocess, dis up he gene’s exp ession, inhibi
angiogenesis, econs uc he umo en i onmen , ep og am, and dec ease d ug
esis ance o umo cells [2] RNA he apy may be di ided in o h ee majo classes:
an isense oligonucleo ides (ASO), RNA in e e ence (RNAi) he apies, and messenge
(mRNA) he apy (Figu e 1) (Table 1). ASO a e single-s anded sequences o 15–25
nucleo ides ha bind speci ically o a ge mRNA by complemen a y base pai ing.
Because o ASO’s weak hyd ophilici y, a common modi ica ion is hioliza ion o inc ease
di usion in issues and abso p ion.
Figu e 1. Main RNA used o cance he apy.
Thei easy di usion and abso p ion h ough issues allows hem o di ec ly bind o
a ge s a e being injec ed in o pa ien s [7].
ASO may ac a hei a ge mRNAs by ac i a ing RNase H o inhibi ing ansla ion
by p e en ing ibosomes’ ac ion h ough a s e ic e ec [2]. ASO may ac by co ec ing an
al e ed spliceosome o p o eins, epai ing de ec i e RNAs, es o ing p o eins, o
down egula ing genes’ exp ession [2]. Among ASO, some ac as miRNA inhibi o s. These
oligonucleo ides bind o he ac i e chains o endogenous miRNAs wi h gene-silencing
Figu e 1. Main RNA used o cance he apy.
Thei easy di usion and abso p ion h ough issues allows hem o di ec ly bind o
a ge s a e being injec ed in o pa ien s [7].
ASO may ac a hei a ge mRNAs by ac i a ing RNase H o inhibi ing ansla ion
by p e en ing ibosomes’ ac ion h ough a s e ic e ec [
2
]. ASO may ac by co ec ing an
al e ed spliceosome o p o eins, epai ing de ec i e RNAs, es o ing p o eins, o down-
egula ing genes’ exp ession [
2
]. Among ASO, some ac as miRNA inhibi o s. These
oligonucleo ides bind o he ac i e chains o endogenous miRNAs wi h gene-silencing
e ec s. The e o e, hey enhance gene exp ession [
2
]. In con as , RNA in e e ence (RNAi)
he apy is igge ed by double-s anded RNA (dsRNA). RNAi he apy ac s by knocking
down he exp ession o he genes o in e es by p omo ing sho in e e ing RNAs (siRNAs).
Some molecules a e o syn he ic manu ac u ing (siRNA and speci ic RNAi sequences).
Mo eo e , RNAi he apy may be deli e ed h ough sho hai pin RNAs (shRNAs) and
Cance s 2022,14, 2677 3 o 19
mic oRNAs (miRNAs) [
2
]. siRNAs a e double-s anded RNA molecules, 20–25 nucleo ides
in leng h. They a e made om cu ing a long dsRNA and dis up ing mRNA be o e ansla-
ion by binding i wi h 100% complemen a i y and high a ge speci ici y. Sho hai pin
RNAs (shRNAs) a e sequences o RNA, ypically abou 80 base pai s in leng h, ha include
a egion o in e nal hyb idiza ion ha c ea es a loop s uc u e. shRNA molecules a e p o-
cessed wi hin he cell upon ansc ip ion o o m a double-s anded siRNA, which knocks
down gene exp ession.
Table 1. Main classes o RNA he apy.
Class o RNA
The apy Fea u es
Example in
Cance The apy
(Fo mula ion)
Ta ge Indica ion Re e ences
ASO
12–25 nucleo ides
Single-s anded
Chemically modi ied
(3 classes)
Dan a i sen STAT3
(down egula ion)
Ad anced/ ecu en
solid umo s o
lymphoma
[8]
siRNAs
20–25 nucleo ides
Double-s anded
Inco po a ed in RISC
ALN-VSP02
(lipid nanopa icle-
o mula ed)
VEGF and KSP
(down egula ion)
Solid umo s wi h
li e in ol emen [9]
miRNAs
18–25 nucleo ides
Single-s anded
Inco po a ed in RISC
miR-29b
(ca ionic
lipoplexes)
CDK6, DNMT3B,
MCL1
(down egula ion)
Lung cance [10]
miRNA
mimics
18–25 nucleo ides
Single-s anded
Inco po a ed in RISC
miR-4689 KRAS,AKT
(down egula ion)
KRAS mu an
colo ec al cance [11]
an i-miR
18–25 nucleo ides
Single-s anded
Inco po a ed in RISC
An i-miR-155 miR-155
(down egula ion) Colo ec al cance [12]
shRNA
80 nucleo ides
Double-s anded wi h a
loop sequence
Inco po a ed in plasmid
ec o s
hTERT-shRNA
(plasmid)
hTERT
(down egula ion) Colo ec al cance [13]
mRNA Single-s anded
Less s able han DNA
AGS-003
(dend i ic cells)
CD40L RNA
umo RNA Renal cance [14]
Legend: RNAs; shRNA: sho hai pin RNA; siRNA: small in e e ing RNA; RISC: RNA-inducing silencing
complex.
The bene i o shRNA is ha i can be inco po a ed in o speci ic plasmid ec o s, pe -
mi ing cell- ype-speci ic o inducible p omo e s in eg a ed in o genomic DNA o longe -
e m o s able exp ession, and hus mo e p olonged knockdown o he a ge mRNA [
3
].
miRNAs a e sho -endogenous non-coding RNA molecules ha limi gene exp ession by
es ic ing mRNA om ansla ion and p omo ing mRNA decay. MiRNAs a e in eg a ed
wi h siRNAs and p o eins in he RNA-induced silencing complex (RISC). They egula e
gene exp ession based on pai ing wi h he a ge mRNA’s 3
0
un ansla ed egion (UTR).
When binding wi h complemen a y mRNA occu s, RISC ac i a es i s RNase componen
and deg ades i s a ge . On he o he hand, miRNAs come om single-s anded RNA.
Folding miRNAs c ea e s em-loops, small, olded a eas o dsRNA [
4
]. Due o impe ec
base pai ing, miRNA ac ion can a ec hund eds o less speci ic genes. Mo eo e , miRNA
may in luence he CpG island me hyla ion o gene p omo e s and egula e gene exp ession
a he ansc ip ional le el [
2
]. Among RNAi echniques, miRNA mimics unc ion simila ly
o endogenous miRNAs. Thei ac ion may es o e al e ed miRNA ma u a ion mechanisms
o enhance he unc ion o speci ic umo supp esso miRNAs [
5
]. In con as , miRNA
compe i i e agonis s block he binding o endogenous miRNAs o RISC. In his way, hey
up egula e he exp ession o ela ed p o eins [
6
]. mRNA he apy is an al e na i e o DNA
Cance s 2022,14, 2677 4 o 19
he apy and consis s o he injec ion o a speci ic RNA messenge in o a pa ien ’s body
o p omo e p o ein syn hesis in cells [
15
]. mRNA is less s able han DNA and is an easy
a ge o endogenous nucleases, wi h possible immune esponses gi en by high numbe s
o neoan igens. To educe hese e en s, modi ica ion o he nucleoside po ion o he u acil
ibose has been e alua ed wi h he c ea ion o an immune-e asi e “pseudou acil” [
16
].
Bo h siRNAs and miRNAs ha e hyd ophilic na u e, nega i e cha ge, and ela i ely high
molecula weigh (14–15 kDa), which make hem poo ly pe meable ac oss biological mem-
b anes. Howe e , encapsula ion o siRNAs in o esicles o conjuga ion o ce ain ligands
can help deli e hem o desi ed issues o cells and, a he same ime, a oid enal clea -
ance [
17
]. Wi h espec o siRNAs and miRNAs, mRNAs ha e highe and he e ogeneous
molecula weigh s and a e nega i ely cha ged. Unlike d ugs ha can c oss he lipid bilaye ,
he as majo i y o RNA-based he apeu ics a e oo cha ged and/o oo la ge o en e
cells, and demand a deli e y agen . Nanopa icles (such as liposomes, polyme s, and
pep ides) ha e been used o shu le mRNA o he cell cy osol [
18
]. In addi ion, he use o
lipid nanopa icles (LNPs) as a deli e y sys em o mRNA allows he ex ended ime o
d ug ac ion, educed d ug oxici y, and imp o ed d ug s abili y [
19
]. Table 1shows he
di e en classes o RNA he apies and gi es examples o each o hem.
3. Nanopa icles o he Deli e y o RNA
The de elopmen o RNA-based he apeu ics has expe ienced a boos since he 1990s
owing o he inc easing knowledge o nucleic acid chemis y and he decline in p oduc ion
cos s o mRNA [20].
The he apeu ic po en ial o RNA la gely depends on i s abili y o each he desi ed
a ge cells and exp ess he p o eins o in e es . Howe e , RNA p esen s limi ed s abili y in
se um, su e s om apid blood clea ance, o - a ge e ec s, and poo cellula up ake, and
may ac i a e immune esponses [
21
–
23
]. The e o e, he e icien and sa e deli e y o RNA
is s ill one o he signi ican challenges hampe ing i s he apeu ic applica ion.
The ansla ion e iciency and s abili y o exogenous RNA can be enhanced by se e al
me hods, such as UTR (un ansla ed egions) manipula ion, codon op imiza ion, and chem-
ical modi ica ion o he poly(A) ail o RNA [
24
,
25
]. Fu he mo e, i s immunogenici y can
be educed h ough high-pe o mance liquid ch oma og aphy pu i ica ion and chemical
manipula ion [
26
–
33
]. Ne e heless, he op imized RNA s ill has o a oid enzyma ic deg a-
da ion, in e ac wi h he a ge cell, c oss he cy oplasmic memb ane, and di use in he
cy oplasm o each he ibosomes. Despi e hese modi ica ions, RNA he apeu ics ail o
show e icien and speci ic up ake by umo cells. Recen ad ances in nano echnology ha e
led o new oppo uni ies in cance p e en ion and ea men . No el o mula ions o RNA
in nanosys ems o ec o s we e de eloped [
34
]. The use o i al and non- i al deli e y
sys ems esul ed in imp o ed s abili y and oxici y, umo -speci ic deli e y, and educed
immunogenici y. Vi al deli e y sys ems ( e o i uses, len i i uses, adeno i uses) make up
abou wo- hi ds o clinical ials wi h nucleic acids pe o med o da e. Al hough e ec i e
in a ge ed cellula deli e y, hese sys ems aise some sa e y conce ns ela ed o immune e-
sponses, ha e a educed ca go capaci y, and a e di icul o scale up manu ac u ing [
35
–
38
].
Non- i al deli e y sys ems ha e eme ged as a sa e al e na i e o cance he apy, as hey
a e less immunogenic, less oxic, and less oncogenic. In addi ion, hei p oduc ion is mo e
cos -e ec i e and easie o scale up [
39
]. Howe e , hese sys ems’ low nucleic acid deli e y
e iciency hampe s hei ansla ion in o clinical p ac ice and ep esen s a p oblem ha s ill
needs o be add essed [
40
]. Nowadays, he e exis s a as a ay o non- i al nanoca ie s
o RNA deli e y, wi h dis inc i e composi ions and hus unique p ope ies. Some essen ial
nanopa icle pla o ms a e liposomes, exosomes, polyme s, dend ime s, nanogels, and
ino ganic nanopa icles, such as ca bon nano ubes and gold and magne ic nanopa icles
(Figu e 2) [41].
Cance s 2022,14, 2677 5 o 19
Cance s 2022, 14, x FOR PEER REVIEW 5 o 19
composi ions and hus unique p ope ies. Some essen ial nanopa icle pla o ms a e
liposomes, exosomes, polyme s, dend ime s, nanogels, and ino ganic nanopa icles, such
as ca bon nano ubes and gold and magne ic nanopa icles (Figu e 2) [41].
Figu e 2. Schema ic ep esen a ion o nanopa icles used in RNA deli e y.
3.1. Lipids o Lipid-Based Nanopa icles
One o he mos ad anced RNA deli e y me hods is co- o mula ion in o lipid
nanopa icles (LNP) [42,43]. LNP o RNA deli e y mainly comp ises a ca ionic o
ionizable lipid bea ing a e ia y o qua e na y ammonium g oup, which encapsula es he
polyanionic RNA, p o ec ing i om deg ada ion and inc easing i s s abili y in blood
ci cula ion (Figu e 3). In addi ion o he ca ionic/ionizable lipid, hese o mula ions
ypically con ain a zwi e ionic lipid (helpe lipid, e.g., 1,2-dioleoyl-sn-glyce o-3-
phosphoe hanolamine, DOPE) mimicking cell memb ane lipids, choles e ol o he
s abiliza ion o he lipid bilaye o he nanopa icle, and a polye hene glycol (PEG), mean
o imp o e colloidal s abili y and educe p o ein abso p ion [44,45]. Ca ionic lipids
equen ly used in lipoplexes include N-[1-(2,3-dioleyloxy)p opyl-N,N,N-
ime hylammonium chlo ide (DOTMA) and N-[1-(2,3-dioleoyloxy)p opyl]-N,N,N-
ime hyl-ammonium chlo ide (DOTAP). The use o ca ionic lipids o lipoplex o ma ion
enhances he up ake o RNA h ough he in e ac ion o he posi i ely cha ged complexes
wi h he nega i ely cha ged cell memb anes. Se e al ca ionic LNP ha e been success ully
used as RNA ca ie s in a ge ed cance he apy, leading o highe accumula ion and
inc eased p o ein exp ession, which esul ed in supp essed/blocked umo g ow h
[10,46].
Figu e 2. Schema ic ep esen a ion o nanopa icles used in RNA deli e y.
3.1. Lipids o Lipid-Based Nanopa icles
One o he mos ad anced RNA deli e y me hods is co- o mula ion in o lipid nanopa -
icles (LNP) [
42
,
43
]. LNP o RNA deli e y mainly comp ises a ca ionic o ionizable
lipid bea ing a e ia y o qua e na y ammonium g oup, which encapsula es he polyan-
ionic RNA, p o ec ing i om deg ada ion and inc easing i s s abili y in blood ci cula ion
(Figu e 3
). In addi ion o he ca ionic/ionizable lipid, hese o mula ions ypically con ain
a zwi e ionic lipid (helpe lipid, e.g., 1,2-dioleoyl-sn-glyce o-3-phosphoe hanolamine,
DOPE) mimicking cell memb ane lipids, choles e ol o he s abiliza ion o he lipid bilaye
o he nanopa icle, and a polye hene glycol (PEG), mean o imp o e colloidal s abili y
and educe p o ein abso p ion [
44
,
45
]. Ca ionic lipids equen ly used in lipoplexes in-
clude N-[1-(2,3-dioleyloxy)p opyl-N,N,N- ime hylammonium chlo ide (DOTMA) and
N-[1-(2,3-dioleoyloxy)p opyl]-N,N,N- ime hyl-ammonium chlo ide (DOTAP). The use
o ca ionic lipids o lipoplex o ma ion enhances he up ake o RNA h ough he in e -
ac ion o he posi i ely cha ged complexes wi h he nega i ely cha ged cell memb anes.
Se e al ca ionic LNP ha e been success ully used as RNA ca ie s in a ge ed cance he -
apy, leading o highe accumula ion and inc eased p o ein exp ession, which esul ed in
supp essed/blocked umo g ow h [10,46].

Cance s 2022,14, 2677 6 o 19
Cance s 2022, 14, x FOR PEER REVIEW 6 o 19
Figu e 3. S uc u es o some common lipids/polyme s used o p epa ing nanopa icles.
The inclusion o helpe lipids, such as DOPE o choles e ol, gene ally inc eases he
in i o ans ec ion e iciency o he lipoplexes [47]. Howe e , mos o hese posi i ely
cha ged sys ems a e highly cy o oxic in i o, and he o mula ions ha e o be ca e ully
adjus ed o main ain cellula iabili y [48–51].
Mo e ecen s udies ha e ocused on using LNP based on pH-dependen ionizable
ca ionic lipids, which ha e been shown o e icien ly ans ec mRNA o exp ess
he apeu ic p o eins. These lipids a e posi i ely cha ged a acidic pH bu neu al a
physiological pH. The esul ing LNP display low su ace cha ge a physiological pH and
a e ela i ely non- oxic and non-immunogenic. The s uc u e o pH-dependen
nanopa icles may be des abilized in en i onmen s wi h pH alues lowe han 6.5,
acili a ing he elease o encapsula ed ca goes wi hin he mo e acidic umo
mic oen i onmen [52–54]. In ac , he i s siRNA d ug app o ed by he FDA, Onpa o,
is based on ionizable lipid (6Z,9Z,28Z,31Z)-hep a iacon a-6,9,28,31- e aen-19-yl-4-
(dime hylamino) bu anoa e (DLin-MC3-DMA, MC3) [55]. Se e al MC3-based LNP ha e
hen been es ed o mRNA he apeu ics [56,57]. E hanolamine was iden i ied as a
a o able headg oup. The inco po a ion o biodeg adable lipids esul ed in nanopa icles
wi h educed oxici y and be e deli e y e icacy [58,59]. Biodeg adabili y may be
con e ed by he p esence o an es e bond on he hyd ophobic ail o on he linke , which
accele a es li e clea ance. In addi ion, he elease o he ca go may be igge ed h ough
clea age o he labile es e bond (pH, nucleases) and consequen modi ica ion o he
agg ega e s uc u e [58–60].
Fu he , he use o unsa u a ed lipid ails in he lipid s uc u e has been shown o
inc ease luidi y and in oduce s uc u al de ec s in he cell memb ane, acili a ing usion
o he LNP wi h he cell memb ane, as well as endosomal escape [61]. Howe e , a a ional
balance o unsa u a ion and biodeg adabili y is o he u mos impo ance, since hese wo
ac o s seem o s ongly a ec he deg ee and si e o p o ein exp ession [62,63].
The de elopmen o lipid nanopa icles based on se ine-de i ed gemini su ac an s
and monoolein (MO) as helpe lipids o siRNA deli e y has also been epo ed. The use
o amino acids as pola headg oups in he design o su ac an s leads o enhanced
biological p ope ies (biocompa ibili y and oxici y) compa ed o con en ional qua e na y
ammonium-based su ac an s [64]. The lipoplexes o med (gemini/MO/siRNA) we e
Figu e 3. S uc u es o some common lipids/polyme s used o p epa ing nanopa icles.
The inclusion o helpe lipids, such as DOPE o choles e ol, gene ally inc eases he
in i o
ans ec ion e iciency o he lipoplexes [
47
]. Howe e , mos o hese posi i ely
cha ged sys ems a e highly cy o oxic
in i o
, and he o mula ions ha e o be ca e ully
adjus ed o main ain cellula iabili y [48–51].
Mo e ecen s udies ha e ocused on using LNP based on pH-dependen ionizable
ca ionic lipids, which ha e been shown o e icien ly ans ec mRNA o exp ess he a-
peu ic p o eins. These lipids a e posi i ely cha ged a acidic pH bu neu al a physio-
logical pH. The esul ing LNP display low su ace cha ge a physiological pH and a e
ela i ely non- oxic and non-immunogenic. The s uc u e o pH-dependen nanopa i-
cles may be des abilized in en i onmen s wi h pH alues lowe han 6.5, acili a ing he
elease o encapsula ed ca goes wi hin he mo e acidic umo
mic oen i onmen [52–54]
.
In ac , he i s siRNA d ug app o ed by he FDA, Onpa o, is based on ionizable lipid
(6Z,9Z,28Z,31Z)-hep a iacon a-6,9,28,31- e aen-19-yl-4-(dime hylamino) bu anoa e (DLin-
MC3-DMA, MC3) [
55
]. Se e al MC3-based LNP ha e hen been es ed o mRNA he a-
peu ics [
56
,
57
]. E hanolamine was iden i ied as a a o able headg oup. The inco po a ion
o biodeg adable lipids esul ed in nanopa icles wi h educed oxici y and be e deli e y
e icacy [
58
,
59
]. Biodeg adabili y may be con e ed by he p esence o an es e bond on he
hyd ophobic ail o on he linke , which accele a es li e clea ance. In addi ion, he elease
o he ca go may be igge ed h ough clea age o he labile es e bond (pH, nucleases) and
consequen modi ica ion o he agg ega e s uc u e [58–60].
Fu he , he use o unsa u a ed lipid ails in he lipid s uc u e has been shown o
inc ease luidi y and in oduce s uc u al de ec s in he cell memb ane, acili a ing usion
o he LNP wi h he cell memb ane, as well as endosomal escape [
61
]. Howe e , a a ional
balance o unsa u a ion and biodeg adabili y is o he u mos impo ance, since hese wo
ac o s seem o s ongly a ec he deg ee and si e o p o ein exp ession [62,63].
The de elopmen o lipid nanopa icles based on se ine-de i ed gemini su ac an s
and monoolein (MO) as helpe lipids o siRNA deli e y has also been epo ed. The
use o amino acids as pola headg oups in he design o su ac an s leads o enhanced
biological p ope ies (biocompa ibili y and oxici y) compa ed o con en ional qua e na y
ammonium-based su ac an s [
64
]. The lipoplexes o med (gemini/MO/siRNA) we e
ound o ha e sizes o 100–250 nm and we e sui able o in a enous adminis a ion.
The sys ems we e e ec i e in RNA complexa ion and gene silencing and p esen ed no
Cance s 2022,14, 2677 7 o 19
signi ican cy o oxici y. The ans ec ion e iciency was shown o be dependen on he
con en o he MO.
Solid lipid nanopa icles (SLN) con aining ca ionic lipids we e also epo ed as RNA
ca ie s o cance he apy. They can be p oduced wi hou he need o o ganic sol en s,
lyophilized, and he dehyd a ed SLN a e s able o up o 9 mon hs when s o ed a em-
pe a u es up o 30
◦
C [
65
]. In addi ion, he lyophilized SLN main ained hei ans ec ion
e icacy o e ime [
66
]. Al hough e ec i e as deli e y sys ems, se e al p oblems associa ed
wi h he posi i e cha ge o he lipids (e.g., oxici y) p ecluded hei de elopmen and
clinical use [
67
]. When neu al lipids we e used o eplace ca ionic ones, signi ican RNA
accumula ion, a ge genes’ down egula ion, and umo g ow h inhibi ion we e achie ed
wi hou inducing oxici y. Howe e , hese neu al lipid SLN su e om lowe loading
capaci y and lowe ans ec ion e iciency compa ed o he ca ionic lipid SLN.
Mo e ecen ly, miRNA-loaded exosomes ha e been enginee ed as cance he apeu ics
(endome ial cance , b eas cance , colo ec al cance , li e cance ) [
68
–
72
]. Exosomes a e
small (50–150 nm) endogenous memb ane esicles sec e ed om se e al mammalian
cell ypes. These ex acellula esicles (EV) can use wi h he memb ane o a ge cells
and deli e exosome su ace p o eins, ca bohyd a es, lipids, and nucleic acids. They a e
non-immunogenic and non-oncogenic, p esen negligible oxici y, and hus s and as a
p omising and inno a i e pla o m o miRNA deli e y [
73
]. Sh am e al. ha e shown ha
exosomes can e icien ly deli e siRNA in o a ge cance cells, leading o gene silencing
and cance cell dea h [
74
]. Howe e , he e a e s ill se e al limi a ions o implemen ing
an EV-media ed miRNA cance he apy, mainly ela ed o he la ge-scale p oduc ion,
isola ion, and cha ac e iza ion o EV sui able o clinical ansla ion s udies. In addi ion,
he de e mina ion o he RNA con en in he EV aces some p oblems. Finally, he dosage
mus be accu a ely de ined, and ou es o adminis a ion mus be be e explo ed since
mos sys emically injec ed EV a e deli e ed o he li e [75].
3.2. Polyme s
Polyme ic-based non- i al ec o s ep esen ano he class o nanoscale pla o ms o
RNA deli e y. Speci ically, ca ionic polyme s can bind o nucleic acids o o m polyplexes.
Polyme s can e icien ly p o ec RNA om nucleases and p omo e cellula up ake and
endosomal escape, leading o highe RNA deli e y e iciency. Rep esen a i e polyme s o
his class include chi osans, polye hyleneimine (PEI), dend ime s, and nanogels (Figu e 2).
Chi osans a e na u ally de i ed ca ionic polysaccha ides, di e ing in he deg ee o
N-ace yla ion
and molecula weigh (50–2000 kDa). They a e eadily a ailable, biodeg ad-
able, easy o modi y, and possess unique biological p ope ies associa ed wi h hei polyca-
ionic na u e [
76
–
78
]. Chi osan is only poo ly wa e -soluble and exhibi s low ans ec ion
e icacy. Howe e , i can be de i a ized o inc ease nucleic acid deli e y e iciency by chi-
osan ec o s. S a egies o de i a iza ion include s uc u al modi ica ions–like (i) copoly-
me iza ion: polye hylene glycol, PEG, and polye hyleneimine, PEI, a e commonly used,
al hough o he chi osan g a copolyme s a e being s udied as nucleic acid ca ie s [
79
];
and (ii) unc ional g oup modi ica ion: N-alkyla ion and qua e niza ion enhance colloidal
s abili y and ans ec ion e icacy o he nanopa icles–and ligand conjuga ion–pep ides,
p o eins, and non-p o einaceous ligands, like ca bohyd a es, olic acid and hyalu onic acid
a e commonly used o chi osan ec o conjuga ion [
80
,
81
]. Al hough non-p o einaceous
ligands a e usually less immunogenic and p oduce mo e s able ec o s, p o einaceous
ligands o e a as di e si y o choices wi h a o able unc ionali ies o ec o conjuga ion
o nucleic acid deli e y [
82
]. Chi osan-based nanopa icles simul aneously encapsula ing
siRNA and he an icance d ug doxo ubicin we e shown o dec ease he iabili y, g ow h,
p oli e a ion, and mig a ion o b eas [
83
] and colo ec al [
84
,
85
] cance cells and induce
hei apop osis.
PEI is a ca ionic polyme ha has been widely used in gene he apy [
86
]. I is
highly e icien in he compac ion o nucleic acids and p omo es endosomal escape ia
he p o on sponge e ec [
87
]. The ans ec ion e iciency o PEI-mRNA polyplexes in
Cance s 2022,14, 2677 8 o 19
se e al ypes o cells has been epo ed, bu cy o oxici y om PEI hinde s i s he apeu ic
applica ion [44,48,88].
Inco po a ing PEI in o polyme s (as abo e o chi osan) has been epo ed o dec ease
i s oxici y. Fo example, PEI-
β
-cyclodex in conjuga es show low oxici y while being
highly e icien in p omo ing cell up ake and endosomal escape, leading o enhanced
ans ec ion e iciency [89].
Chemical modi ica ion o low molecula weigh PEI (<2 kDa) wi h salicylamide o
s ea ic acid has also been epo ed o educe he oxici y and enhance ans ec ion e iciency
o he esul ing polyplexes/micelles [90,91].
Dend ime s a e polyme ic ma e ials wi h a highly b anched 3D s uc u e [
92
]. They
consis o a cen al co e, many laye s o epea ing uni s, and mul iple unc ional g oups
on he su ace. Due o hei unique s uc u e, hey possess a ious in e es ing physical
p ope ies, such as good wa e -solubili y, nanoscale uni o m size, symme ical shapes,
in e nal ca i ies, good biocompa ibili y, s abili y, and high d ug-loading capaci y. Thei
oxici y is gene ally low bu depends on he numbe o e minal amino g oups and posi i e
cha ge densi y. Dend ime -based o ganic and ino ganic nanopa icles ha e been widely
s udied and exhibi ed high po en ial in cell a ge ing and d ug deli e y [
93
–
95
]. Dend ime -
based i on oxide nanopa icles (IONP) show educed oxici y, enhanced biocompa ibili y
and sa e y, enhanced escape om he e iculoendo helial sys em (RES), and enhanced
MRI p ope ies compa ed o non- unc ionalized IONP. Dend onized magne ic IONP ha e
been applied in gene deli e y as magne oplexes o magne o ec ion, leading o high-le el
ansgene exp ession a e a sho incuba ion ime using low doses o nucleic acid and a
small amoun o NP [96–98].
Dend ime s ha e also been used as empla es o gold nanopa icles (AuNPs) o
con ol hei size and shape, which a e known o in luence he e iciency o AuNPs in
biological sys ems highly [99,100].
Nanogels (NG) a e gel pa icles wi h a h ee-dimensional hyd ophilic ne wo k s uc u e.
They a e commonly made up o polyac ylic acid, polyac ylamides, polyaminoacids, and
o he high molecula weigh polyme s. NG-based d ug deli e y sys ems possess a la ge
su ace a ea, s uc u al s abili y, and he abili y o swell. They p esen a high loading
capaci y, encapsula ing ei he hyd ophobic o hyd ophilic d ugs. Thei shape and size
can be inely uned, and hey a e sensi i e o pH, empe a u e, ionic s eng h, and o he
ex e nal s imuli, which con e s hem adequa e con olled d ug elease capaci ies [
101
–
103
].
A hiola ed PEI-dex in NG was shown o e icien ly deli e siRNA o cance cells
wi hou inducing comp omising oxici y. In any case, he a iable mo phology o he
nanopa icles, as well as hei size dis ibu ion, allied o he lack o da a on hei clinical
sa e y and e icacy, a e issues o be o e come o a b oad applica ion o his nanopla o m
o be easible [104].
3.3. Ino ganic Nanopa icles
Ino ganic nanoma e ials (de i ed om gold, ca bon, silica, e c.) a e p omising ca ie
pla o ms o RNA deli e y due o hei unique physicochemical p ope ies, which endow
hem exci ing a ibu es, such as long- e m s abili y, high loading capaci y, and op ical
esponsi eness. These ino ganic nanopa icles a e usually easie o syn hesize and scale
up han o ganic-based ones and ha e been he subjec o much esea ch conce ning hei
po en ial as nanoca ie s o nucleic acid deli e y.
The calcium phospha e (CaP) composi e is he oldes non- i al gene ca ie , in oduced
in 1973. I is biocompa ible and biodeg adable and o ms complexes wi h nucleic acids,
success ully deli e ing hem o cells [
105
]. Howe e , he size o he CaP p ecipi a es is
challenging o con ol, which cons i u es a se e e limi a ion o hei use. Pegyla ion and
lipid coa ing imp o e hese nanoca ie s’ colloidal s abili y, which shows ele an
in i o
e icacies [106–109]. The calcium-phospha e co e is esponsible o endosomal escape and
ca go elease in o he cy osol. Fu he mo e, hese nanopa icles can e icien ly co-deli e
siRNA and mRNA [110].
Cance s 2022,14, 2677 9 o 19
Gold nanopa icles, AuNPs, possess high chemical s abili y and a ac i e op ical p ope -
ies, a e easy o unc ionalize, and a e o pa icula in e es o biomedical applica ions [
111
].
They ha e been applied as he apeu ics in d ug deli e y, diagnos ics, and imaging [
112
].
Thei e iciency in biological sys ems depends mainly on hei shape, size, and size dis i-
bu ion, and many app oaches ha e been made o op imize hese pa ame e s [
99
,
100
,
113
].
Gold can be di ec ly conjuga ed ia elec os a ic and/o co alen in e ac ions o hiola ed
compounds o o m s abilized monolaye -coa ed NP, whose p ope ies can easily be uned
h ough unc ionaliza ion o mee speci ic needs. Fo example, Ghosh e al. de eloped
e icien nanoca ie s based on cys eamine- unc ionalized AuNP o deli e miRNA o
cance cells, using neu oblas oma and o a ian cance cell lines [114].
Ca bon nano ubes (CNT) can be ca ego ized in o single-, double-, o mul i-walled
(SWNT, DWNT, and MWNT) acco ding o he numbe o g aphene laye s in hei s uc-
u e. They a e p omising nanoca ie s o nucleic acid deli e y, as hey can a oid he
endosomal ba ie h ough an endocy osis-independen cell pene a ion pa hway. CNT
a e poo ly wa e -soluble; howe e , hey o e a la ge su ace a ea, which can be modi-
ied wi h unc ional g oups and loaded wi h d ugs o nucleic acids, hus enhancing hei
aqueous dispe sibili y [
115
,
116
]. CNT-media ed deli e y o siRNA may be accomplished
ei he by chemical conjuga ion o he nucleic acid o he CNT o he ma e ial used o coa
hem [
117
] o h ough o ma ion o non-co alen complexes be ween chemically unc ion-
alized ca ionic CNT and he nega i ely cha ged siRNA [
118
–
121
]. Su ace modi ica ion o
CNT using dend ime s has been epo ed o enhance hei aqueous dispe sibili y. These
nanoca ie s ha e been shown o e icien ly complex siRNA and media e i s in acellula
deli e y wi h minimal induced cy o oxici y, and may hus be good candida e ec o s o
in i o gene silencing [122].
Mesopo ous silica-based nanopa icles (MSNP) we e i s applied o d ug deli e y in
2001 [
123
]. They p esen low oxici y, la ge su ace a ea, and hus an enhanced loading
capaci y. Howe e , hey canno induce endosomal escape and mus be chemically modi ied
o enhance hei ans ec ion e icacy
in i o
[
124
,
125
]. Ngamche d akul e al. modi ied
siRNA loaded MSNP by adding PEI o p omo e endosomal escape, and PEG, o p o ec
he siRNA om deg ada ion, and educe he oxici y induced by he PEI. The modi ied
nanopa icles we e shown o induce apop osis in b eas cance cells
in i o
[
126
]. Func-
ionaliza ion o MSNP wi h cyclodex in-g a ed PEI has also been epo ed o enhance
he loading capaci y o siRNA and enable i s e ec i e endosomal escape [
127
]. Fu he -
mo e, silica nanopa icles conjuga ed o a disialoganglioside an ibody we e used o deli e
miRNA o neu oblas oma, wi h p omising esul s [128].
I on oxide magne ic nanopa icles ha e also been p oposed as ehicles o he deli e y
o nucleic acids [
129
]. Magne ic nanopa icles coa ed wi h posi i ely cha ged polyme s,
such as PEI, ha e been shown o signi ican ly inc ease ans ec ion e iciency compa ed o
ca ionic polyme s/su ac an s o lipids alone [
130
]. This echnique, known as magne o ec-
ion, elies on he combina ion o magne ic nanopa icles wi h a posi i ely cha ged coa ing
and nucleic acids o o m he co esponding complexes, which upon applying a magne ic
ield adhe e o he cell su ace and in e nalize by endocy osis [
131
,
132
]. Nanopa icle
coa ing has been epo ed o p o ec nucleic acid om deg ada ion by nucleases. Supe -
pa amagne ic i on oxide nanopa icles (SPION) a e e ec i e ehicles o he deli e y o
nucleic acids and o e he possibili y o moni o ing biodis ibu ion. Luo e al. epo ed he
use o a olic acid unc ionalized polye hyleneimine SPION o siRNA deli e y o gas ic
cance cells [133].
The me hods desc ibed abo e ep esen some o he mos used nanopla o ms o
RNA deli e y. S ill, o he s exis ha we e no e e ed o, as he aim was o p o ide a
gene al, al hough no ex ensi e, o e iew o he wide a ie y o a ailable deli e y sys ems,
speci ically o RNA deli e y. The classi ica ions o he deli e y sys ems may some imes
o e lap, as, e.g., in he case o ino ganic gold nanopa icles unc ionalized wi h polyme s.
Whe he hey i in he class o AuNP, o o polyme s, o may be conside ed as hyb id
nanopa icles, he mos impo an hing is o be awa e o he many conside a ions ha
Cance s 2022,14, 2677 16 o 19
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