Nanomedicine for the delivery of RNA in cancer

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 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 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 56. Veiga, N.; Goldsmi h, M.; G ano , Y.; Rosenblum, D.; Dammes, N.; Kedmi, R.; Ramishe i, S.; Pee , D. Cell speci ic deli e y o modi ied mRNA exp essing he apeu ic p o eins o leukocy es. Na . Commun. 2018,9, 4493. [C ossRe ] 57. 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