Supramolecular Cyclodextrin-Based Hydrogels for Controlled Gene Delivery

polyme s Re iew Sup amolecula Cyclodex in-Based Hyd ogels o Con olled Gene Deli e y Ana Rey-Rico 1,* and Magali Cucchia ini 2 1Cell The apy and Regene a i e Medicine Uni , Cen o de In es igacións Cien í icas A anzadas (CICA), Uni e sidade da Co uña, Campus de A Co uña, 15071 A Co uña, Spain 2Cen e o Expe imen al O hopaedics, Saa land Uni e si y Medical Cen e , D-66421 Hombu g/Saa , Ge many; [email p o ec ed] *Co espondence: ana. ey[email p o ec ed]; Tel. +34-881-01-5543 Recei ed: 6 Ma ch 2019; Accep ed: 17 Ma ch 2019; Published: 19 Ma ch 2019   Abs ac : Con olled deli e y o gene ans e ec o s is a powe ul s a egy o enhance he empo al and spa ial p esen a ion o he apeu ic agen s in a de ined a ge . Hyd ogels a e adap ed bioma e ials o gene deli e y capable o ac ing as a localized depo o genes while main aining he long e m local a ailabili y o DNA ec o s a a speci ic loca ion. Sup amolecula hyd ogels based on cyclodex ins (CDs) ha e a ac ed conside able a en ion as po en ial bioma e ials in a b oad ange o d ug deli e y applica ions. Thei unique cha ac e is ics o hixo opici y and low cy o oxici y due o hei p oduc ion unde mild condi ions make hem po en ial candida es o o m injec able deli e y sys ems. This wo k aims o p o ide an o e iew o he use o CD-based polypseudo o axane hyd ogels as con olled gene deli e y sys ems o di e en applica ions in egene a i e medicine. Keywo ds: sup amolecula hyd ogels; cyclodex in-based polypseudo o axane hyd ogels; con olled gene deli e y; non i al ec o s; i al ec o s; gene ans e 1. In oduc ion 1.1. Cyclodex ins Cyclodex ins (CDs) ep esen a g oup o cyclic oligosaccha ides consis ing o a ela i ely hyd ophobic ca i y and a hyd ophilic ex e nal ace ob ained om enzyma ic ans o ma ion o s a ch wi h a o us-like molecula shape [ 1 ]. CDs a e classi ied as α -, β , o γ -CDs, depending on he numbe o D(+)-glucose uni s linked by α -1,4-linkages, being o 6, 7, and 8, espec i ely [ 2 ] (Figu e 1). Mo eo e , he p esence o 2-, 3-, and 6-hyd oxyl g oups on he ing makes CDs e sa ile pla o ms o s uc u al modi ica ions o imp o e hei na u al p ope ies in di e en applica ions [ 3 ]. In addi ion, CDs a e gene ally conside ed as sa e (GRAS) subs ances by he Food and D ug Adminis a ion (FDA) [1]. CDs ha e he abili y o h ead along ce ain polyme egions (main-chain complexes) o la e al chains (side-chain complexes), leading o he o ma ion o sup amolecula assembled s uc u es. These sup amolecula s uc u es a e no mally e e ed o as polypseudo o axane when CDs can e e sibly a el along he polyme backbone o la e al chains. Con e sely, when bo h ends o he polyme chains in polypseudo o axanes a e co alen ly capped wi h bulky molecules, CDs a e en apped and canno be de- h eaded om he assembly, gi ing so-called poly o axanes [4,5]. Polyme s 2019,11, 514; doi:10.3390/polym11030514 www.mdpi.com/jou nal/polyme s Polyme s 2019,11, 514 2 o 9 Polyme s 2018, 10, x FOR PEER REVIEW 2 o 10 Figu e 1. S uc u e o cyclodex ins. Two-dimensional sequences o α-CDs (CID: 444913) (A), β-CDs (CID: 444041) (B), and γ-CDs (CID: 5287407) (C) we e ob ained om PubChem Compound. Th ee-dimensional (3D) s uc u es o he di e en compounds we e d awn wi h ChemBioO ice 2012 (Chem3D P o 13; Pe kinElme In o ma ics). 1.2. CD-based Polypseudo o axane Hyd ogels Sup amolecula hyd ogels ep esen a ype o bioma e ial consis ing o a solid h ee-dimensional ne wo k o med ia nonco alen bonds, such as a hyd ogen bond, hyd ophobic in e ac ion, and ca ion–π and π–π in e ac ions [6]. CD-based polypseudo o axanes gene ally consis o polyme s, such as poly(e hylene oxide) (PEO) o poly(e hylene glycol) (PEG) [7], poly(p opylene oxide) (PPO) [8], o copolyme s ha ing blocks o PEO and/o PPO, alone as PEO-PPO-PEO (Plu onics) [9–11], o combined wi h o he blocks, such as poly[(R)-3-hyd oxybu y a e] [12] o poly(cap olac one) (PCL) [13–15]. Sup amolecula hyd ogels based on polypseudo o axanes ha e al eady been epo ed as po en ial candida es o di e en issue enginee ing applica ions due o hei hixo opic na u e and excellen biocompa ibili y, showing as po en ial as injec able hyd ogel ca ie s ha may be adminis e ed ia non-in asi e implan a ion [16]. Likewise, he mild condi ions in he absence o o ganic sol en s unde which polypseudo o axane gels a e o med allow o he inco po a ion o a a ie y o biomolecules, also encompassing hyd ophobic molecules ha bene i om he p esence o ee CDs o micelle-like s uc u es o d ug hos ing [1,16,17]. 2. Gene T ans e Vec o s: Basic Concep s 2.1. Non i al Vec o s Gene ans e ia non i al ec o s ( ans ec ion) is based on he inco po a ion o DNA, ei he naked bu mos ly complexed wi h ca ionic polyme s o wi h ca ionic lipids (in polyplexes and lipoplexes) in o a a ge popula ion [18]. As a esul o his complexa ion, DNA ca go may be p o ec ed agains deg ada ion by nucleases and se um componen s by c ea ing a less nega i e su ace cha ge [19]. S ill, and unlike i al coun e pa s ha ha e e ol ed o o e come cellula and immune de ense mechanisms, non i al ca ie s exhibi educed ans ec ion e iciencies as hey a e p ecluded by nume ous ex a- and in acellula obs acles [18]. The e o e, he ob aining o an e icien non i al-media ed ans e migh need epea ed adminis a ion o achie e sa is ac o y gene he apeu ic e ec s, due o hei sho e en ion ime o low he apeu ic e icacy in a ge issues [15]. Howe e , he main ad an age o hese ypes o ec o s is hei biosa e y as hey a oid he isk o acqui ing eplica ion compe ence and o inse ional mu agenesis commonly associa ed wi h i al ec o s. Likewise, i s po en ial o la ge scale p oduc ion a ela i ely low expense makes hese ec o s a ac i e ools o gene he apy [20]. 2.2. Vi al Vec o s Figu e 1. S uc u e o cyclodex ins. Two-dimensional sequences o α -CDs (CID: 444913) ( A ), β -CDs (CID: 444041) ( B ), and γ -CDs (CID: 5287407) ( C ) we e ob ained om PubChem Compound. Th ee-dimensional (3D) s uc u es o he di e en compounds we e d awn wi h ChemBioO ice 2012 (Chem3D P o 13; Pe kinElme In o ma ics). 1.2. CD-Based Polypseudo o axane Hyd ogels Sup amolecula hyd ogels ep esen a ype o bioma e ial consis ing o a solid h ee-dimensional ne wo k o med ia nonco alen bonds, such as a hyd ogen bond, hyd ophobic in e ac ion, and ca ion–πand π–πin e ac ions [6]. CD-based polypseudo o axanes gene ally consis o polyme s, such as poly(e hylene oxide) (PEO) o poly(e hylene glycol) (PEG) [ 7 ], poly(p opylene oxide) (PPO) [ 8 ], o copolyme s ha ing blocks o PEO and/o PPO, alone as PEO-PPO-PEO (Plu onics) [ 9 – 11 ], o combined wi h o he blocks, such as poly[(R)-3-hyd oxybu y a e] [12] o poly(cap olac one) (PCL) [13–15]. Sup amolecula hyd ogels based on polypseudo o axanes ha e al eady been epo ed as po en ial candida es o di e en issue enginee ing applica ions due o hei hixo opic na u e and excellen biocompa ibili y, showing as po en ial as injec able hyd ogel ca ie s ha may be adminis e ed ia non-in asi e implan a ion [ 16 ]. Likewise, he mild condi ions in he absence o o ganic sol en s unde which polypseudo o axane gels a e o med allow o he inco po a ion o a a ie y o biomolecules, also encompassing hyd ophobic molecules ha bene i om he p esence o ee CDs o micelle-like s uc u es o d ug hos ing [1,16,17]. 2. Gene T ans e Vec o s: Basic Concep s 2.1. Non i al Vec o s Gene ans e ia non i al ec o s ( ans ec ion) is based on he inco po a ion o DNA, ei he naked bu mos ly complexed wi h ca ionic polyme s o wi h ca ionic lipids (in polyplexes and lipoplexes) in o a a ge popula ion [ 18 ]. As a esul o his complexa ion, DNA ca go may be p o ec ed agains deg ada ion by nucleases and se um componen s by c ea ing a less nega i e su ace cha ge [ 19 ]. S ill, and unlike i al coun e pa s ha ha e e ol ed o o e come cellula and immune de ense mechanisms, non i al ca ie s exhibi educed ans ec ion e iciencies as hey a e p ecluded by nume ous ex a- and in acellula obs acles [ 18 ]. The e o e, he ob aining o an e icien non i al-media ed ans e migh need epea ed adminis a ion o achie e sa is ac o y gene he apeu ic e ec s, due o hei sho e en ion ime o low he apeu ic e icacy in a ge issues [ 15 ]. Howe e , he main ad an age o hese ypes o ec o s is hei biosa e y as hey a oid he isk o acqui ing eplica ion compe ence and o inse ional mu agenesis commonly associa ed wi h i al ec o s. Likewise, i s po en ial o la ge scale p oduc ion a ela i ely low expense makes hese ec o s a ac i e ools o gene he apy [20]. 2.2. Vi al Vec o s Vi al gene ans e ( ansduc ion) is based on he na u al cellula en y pa hways o i uses om which hey a e de i ed [ 20 ]. The mos common i uses manipula ed o gene ans e pu poses Polyme s 2019,11, 514 3 o 9 include he pes simplex i us (HSV) [ 21 , 22 ], adeno i uses [ 23 , 24 ], e o- and len i i uses [ 25 – 27 ], and adeno-associa ed i us (AAV) [ 28 – 30 ]. While gene ans e ia i al ec o s is highly e icien , he exis ence o pa ien -associa ed ac o s and physiological ba ie s (high immunogenici y, inhibi ion o ansduc ion in he p esence o speci ic an icoagulan s) may hinde he e ec i e deli e y, p ocessing, and exp ession o ansgenes in he a ge cells [31–33]. 3. Con olled Deli e y o Gene T ans e Vec o s ia CD Hyd ogels 3.1. P inciples o Con olled Gene Deli e y The con olled deli e y o gene ans e ec o s ep esen s a powe ul ool o add ess he issues associa ed wi h he use o gene ans e ec o s in clinical se ings (i.e., educed e iciency, apid deg ada ion, physiological ba ie s, and/o ec o - and pa ien -speci ic- immune esponses) [ 33 ]. In addi ion, sus ained gene exp ession has been epo ed o be mo e e ec i e compa ed wi h he adminis a ion o ecombinan molecules [ 34 ], p o iding high le els o he apeu ic genes o a ge a ious physiological mechanisms, such as angiogenesis [ 35 ] o chond ogenesis [ 11 ], o du ably enhance he epai o inju ed issues and o gans [ 36 ]. He eo , he p olonged p esence o he gene ans e ec o s in he cellula mic oen i onmen may imp o e he e icacy o he he apeu ic ca go by p o iding a long e m, sus ained a a speci ic place ea men while minimizing he exposi ion o non- a ge issues [15,34,37]. Among a a ie y o bioma e ials, hyd ogels ha e been epo ed as po en ial ools o gene deli e y, a o ding bo h p o ec ion o he gene ans e ec o s agains ex acellula deg ada ion and p ema u e agg ega ion and con olled supply o he he apeu ic molecules a he a ge place. 3.2. Con olled Deli e y o Gene T ans e Vec o s ia Sup amolecula -Based CD Hyd ogels While polypseudo o axane hyd ogels ha e been ex ensi ely s udied o d ug deli e y app oaches [ 1 , 4 ], hei applica ion in gene deli e y app oaches has been less explo ed. In his ega d, mos applica ions using CDs o gene he apy pu poses ocused on he use o poly o axanes based on CD-con aining ca ionic polyme s ac ing as ca ie s o plasmid DNA. Gene deli e y om polypseudo o axanes hyd ogels has been chie ly associa ed wi h he shedding o CDs om he linea polyme backbone, being he de- h eading a e p opo ional o he olume o he dissolu ion medium [ 38 ]. The e o e, when injec ed in o he body, polypseudo o axane hyd ogels migh g adually dilu e upon con ac wi h physiological luids leading o CD all-o and he eleased genes being abso bed by su ounding cells h ough endocy osis [38]. He e, we will explo e he use o polypseudo o axane hyd ogels as con olled deli e y sys ems o gene ans e ec o s and hei po en ial applica ion in issue enginee ing and egene a i e medicine app oaches. 3.2.1. Con olled Deli e y o Non i al Vec o s Sup amolecula hyd ogels based on block copolyme composed o poly(L-lysine) (PLL) segmen s o complexa ion o plasmid DNA encoding o he g een luo escen p o ein (GFP) and Plu onic ® F68 (PF68-PLL) o o m inclusion complexes α -CD we e p oduced [ 9 ] (Table 1). Bo h gela ion ime and mechanical s eng h could be modula ed by uning he amoun s o F-68-PLL and α -CD. Likewise, he sys ems eleased DNA complexes o 3 days allowing o sus ained ansgene exp ession in a ib oblas cell line wi h educed cy o oxici y. Polyme s 2019,11, 514 4 o 9 Table 1. Con olled deli e y o non i al ec o s ia sup amolecula -based CD hyd ogels. Polyme s/CDs Vec o s Ou comes App oaches Ta ge s Re e ences PF68-PLL/α-CD pDNA-GFP Sus ained pDNA deli e y o 80 h; ans ec ion e iciency ~14% mouse ib oblas cells 3T3 n.s. [9] MPEG-PCL-PDMAEMA/α-CD pDNA-luc Sus ained elease o pDNA up o 6 days; ans ec ion e iciency compa able o eshly p epa ed PEI polyplexes COS-7 cells n.s. [13] PEG-α-CD-c oss-linked PVDT pDNA-luc E icien e e se gene ans ec ion o cells cul u ed on he gel su ace COS-7 cells n.s. [39] MPEG-PCL-PEI/α-CD MPEG-PCL-PEIFA/α-CD pDNA-GFP pDNA-Nu 77 Sus ained elease o pDNA o 7 days; ans ec ion e iciency o 63% a op imal weigh a io o 1.5; signi ican inhibi ion o he apeu ic esis an umo g ow h wi h high exp ession o Bcl-2 p o eins Highe e iciency when combining he chemo he apeu ic agen pacli axel and he a ge ing abili y o FA HEK293 cells, umo model (BALB/c nude mice) umo [15,40] MPEG-PLLD-A g/α-CD pMMP-9 Con olled elease o 6 days; ans ec ion e iciency up o 72%; sus ained umo g ow h inhibi ion a e 21 days wi h good biocompa ibili y HNE-1 cells, nude mice bea ing HNE-1 umo s umo [37] Abb e ia ions: CD: cyclodex in; Plu onic ® F68; PLL: poly(L-lysine); α -CD: alpha-CD; MPEG-PCL-PDMAEM:. me hoxy-poly(e hylene glycol)-b-poly( ε -cap olac one)-b- poly[2-(dime hylamino)e hyl me hac yla e]; PEG: poly-e hylene glycol; β -CD: be a-CD; PVDT: poly-2- inyl-4,6-diamino-1,3,5- iazine; PEI: poly(e hylene imine); PLLD-A ginine- unc ionalized PLL dend on; pDNA: plasmid DNA; GFP: g een luo escen p o ein; luc: luci e ase; Nu 77: Bcl-2 (B-cell lymphoma 2) con e sion Nu 77 gene; FA: olic acid; MMP-9: ma ix me allop o einase 9; HNE-1: human nasopha yngeal ca cinoma HNE-1 cells; n.s.: no speci ied. Polyme s 2019,11, 514 5 o 9 Li e al. p epa ed iblock copolyme s o me hoxy-poly(e hylene glycol)-b-poly( ε -cap olac one)-b- poly[2-(dime hylamino)e hyl me hac yla e] (MPEG-PCL-PDMAEMA) wi h well-de ined ca ionic block leng hs o condense pDNA in polyplexes. O no e, MPEG impa ed s abili y o he pDNA polyplexes and also se ed as an ancho ing segmen when he pDNA polyplexes we e encapsula ed in α -CD-based sup amolecula polypseudo o axane hyd ogels. In addi ion, he sys ems eleased pDNA in a sus ained way o up o 6 days, leading o ans ec ion le els compa able o hose achie ed wi h eshly p epa ed poly(e hylene imine) (PEI) polyplexes [13]. Sup amolecula based CD hyd ogels ha e also been manipula ed as sca olds o ma ix-media ed gene ans ec ion. By hese lines, hyd ogen bonding s eng hened hyd ogels we e p epa ed by adical copolyme iza ion o PEG me hac yla ed β -CD (PEG- β -CD) and 2- inyl-4,6- diamino-1,3,5- iazine (VDT) monome [ 39 ]. Immobiliza ion o plasmid DNA on o he su ace o hyd ogels was achie ed by hyd ogen bonding be ween he base pai s and diamino iazine, esul ing in an e icien e e se gene ans ec ion o he luci e ase gene in a kidney cell line (COS-7) cul u ed on he gel su ace. CD-based polypseudo o axane hyd ogels ha e also been desc ibed as po en ial candida es o design injec able hyd ogels o cance he apy by p o iding a long e m, sus ained a umo si es ea men while minimizing he exposi ion o non- a ge issues [15,37]. Injec able sup amolecula hyd ogel sys ems we e o med by complexa ions be ween α -CD and ca ionic MPEG-PCL-PEI copolyme . The esul ing hyd ogels we e capable o o ming polyplexes wi h epo e plasmid DNA and o p o iding a sus ained elease o pDNA in he o m o polyplexes o up o 7 days. O no e, he inco po a ion o he an ipop o ic Bcl-2 con e sion gene in he sys ems esul ed in an e ec i e inhibi ion o umo g ow h a e 7 days in i o when injec ing in o a solid umo o nude mice [ 15 ]. Mo e ecen ly, he same au ho s in ol ed a simila sys em inco po a ing a olic acid a ge ed g oup (MPEG-PCL-PEI-FA/ α -CD) o co-deli e he chemo he apeu ic pacli axel and he an ipop o ic Bcl-2 con e sion gene in a umo o mice [ 40 ]. Rapid solidi ica ion o he sys ems was no ed a e adminis a ion ia pe i umo al injec ion. Likewise, a signi ican p e en ion o he in i o g ow h o he apeu ic- esis an H460/Bcl-2 umo was obse ed as a consequence o he sus ained elease o sup amolecula hyd ogel and a ge ing abili y media ed by FA ligand. A simila endency was obse ed upon encapsula ion o an MMP-9 shRNA plasmid (pMMP-9) in o α-CD and PEGyla ed a ginine- unc ionalized PLL dend on hyd ogels [37]. 3.2.2. Con olled Deli e y o Vi al Vec o s While less explo ed han hei non i al ec o coun e pa s, mos o he a emp s o design con olled deli e y sys ems o i al ec o s ocused on he design o mic ocapsules o biodeg adable polyme s o he sus ained elease o he ec o a he a ge place, and polyme conjuga es ha p o ide s eal h, cell- a ge ed shells o he i al ec o s [ 41 – 43 ]. In his ega d, al hough i al gene ans e is highly e icien , i s ou come can s ill be p ecluded by some ba ie s, such as high immunogenici y and inhibi ion o ansduc ion in he p esence o speci ic an icoagulan s [31–33]. While ecombinan adeno-associa ed i us ( AAV) ec o s a e conside ed he sa es ec o s o i al gene ans e , hei ansla ional use in pa ien s migh be impeded by he p esence o neu alizing an ibodies agains he AAV capsid p o eins in he hos [ 32 ], especially by hose p esen in he syno ial luid o pa ien s wi h join diseases [ 44 ]. Con olled deli e y o AAV ec o s ia polyme ic bioma e ials has al eady shown o be a po en way o o e come hese issues [ 33 ]. Addi ionally, sy ingeable hyd ogels ha can be p ecisely placed in a speci ic si e o he body using minimally in asi e ways and ha ans o m in o depo s o sus ained elease o ac i e subs ances a oiding hei di usion o non- a ge places, ha e been la gely pu sued o di e en egene a i e medicine app oaches. The e o e, we ecen ly gene a ed sy ingeable polypseudo o axane gels o p oduce ma e ials ha can du ably deli e AAV ec o s o applica ions in ca ilage egene a ion [ 11 ] (Table 2). To achie e his goal, dispe sions o Plu onic ® F68 (PF68) o Te onic ® 908 (T908) con aining ei he hyalu onic acid (HA) o chond oi in sul a e (CS) we e p epa ed in PBS. α -CD was nex added o o m polypseudo o axane gels. Compa ed wi h ee ec o s, he gels allowed o p omo e highe le els o ansgene exp ession. CS (o Polyme s 2019,11, 514 6 o 9 HA)/PF68/ α -CD gels apidly eleased AAV ec o s while CS (o HA)/T908/ α -CD gels p o ided sus ained elease, p obably due o di e en in e ac ions wi h he i al ec o s. Inco po a ion o α -CD in o CS (o HA)/PF68 gels esul ed in highe AAV concen a ions and sus ained le els o ansgene exp ession in monolaye cul u es o p ima y human bone ma ow-de i ed mesenchymal s em cells (hMSCs) o e ime. In addi ion, HA inc eased bo h bioac i i y and cy ocompa ibili y o he gels [11]. Table 2. Con olled deli e y o i al ec o s ia sup amolecula -based cyclodex ins (CD) hyd ogels. Polyme s/CDs Vec o s Ou comes App oaches Ta ge s Re e ences CS (o HA)/PF68/ α -CD CS (o HA)/T908/ α -CD AAV-lacZ Sus ained elease o 21 days; CS (o HA)/PF68/α-CD gels esul ed in he highes AAV concen a ions and sus ained le els o ansgene exp ession o e ime hMSCs ca ilage epai [11] Abb e ia ions: CD: cyclodex in; CS: chond oi in sul a e; HA: hyalu onic acid; PF68: Plu onic ® F68; T908: Te onic ® 908; α-CD: alpha-CD; hMSCs: human bone ma ow-de i ed mesenchymal s em cells. Likewise, o s udy he po en ial o he sys ems o ca ilage egene a ion app oaches, hyd ogels we e cul u ed o 21 days upon con ac wi h hMSCs in a 3D agg ega e cul u e model. O no e, no dele e ious e ec s om he hyd ogels we e no ed on he chond ogenic po en ial o he cells exhibi ing no di e ences wi h hose cells cul u ed in he absence o polyseudo o axane sys ems (Figu e 2A). No ewo hy, con olled deli e y o a epo e gene ( AAV-lacZ) ia HA/PF68/ α -CD hyd ogels esul ed in he mos e ec i e gene ans e (Figu e 2A). Simila ly, supe io chond ogenic di e en ia ion was no ed by deli e y o he chond ogenic ac o SOX9 ( AAV-FLAG-hsox9) ia hese hyd ogel sys ems (Figu e 2B). These sys ems migh also be en isioned o he deli e y o o he mo phogens capable o s imula ing MSC di e en ia ion o ano he lineages, such as os eoblas s. Polyme s 2018, 10, x FOR PEER REVIEW 7 o 10 Figu e 2. Con olled deli e y o AAV-lacZ (A) o AAV-FLAG-hsox9 (B) ia polypseudo a axane hyd ogels. Pelle s we e cul u ed in he absence (posi i e con ol; ee AAV o m) o p esence o AAV-loaded hyd ogels sys ems (Plu onic® F68 (PF68)/ chond oi in sul a e (CS)/alpha-cyclodex ins (α-CD) o PF68/ hyalu onic acid (HA)/α-CD) o 21 days and moni o ed o chond ogenic di e en ia ion (A, B: Toluidine blue s aining: magni ica ion x4, all ep esen a i e da a) and β-galac osidase ac i i y (A: β-gal immunouno eac i i y: magni ica ion x20, all ep esen a i e da a). 4. Conclusions O e he pas decades, CD-based sup amolecula hyd ogels aised g owing in e es as bioma e ials o d ug and gene deli e y app oaches. Because o hei unique p ope ies o hixo opici y, biosa e y, and easy modi ica ion, CD-based polypseudo o axane hyd ogels can be used as p omising injec able deli e y sys ems o con olled gene deli e y. Likewise, he dilu ion p ocess o hese sys ems in con ac wi h body luids may be modula ed by adjus ing injec ion imes o changing he molecula weigh o he polyme ic backbone [38]. Di e en CD-based polypseudo o axane hyd ogels ha e been p oduced o design sys ems able o p o ide a long e m local a ailabili y o DNA ec o s a a speci ic loca ion and o s imula e se e al physiological mechanisms able o enhance he epai o inju ed issues. Au ho Con ibu ions: A.R.-R. and M.C. con ibu ed equally o he concep ion and w i ing o he wo k. Acknowledgmen s: Ana Rey-Rico hanks he InTalen p og am om UDC-Indi ex o he esea ch g an and Magali Cucchia ini he Deu sche A h ose-Hil e e.V. Con lic s o In e es : The au ho s decla e no con lic s o in e es . Re e ences 1. Simoes, S.M.; Rey-Rico, A.; Conchei o, A.; Al a ez-Lo enzo, C. Sup amolecula cyclodex in-based d ug nanoca ie s. Chem. Commun. 2015, 51, 6275–6289. Figu e 2. Con . Polyme s 2019,11, 514 7 o 9 Polyme s 2018, 10, x FOR PEER REVIEW 7 o 10 Figu e 2. Con olled deli e y o AAV-lacZ (A) o AAV-FLAG-hsox9 (B) ia polypseudo a axane hyd ogels. Pelle s we e cul u ed in he absence (posi i e con ol; ee AAV o m) o p esence o AAV-loaded hyd ogels sys ems (Plu onic® F68 (PF68)/ chond oi in sul a e (CS)/alpha-cyclodex ins (α-CD) o PF68/ hyalu onic acid (HA)/α-CD) o 21 days and moni o ed o chond ogenic di e en ia ion (A, B: Toluidine blue s aining: magni ica ion x4, all ep esen a i e da a) and β-galac osidase ac i i y (A: β-gal immunouno eac i i y: magni ica ion x20, all ep esen a i e da a). 4. Conclusions O e he pas decades, CD-based sup amolecula hyd ogels aised g owing in e es as bioma e ials o d ug and gene deli e y app oaches. Because o hei unique p ope ies o hixo opici y, biosa e y, and easy modi ica ion, CD-based polypseudo o axane hyd ogels can be used as p omising injec able deli e y sys ems o con olled gene deli e y. Likewise, he dilu ion p ocess o hese sys ems in con ac wi h body luids may be modula ed by adjus ing injec ion imes o changing he molecula weigh o he polyme ic backbone [38]. Di e en CD-based polypseudo o axane hyd ogels ha e been p oduced o design sys ems able o p o ide a long e m local a ailabili y o DNA ec o s a a speci ic loca ion and o s imula e se e al physiological mechanisms able o enhance he epai o inju ed issues. Au ho Con ibu ions: A.R.-R. and M.C. con ibu ed equally o he concep ion and w i ing o he wo k. Acknowledgmen s: Ana Rey-Rico hanks he InTalen p og am om UDC-Indi ex o he esea ch g an and Magali Cucchia ini he Deu sche A h ose-Hil e e.V. Con lic s o In e es : The au ho s decla e no con lic s o in e es . Re e ences 1. Simoes, S.M.; Rey-Rico, A.; Conchei o, A.; Al a ez-Lo enzo, C. Sup amolecula cyclodex in-based d ug nanoca ie s. Chem. Commun. 2015, 51, 6275–6289. Figu e 2. Con olled deli e y o AAV-lacZ ( A ) o AAV-FLAG-hsox9 ( B ) ia polypseudo a axane hyd ogels. Pelle s we e cul u ed in he absence (posi i e con ol; ee AAV o m) o p esence o AAV-loaded hyd ogels sys ems (Plu onic ® F68 (PF68)/ chond oi in sul a e (CS)/alpha-cyclodex ins ( α -CD) o PF68/ hyalu onic acid (HA)/ α -CD) o 21 days and moni o ed o chond ogenic di e en ia ion ( A , B : Toluidine blue s aining: magni ica ion × 4, all ep esen a i e da a) and β-galac osidase ac i i y (A:β-gal immunouno eac i i y: magni ica ion ×20, all ep esen a i e da a). 4. Conclusions O e he pas decades, CD-based sup amolecula hyd ogels aised g owing in e es as bioma e ials o d ug and gene deli e y app oaches. Because o hei unique p ope ies o hixo opici y, biosa e y, and easy modi ica ion, CD-based polypseudo o axane hyd ogels can be used as p omising injec able deli e y sys ems o con olled gene deli e y. Likewise, he dilu ion p ocess o hese sys ems in con ac wi h body luids may be modula ed by adjus ing injec ion imes o changing he molecula weigh o he polyme ic backbone [38]. 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