Divergent approach to nanoscale glycomicelles and photo-responsive supramolecular glycogels. Implications for drug delivery and photoswitching lectin affinity

This jou nal is © The Royal Socie y o Chemis y 2023 J. Ma e . Chem. B, 2023, 11, 10189–10205 | 10189 Ci e his: J. Ma e . Chem. B, 2023, 11, 10189 Di e gen app oach o nanoscale glycomicelles and pho o- esponsi e sup amolecula glycogels. Implica ions o d ug deli e y and pho oswi ching lec in affini y† Elena Rome o-Ben, ‡ a M Ca men Cas illejos, ‡ a C is ian Rosales-Ba ios, a Ma ı ´a Expo ´si o, a Pila Ruda, a Paula M. Cas illo, a S e ania Na decchia, b Juan de Vicen e b and Nou eddine Khia * a The ield o s imuli- esponsi e sup amolecula bioma e ials has apidly ad anced in ecen yea s, wi h po en ial applica ions in di e se a eas such as cance he anos ics, issue enginee ing, and ca alysis. Howe e , designing molecula ma e ials ha exhibi p ede e mined hie a chical sel -assembly o con ol he size, mo phology, su ace chemis y, and esponsi eness o he inal nanos uc u es emains a signi ican challenge. In his s udy, we p esen a di e gen syn he ic app oach o he ab ica ion o sphe ical micelles and unc ional 1D-glyconano ube-based pho o esponsi e gels om s uc u ally ela ed diazobenzene/diace ylene glycolipids. The esul ing nanos uc u es we e cha ac e ized using NMR, TEM, and SEM, con i ming he o ma ion o sphe ical and ubula nanos uc u es in bo h he gel and solu ion s a es. Upon UV i adia ion, a e e sible gel–sol ansi ion was obse ed, esul ing om he pho oswi ching o he azobenzene uni om he s e ched ans o m o he compac , me as able cis o m. Ou gels we e shown o enable spa io- empo al con ol o he adhesion and elease o he lec in Concana alin A, demons a ing po en ial use as egene able bioma e ials o igh agains in ec ions wi h oxins and pa hogens. Addi ionally, ou micelles and gels we e e alua ed as nanocon aine s o loading and con olled elease o hyd ophobic dyes and an i umou al agen s, sugges ing hei possible use as sma he anos ic d ug deli e y sys ems. In oduc ion The de elopmen o complex nanoscale sys ems able o ecei e and execu e commands om ex e nal s imuli has ecei ed inc eased a en ion in ecen yea s due o hei po en ial use in d ug deli e y, issue enginee ing,biomine aliza ion,molecula elec onics, and ca alysis. 1–7 O ele ance o he design o hese sys ems as cance he anos ics, s uc u e–ac i i y ela ionship s udies ha e shown ha opology and size a e key ac o s o hei cellula up ake, ci cula ion ime and in e ac ion wi h speci ic ecep o s. 8–12 Indeed, while sphe ical nanoma e ials a e well sui ed o p ocesses in ol ing apid cellula up ake, 13 1D- od s uc u esa emo eapp op ia e o hose equi ingalonge ci cula ion ime. 14 On he o he hand, i has been shown ha sphe ical micelles a e po en inhibi o s o globula ecep o s, while 1D-nano ibe s as well as 3D-gels a e mo e sui able o adhesion and inhibi ion o bac e ial mo ili y. 15–18 The e o e, de eloping di e gen and cos -e ec i e syn he ic app oaches allowing he syn hesis o sma unc ionalized o ganic ma e ials and modula ing hei sizes, opology and unc ionali y om well- designed molecula monome s is highly desi able, al hough i emains an ou s anding challenge. 19 One amily o molecules sui able o he c ea ion o sup amolecula di e si y a e he amphiphiles, whose sel -o ganiza ion can lead o he o ma ion o species wi h a wide ange o sizes and whose unc ionali y can ange om o dina y soap o ex acellula ma ix mimics., 20,21 A pa adigma ic example a e sup amolecula gels, 22,23 ob ained om low molecula weigh o ganogela o s (LMOGS), 24–26 whose hie a chical sel -o ganiza ion lead o lexible 1D-nano ibe s wi h a high aspec a io ha ing sizes anging om submic on o hund eds o mic ome e s which by en anglemen , o m he mac oscopic sup amolecula gels o millime e size. 27,28 This hie a chy p o ides an excellen oppo uni y o design in elligen sup amolecula a Asymme ic Syn hesis and Func ional Nanosys ems G oup, Ins i u o de In es igaciones Quı ´micas (IIQ), CSIC-Uni e sidad de Se illa, A da. Ame ´ ico Vespucio 49, 41092, Se ille, Spain. E-mail: [email p o ec ed] b Depa men o Applied Physics and Excellence Resea ch Uni ‘Modeling Na u e’ (MNa ), Facul y o Sciences, Uni e si y o G anada, C/Fuen enue a s/n, 18071 – G anada, Spain †Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: h ps://doi.o g/ 10.1039/d3 b01713c ‡These au ho s con ibu ed equally. Recei ed 28 h July 2023, Accep ed 7 h Oc obe 2023 DOI: 10.1039/d3 b01713c sc.li/ma e ials-b Jou nal o Ma e ials Chemis y B PAPER Open Access A icle. Published on 09 Oc obe 2023. Downloaded on 6/11/2024 11:45:18 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online View Jou nal | View Issue 10190 | J. Ma e . Chem. B, 2023, 11, 10189–10205 This jou nal is © The Royal Socie y o Chemis y 2023 sys ems wi h unc ional esponses by inco po a ing mo i s esponsi e o ex e nal s imuli in o he molecula s uc u es o amphiphiles. 29–31 Among he diffe en s imuli, using ligh offe s he ad an age o being easily u ned on and off, employing adjus able wa e- leng h and in ensi y, and offe ing high spa ial and empo al con ol. 29,30,32–36 Mo eo e , an in e es ing class o amphiphiles a e glycolipids whose hie a chical sup amolecula o ganiza ion leads o complex sys ems endowed wi h impo an biological ac i i ies due o he in ol emen o ca bohyd a es in i al p ocesses such as pa hogen adhesion, ansplan ejec ion, e iliza ion, and cell p oli e a ion/di e en ia ion. 29,37–40 Based on hese p emises and wi hin ou in e es in he de elopmen o unc ional so glyconanoma e ials, 41–44 we epo , he ein, a new modula syn he ic app oach o sel -associa i e mul i unc- ional neoglycolipids 1–3 o he di e gen nano ab ica ion o unc ional sphe ical micelles, 1D-lipid glyconano ubes, and pho o esponsi e 3D-gels, Fig. 1. Resul s and discussion Syn he ic design In he design o he sel -associa i e monome s, diazobenzene, linked di ec ly o he bio ecogni ion elemen mannose, was used as a ligh - esponsi e g oup. 29,34 The design also includes a a iable oligoe hylene glycol o ine- une he hyd ophobic– hyd ophilic balance o he neoglycolipid. Fu he mo e, as hyd o- phobic ail a 25 ca bon chain bea ing a pho o-polyme izable diace ylenic unc ion, known o i s abili y o o m nanoma e ials wi h in e es ing ch oma ic p ope ies was used. 44–49 The design was alida ed by syn hesizing unc ional sphe ical micelles (Fig.1a),and1D-lipidglyconano ubes(Fig.1b), ha en angle o o m ligh esponsi e 3D-gels (Fig. 1c). Syn hesis o he monome s The syn hesis o he h ee compounds (1,2and 3), use e a-O- ace yl-a-D-mannopy anosyl ichlo oace imida e 4, ob ained in wo s eps om mannose pen aace a e, as s a ing ma e ial, Scheme 1. Monoglycosyla ion o 4,40-dihyd oxyazobenzene 5 accep o , 50 using dono 4and bo on i luo ide as an ac i a o gi es he desi es compound 6wi h 86% yield. Fo he modula ion o he hyd ophilic–hyd ophobic balance, as well as o un a elling he e ec o he connec ing g oup be ween he pola head g oup and he apola ail, bi unc ional space s 7–9, de i ed om e a- e hyleneglycol (7,8) o eicosae yleneglycol (9), we e used, Scheme 1. The condensa ion o alcohol 6wi h osyla ed space 7in he p esence o K 2 CO 3 , LiCl in ace oni ile gi es he co esponding de i a i e 10 in 60% yield, Scheme 1a. Nex , he dep o ec ion o he NHBoc de i a i e 10 using i luo oace ic acid in me hylene chlo ide gi es he ee amine 11. Amida ion wi h pen acosadiinoic acid (PCDA) in he p esence o TBTU and DIPEA in DMF gi es he co esponding amide 12 wi h 60% yield and, inally, Zemplen deace yla ion gi es he desi ed neoglycoli- pid 1, Scheme 1a. Fo he syn hesis o he neoglycolipids 2and 3, Scheme 1b, condensa ion o po assium alcohola e o 6wi h he osyla ed azido space s de i ed om e ae hyleneglycol 8o Fig. 1 Di e gen syn hesis o mannose-coa ed micelles (a) and pho o esponsi e mannose-coa ed gel (b) h ough sup amolecula sel -o ganiza ion o suga -coa ed diazobenzene/diace ylene amphiphiles. Amhiphile 3wi h a la ge PEG 20 chain sel -o ganize in o sphe ical micelle able o hos and solubilize he hyd ophobic dye Nile ed (a), while amphiphile 1wi h a sho e ae hyleneglycol chain sel -o ganize in o 1D-nano ubes (b) which e ol e o idimensional gels, able o es ablish selec i e in e ac ions wi h mannose-speci ic lec in Texas ed s concana alin A (d), and o pe o m a e e sible gel– sol ansi ion in esponse o ligh i adia ion (c and e). Pape Jou nal o Ma e ials Chemis y B Open Access A icle. Published on 09 Oc obe 2023. Downloaded on 6/11/2024 11:45:18 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online This jou nal is © The Royal Socie y o Chemis y 2023 J. Ma e . Chem. B, 2023, 11, 10189–10205 | 10191 eicosae hyleneglycol space 9, in ace oni ile gi es he e he de i a i es 13 and 14 in 67% and 70% yields espec i ely. Cu(I)-ca alyzed azide alkyne 1,3-dipola cycloaddi ions (CuAAC) be ween azides 13 o 14 and he N-(2-p opynyl)pen acosa-10,12- diynamide alkyne 15, i sel ob ained in one s ep om PCDA and p opa gylamine, in he p esence o sodium asco ba e and in a me hylene chlo ide/wa e mix u e, a o ded egioselec i ely he 1,2,3- iazole de i a i es 16 and 17 wi h 60 and 80% yield espec i ely. Finally, Zemplen deace yla ion gi es he desi ed compounds 2and 3wi h 97 and 99% chemical yield. The s uc u es o he 3 monome s we e con i med by monodimen- sional and bidimensional NMR as well as by mass spec oscopy (see expe imen al pa and ESI†). Pho oisome iza ion s udy o he monome s Azobenzene compounds a e well-known o hei e e sible pho o- isome iza ion be ween he ans and cis o ms unde al e na ing UV and isible ligh i adia ion. 51 The ligh - esponsi e beha io o h ee neoglycolipids 1–3,Fig.1,wass udiedby 1 HNMR(Fig.2b and c), UV-Vis (Fig. 2d and e), and HPLC-MS spec oscopies ( ide in a). The mos signi ican p o ons o s udying he isome iza ion kine ics o neoglycolipids 1–3 by 1 H NMR a e he diazobenzene p o ons, hep o on5o he iazoleand heanome icp o ono suga . In he s able ans (E) isome s he signals o p o ons 3, 5 and 30,5 0o hediazobenzenemoie yappea as wodouble sa 6.90 ppm and 7.10 ppm espec i ely, while p o ons 2.6 and 20.60 appea oge he a a lowe ield (abou 7.75 ppm). The iazole p o on is obse ed as a single a 7.90 ppm, and he anome ic p o on appea s ei he as a b oad single (in he case o 1- ans and 2- ans) o as a double wi h a small coupling cons an (1.34 Hz) in he case o 3- ans, a app oxima ely 5.60 ppm. In he cis (Z) isome s [1-cis,2-cis (see ESI†), and 3-cis], he signals o he 3, 5 and 30,5 0p o ons unde go a sligh shi a 7.00 and 6.90 ppm, while he 2, 6 and 20,6 0p o ons alpha o he diazo unc ion unde go a p onounced shielding e ec , om 8.00 ppm o 6.80 ppm. Al hough he anome ic p o on unde goes a sligh chemical shi change ( om 5.60 ppm o 5.50 ppm), i s loca ion in a posi ion whe e he e a e no o he signals allows i o be used as an addi ional con ol o moni o and quan i y he ans–cis isome s. To gain u he s uc u al insigh s, we conduc ed selec i e 1D-NOESY and 1H/1H EASY ROESY 2D expe imen s (see ESI†). In he case o he 2- ans de i a i e, ou NOESY 1D expe imen s acili a ed mo e p ecise signal assignmen s o bo h a oma ic ings (see Fig. S53–S56 in he ESI†). Addi ionally, 2D 1H/1H EASY- ROESY expe imen s, ca ied ou on a p edominan ly 2-cis con igu- a ion, e ealed con ac NOEs be ween he H3 and H5 p o ons o he a oma ic ing linked o he suga and a me hylene p o ons o hePEGchain(seeFig.S60in heESI†). Ou s udy allowed us o quan i y he pho os a iona y (Z:E) s a e o he h ee neoglycolipids in DMSO. Neoglycolipid 1 exhibi ed a pho os a iona y s a e o 75% cis (Z) and 25% ans (E) isome s (Fig. 2b), while neoglycolipid 2displayed a pho o- s a iona y s a e o 82% cis (Z) and 18% ans (E) isome s (see ESI†). Rema kably, and con a y o mos epo ed diazobenzene glycolipids, neoglycolipid 3showed a unique beha io as i s pho oswi ching was independen o he sol en and comple e e en in me hanol, wi h a pho os a iona y s a e o 100% cis (Z) isome (Fig. 2c). The comple e isome ic con e sion o neogly- colipid 3is o g ea in e es in s udying he in luence o ca bohyd a e o ien a ion on lec in–ca bohyd a e in e ac ions. The slow he mal e u n om pu e 3-cis o pu e 3- ans isome , occu ing o e a pe iod o 96 hou s in he da k, u he adds o he signi icance o neoglycolipid 3. Mo eo e , unde blue ligh i adia ion (460 nm), he e u n om he 3-cis o 3- ans isome in neoglycolipid 3was signi ican ly accele a ed, aking only 2 hou s (Fig. 2c). UV/Vis spec oscopic s udies we e conduc ed using DMSO as he sol en and unde gela ion condi ions (see de ails below). Glycolipids exhibi a b oad abso p ion peak a ound 320–350 nm, p ima ily a ibu ed o he ans o m o he azobenzene g oup (Fig. 2d and e). Upon i adia ion, he pho o-induced ans–cis isome iza ion was obse ed o be e e sible, as he cis o m o he azo-su ac an s elaxed back o he ans o m a e exposu e o isible ligh o slowly in he absence o ligh (Fig. 2e). Fu he mo e, a se ies o successi e ‘‘on–o ’’ swi ching expe i- men s on he abso p ion bands o glycolipid 1was ca ied ou . The esul s o hese expe imen s demons a ed ha he p ocess was e e sible o a leas nine cycles, indica ing ha sample 1 exhibi s good s abili y (Fig. 2 ). Sup amolecula sel -assembly o amphiphile 1 and cha ac e iza ion o he pho o esponsi e-glycogel In o de o de e mine he sup amolecula sel -associa ion capa- ci ies o he monome s, we de e mined i s hei wa e and o ganic sol en s solubili ies. Al hough monome s 1and 2a e insolubleinmos sol en s, hemo ehyd ophilicmonome 3is Scheme 1 Syn hesis o sel -associa i es neoglycolipid 1(a), 2and 3(b). (i) BF 3 E 2 O, CH 3 CN, 0 1C; (ii) K 2 CO 3 , LiCl, CH 3 CN; (iii) TFA, CH 2 Cl 2 ; (i ) TBTU, DIPEA, DMF; ( ) MeONa, MeOH; ( i) CuSO 4 , AsCNa, CH 2 Cl 2 /H 2 O. Jou nal o Ma e ials Chemis y B Pape Open Access A icle. Published on 09 Oc obe 2023. Downloaded on 6/11/2024 11:45:18 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online 10192 | J. Ma e . Chem. B, 2023, 11, 10189–10205 This jou nal is © The Royal Socie y o Chemis y 2023 soluble in a ious sol en s including wa e ( ide in a). The use o mix u e o sol en s e eals wa e –e hanol as he bes mix u e. Indeed, in 1 : 1 o 1 : 2 wa e –e hanol mix u e, monome 1showed a g ea abili y o o m gels. In he same condi ions monome 2 was insoluble while monome 3ga e a clea yellowish solu ion. In e es ingly, while in he 1 : 2 wa e e hanol mix u e, he gela ion o 1 akes place a a concen a ion o 1% (w/ ), in 50% aqueous e hanol solu ion i akes place a a concen a ion as low as 0.1%. Addi ionally, in he la e case he compound geli ica e ins an aneously, Fig. 3, a oom empe a u e wi h no need o hea ing–cooling o sonica ion. The s uc u e o he as- o med gel as well as he xe ogel o med om monome 1a 1% was cha ac e ized by ansmission elec on mic oscopy (TEM) and ield emission scanning elec on mic oscopy (FESEM) (Fig. 3). In e locked agglome a ed ubules wi h a uni o m diame e o 31 nm, a hickness o 9 nm, and mic on-scale leng hs we e egula ly ound in he glycogel-1 unde TEM analysis (Fig. 3a). The obse a ion o xe ogel-1 unde SEM a he ens o mic ome e scale showed in e es ing in e wined and wis ed s uc u es in a bush- o m (see Fig. S32, ESI†). Mo eo e , FESEM analysis allowed obse ing he de ailed ib illa agg ega es o ming he bundles shown in he s anda d SEM images (Fig. 3b). Small-angle X- ay sca e ing (SAXS) s udies allowed us o ad ance in he exac s uc u al de e mina ion o he ib illa glycogel-1 (Fig. 3c). Maximum peaks co esponding o a e aged epea ing dis ances o abou 9.5 nm we e ob ained (Fig. 3c). I should be no ed ha he s a ing monome is app oxima ely 5 nm in leng h when ex ended, as de e mined by Chem3D analysis (Fig. 3 ). This sugges s ha a single bilaye would ex end o a maximum o 10 nm, indica ing ha he ube walls a e likely o med by a single amphiphilic bilaye wi h some in e pene a ion o he lipid ail (as shown in Fig. 3d– ). A second peak in he SAXS spec um a 2y=0.51, co esponding o a sepa a ion o 16.6 nm, can be assigned o he inne diame e o he lipid nano ube, which is likely in luenced by he packing o he amphiphilic bilaye . Unde such sup amolecula sel -o ganiza ion, he ube in he gel would expose he mannose esidue usually used o he adhesion o pa hogens o he ex e nal phase, whose speci ic ecep o is in ol ed in se e al biological p ocesses o in e es . 51,52 On he o he hand, he in e nal ca i y o he ube, la ge han 10 nm, could accom- moda e molecules o in e es such as cy o oxic compounds, dyes o couldalsose easacon inedmedium o o ganic eac ions. 53 The mechanical p ope ies o gel-1 we e cha ac e ized h ough heological s ain sweep and equency sweep Fig. 2 Schema ic ep esen a ion o he pho oisome iza ion o neoglycolipids 1–3 (a). 1 HNMR s udies o ans–cis and cis– ans pho o and he mal isome iza ion o he neoglycolipid 1in DMSOd 6 (b), and 3in MeOD (c). UV-Vis s udies o ans–cis (d) pho oisome iza ion and cis– ans eco e y unde blue ligh i adia ion (e) o neoglycolipid 1(0,005 w / % in DMSO). Cycle o abso p ion a e al e na e i adia ion wi h UV (360 nm, g een ba ) and isible ligh (460 nm, yellow ba ) ( ). Pape Jou nal o Ma e ials Chemis y B Open Access A icle. Published on 09 Oc obe 2023. Downloaded on 6/11/2024 11:45:18 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online This jou nal is © The Royal Socie y o Chemis y 2023 J. Ma e . Chem. B, 2023, 11, 10189–10205 | 10193 expe imen s, e ealing i s high low-shea iscosi y and shea - hinning beha io . The gel exhibi ed a hys e esis loop in he low cu e, indica ing hixo opic beha io . Dynamic oscilla o y shea es s showed ha he gel beha ed as a solid-like ma e ial wi h a linea iscoelas ic ange, ollowed by non-linea beha io and yielding a highe s ain ampli udes. The ull s udy wi h de ailed esul s can be ound in he ESI.† Diace ylenic amphiphiles (DA) possess pho o esponsi e p ope ies as hey can unde go polyme iza ion h ough a 1,4- addi ion eac ion o di adicals gene a ed by UV o gi adia ion. This p ocess leads o he o ma ion o sma polydiace ylene (PDA)-nanoma e ials, exhibi ing in iguing ch oma ic p ope ies. P io o in es iga ing he unc ionali y o gel 1, we examined he abili y o he sel -associa ed DA monome 1 o pho opolyme ize in he gel s a e using Raman spec oscopy. The esul s o hese s udies e ealed ha pho opolyme iza ionoccu sonlyin hed ys a eand no in he gel s a e ( o de ailed in o ma ion, see he ESI†). S udies on he UV-induced e e sible gel–sol ansi ion To in es iga e he ligh - esponsi e beha io o glycogel 1, we, i s , conduc ed UV/Vis spec oscopic s udies using dilu ion solu ions in e hanol/wa e (Fig. 4). The esul s e ealed wo dis inc abso p ion peaks a 365 nm and 445 nm, which co espond o he p–p* and an n–p* ansi ions, espec i ely. Fig. 3 Cha ac e iza ion o he glycogel o med by sup amolecula sel -o ganiza ion o neoglycolipid 1. La ge scale high (a) and enla ged (1) TEM images. (1). La ge-scale heigh (b) and enla ge mic og aph o xe ogel. Small angle X- ay sca e ing (c). Model o he molecula bilaye nano ube de i ed om SAXS s udy (d). Schema ic illus a ion o he molecula packing in he sel -assembled s a e (e). Chem 3D schema ic illus a ion o he size o sel -assembled amphiphile 1. Jou nal o Ma e ials Chemis y B Pape Open Access A icle. Published on 09 Oc obe 2023. Downloaded on 6/11/2024 11:45:18 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online 10194 | J. Ma e . Chem. B, 2023, 11, 10189–10205 This jou nal is © The Royal Socie y o Chemis y 2023 Upon exposu e o UV ligh , a no iceable dec ease in he p–p* ansi ion band was obse ed, accompanied by a sligh inc ease in he n–p* ansi ion, indica ing he occu ence o ans–cis pho oisome iza ion o monome 1upon UV i adia ion (Fig. 4a). A e 20 minu es o exposu e, he abso p ion bands s abilized, indica ing ha he pho os a iona y s a e had been eached. Fu he mo e, we conduc ed High-Pe o mance Liquid Ch oma og aphy (HPLC) o sepa a e he wo isome s, and he esul s showed ha 1-cis elu ed a 11.3 minu es, while 1- ans elu ed a 11.7 minu es (Fig. 4c and d). This sepa a ion allowed us o quan i y he p opo ions o bo h isome s a di e en ime poin s. The analysis e ealed ha neoglycolipid 1exhibi ed a Fig. 4 Re e sible gel–sol ansi ion and hodamine elease s udies. UV-Vis s udies o ans–cis (a) pho oisome iza ion and cis– ans eco e y unde blue ligh i adia ion (b) o 1(0,005% in E OH/H 2 O). HPLC ch oma og ams a e exposu e o 1 o UV ligh (c) and o whi e ligh om 0 o 20 minu es (d). Pho og aphs o ials showing he gel–sol ansi ion and colo change unde UV i adia ion (e). La ge-scale heigh ( ) and enla ge phase (1) TEM imageso sphe ical nano ubes in he solu ion. Rhodamine elease in he da k (g) and unde UV i adia ion (h). Pape Jou nal o Ma e ials Chemis y B Open Access A icle. Published on 09 Oc obe 2023. Downloaded on 6/11/2024 11:45:18 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online This jou nal is © The Royal Socie y o Chemis y 2023 J. Ma e . Chem. B, 2023, 11, 10189–10205 | 10195 pho os a iona y s a e wi h 88% in he cis (Z) isome and 12% in he ans (E) isome (Fig. 4c). An impo an obse a ion o p ac ical applica ions is ha his pho o-induced isome iza ion is e e sible. When he glycolipid is in he cis o m, i elaxes back o he ans o m a e exposu e o isible ligh (Fig. 4d) o g adually in he da k (see ESI† o mo e de ails). Pho oexci a ion o glycogel 1 wi h UV ligh a 365 nm causes a gel–sol ansi ion accompanied by a colo change om yellow o o ange, (Fig. 4e). Rema kably, he gel–sol ansi ion akes place as e (a e a ew minu es, see ideo in ESI†), in he case o gel o med in a 33% compa ed o ha o med in 50% aqueous e hanol mix u e (10– 12 h). The esul ing solu ion was analysed by ansmission elec on mic oscopy, which e ealed hep esencedispe sedlipid nano ubes (Fig. 4 ). In e es ingly, he diame e o he ubes (abou 39 nm) was ound o be sligh ly la ge han ha o he ube in he gel s a e. I is belie ed ha he inc ease in diame e is mainly due o he widening o he inne ca i y o he ube, as he walls emained he same size. The p ecise cause o his change is challenging o elucida e, bu i is likely a ibu ed o al e a ions in he cu a u e o he lipid laye esul ing om he isome iza ion o he glycogel-1 molecules. The analysis o cis-glycogel-1 h ough SAXS s udies (see ESI†) indica es ha he size o he ube walls, o med by a single amphiphilic bilaye , emained unchanged. Howe e , he inne diame e o he lipid nano ubes exhibi ed an inc easeandshowednon-uni o mi y. Speci ically, we obse ed h ee dis inc peaks co esponding o a e aged epea ing dis- ances o app oxima ely 9.17 nm, 17.86 nm, and 20.18 nm. These indings sugges ha he diame e inc emen p ima ily a ises om he widening o he inne ca i y o he ube, a he han he widening o he ube walls. The gel is also capable o main ain se e al cycles o o ma ion and disin eg a ion p omo ed by ligh s imuli wi h no no iceable deg ada ion (da a no shown). S udy o hodamine s o age and con olled elease om glycogel-1 Nex , in o de o in es iga e he capabili y o glycogel-1 o small molecules s o age, as well as passi e and ligh -con olled elease we measu ed he diffusion o hodamine B (a common biological dye) om gel-1 o med a 0.5% in he da k and upon UV ligh i adia ion (Fig. 4g and h). Fo his, eshly p epa ed Rho/gel-1 was co e ed wi h he same hyd oalcoholic solu ion used o i s p e- pa a ion and, a diffe en ime in e als, he en i e supe na an was emo ed, eplaced, and he abso p ions a 550 nm eco ded. As deduced om sigmoid cu e o Fig. 5d and he each o a pla eau, a o al hodamine elease om Rho/gel-1 was a ained a 50h(Fig.4g)in heda k.Incon as ,i adia iona 365nmled o he elease o 100% o he d ug wi hin 15 h, Fig. 4h, indica ing ha he diffusion o hodamine om he gel-1 in he da kness is a leas 3- old slowe han i s elease unde UV ligh i adia ion. Spa io empo al con ol o adhesion and elease o he lec in concana alin A Once demons a ed he abili y o he gel o en ap and elease molecules o in e es in a con olled manne , we ocused on he addi ional unc ionali y con e ed by spa ial o ien a ion o he mannose esidue. I is pe inen o ecall a his poin ha spec oscopic and mic oscopic s udies ( ide sup a), suppo he p oposi ion o a model acco ding o which glycogel-1 de i es om he in e connec ion o nano ubes whose walls a e o med by a single bilaye o he amphiphile. I his model is co ec , he ex e nal ace o he nano ubes will expose mannose esidues, p o iding an excellen oppo uni y o s udy he p esen a ion and o ien a ion o ca bohyd a e moie ies in hei mul i alen in e ac ions wi h speci ic ecep o s o he i s ime in he gel s a e. The in e ac ion o he plan lec in concana alin A (ConA), ob ained om jack bean (Conca alia ensi o mis), wi h mul i alen mannosyla ed ma e ials is well s udied and p o ides an excellen ool o alida e ou model. 54–56 ConA is known o selec i ely ecognizes a-manno- py anoside, a-glucopy anoside and o a lesse ex en a-N- ace ylglucosamine. 57–59 As a lec in con ol we used he Peanu agglu inin lec in (PNA) om A achis hypogaea, known o selec i ely ecognize b-galac ose bu no a-mannose epi opes. The s udies we e conduc ed by luo escence spec oscopy using Texas Red s -labelled ConA and FITC-labelled PNA. Conside ing ha PDA de i a i es can exhibi au o luo escence, we con- duc ed a lec in- ee con ol assay (Fig. 5a and b), which showed Fig. 5 Fluo escence s udy o he selec i e in e ac ion o mannose- coa ed glycogel-1 wi h lec ins. The images o he de ec ion o he g een luo escence we e acqui ed upon exci a ion a 480/40 nm wi h a band pass il e a 527/30 nm. The images o he de ec ion ed luo escence we e acqui ed a e exci a ion a 560/40 nm wi h a band il e 645/75. Images o he glycogel-1 in he ans o m alone (a) and (b), a e incuba ion wi h PNA-FITC (c), a e in e ac ion wi h ConA-Texas Red (d), a e incuba ion wi h e e sely labelled lec ins ConA-FITC (e), and wi h PNA-Alexa Fluo 594 ( ). Jou nal o Ma e ials Chemis y B Pape Open Access A icle. Published on 09 Oc obe 2023. Downloaded on 6/11/2024 11:45:18 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online 10196 | J. Ma e . Chem. B, 2023, 11, 10189–10205 This jou nal is © The Royal Socie y o Chemis y 2023 ha glycogel-1 lacks bo h g een (Fig. 5a) and ed luo escence (Fig. 5b). Howe e , an image showing a gel ha ing a ed luo escence was obse ed upon ea men o glycogel-1 wi h ConA-Texas Red s (Fig. 5d), indica ing an effec i e in e ac ion be ween lec in and mannose on he su ace o he ibe s. Con e sely, no luo escence was obse ed when glycogel-1 was ea ed wi h PNA-FITC (Fig. 5c), indica ing ha glycogel-1 is able o es ablish selec i e in e ac ions and is esis an o non-speci ic in e ac ions. As an addi ional con ol, he same assay was pe o med using e e se labelled lec ins (ConA-FITC and PNA-AlexaFluo ). In his case (Fig. 5e and ), and as expec ed, glycogel-1 exhibi ed luo es- cence only in g een. I is also ema kable ha his speci ic ecogni ion akes place in a e y sho ime (30 s), compa able o he bes sys ems epo ed in he li e a u e. 60 This efficiency can be explained by he so-called glycoside clus e effec , 61–63 consequence o mannose mul i alen exposi ion on he 1D-glyco- nano ubes in glycogel-1. In his sense we ecen ly epo ed ha mannose-coa ed single-walled ca bon nano ubes a e among he mos e icien binde o ConA. 31 Taking ad an age o he possibili y o isualizing suga - lec in in e ac ions by luo escence mic oscopy, we conduc ed a s udy on he effec o he spa io empo al a angemen o mannose on hese in e ac ions (Fig. 6) in he gel s a e. Fo his, we ca ied ou wo diffe en es s. The ini ial one consis ed o isome izing he gel om he s able ans o m o he cis o m, ollowed by incuba ion wi h lec in. A e ca ying ou successi e washes, we analysed he luo escence o he agg ega es ob ained (me hod 1, Fig. 6). No luo escence was de ec ed in his case (Fig. 6a), highligh ing he absence o ConA-mannose in e ac ion. This esul indica es o he i s ime, ha a e isome iza ion, he mannose wi hin gel-1 in he cis o m is no longe accessible and he e o e canno es ablish effec i e in e ac ions wi h ConA. This obse a ion is in line wi h p e ious esea ch conduc ed by he Jaya aman g oup, 64 he Ha mann g oup, 65 and he Lindho s g oup. 66,67 These s udies ha e high- ligh ed he c i ical ole o ca bohyd a e ligand o ien a ion in lec in ecogni ion, mainly in solu ion. The Jaya aman g oup pionee ed he de elopmen o pho oswi chable mul i alen ca bohyd a e ligands, showcasing pho oinduced a ia ions in binding affini ies. 64 Simila ly, he Ha mann g oup epo ed changes in inhibi o y concen a ions upon pho oswi ching, unde sco ing he impo ance o ligand s uc u e and o ien a ion. 65 Addi ionally, he Lindho s g oup demons a ed ligh -dependen diffe ences in bac e ial adhesion on immobilized glycosyla ed azo-benzene de i- a i es, bo h on mic o i e pla es 66 and human cell memb anes. 67 In e es ingly, in he second es , once he glycogel-1-ConA Texas Red agg ega e was ob ained (Fig. 6b), UV i adia ion (360 nm, 30 min), ollowed by successi e washes, led o he loss o he ini ial ed luo escence (me hod 2, Fig. 6c). This es indica es ha glycogel-1 is able o elease he adhe en lec in by changing om he ans o m o he cis o m. The abili y o glycogel-1 o cap u e and elease soluble lec ins highligh s i s po en ial o he de elopmen o egene able bioma e ials o igh agains oxins and pa hogen in ec ions. Sup amolecula sel -assembly o he mo e hyd ophilic monome 3 Finally, we ocused on he sup amolecula sel -o ganiza ion o monome 3, which unlike he o he wo neoglycolipids is wa e soluble. We we e especially in e es ed in knowing i his monome is capable o o ming micelles o liposomes due o hei impo ance as sma d ug deli e y sys ems. The amphiphilic neoglycolipid 3showed a c i ical micella concen a ion (CMC) in wa e o 22 mM as de e mined by DLS echnique using a Ze asize Nano ZS sys em. Fo ma ion o he nanomicella sys em ManMic-3 wasca iedou byasimpledispe sion o he neoglycolipid 3in wa e a a concen a ion abo e he CMC. The pho o-polyme i- za ion o he diace ylene unc ion upon ul a iole i adia ion (254 nm) affo ded a pa ially conjuga ed polydiace ylene back- bone o al e na ing enyne g oups (Fig.7a).Thecha ac e is ic size and mo phology o he o med nanosys ems we e de e mined by DLS and TEM. The TEM mic og aph shows he o ma ion o se e al en i ies, being he sphe ical micelles ManMic-3 wi h sizes o 15 nm he mos abundan (Fig. 7b). TEM analysis also shows he p esence o liposome wi h app oxima ely 150 nm in size (Fig. 7c). DLS analysis con i ms he p esence o esicle ha ing an a e age diame e o 184 nm, uni o m in shape and mono- dispe se as indica ed by he PDI o 0.05(Fig.7d).Howe e , he micelles de ec ed by TEM, could no be de ec ed by DLS, since he signal is domina ed by he powe ul sca e ing o he liposome hus hiding he micelles p esen . Indeed, being micelles mo e han one o de o magni ude smalle , sca e mo e han h ee o de s o magni ude less o simila olume phase. As o he gel, we s udied he pho opolyme iza ion o he s a ing DA monome s in he micelle by Raman spec oscopy (Fig. 7g). In his case he Raman spec um shows he p esence o he peak a 2268 cm 1 co esponding o he iple bond in he monome oge he wi h he peaks a 2084 cm 1 and 1454 cm 1 co esponding o he conjuga ed ene-yne sys em, indica ing an incomple e pho opolyme iza ion o he DA monome s in he micelles (Fig. 7g). The ob ained micelles ManMic-3 p esen a shel e ed hyd ophobic inne ca i y o med by he hyd oca bon ails, a hyd ophilic PEG chain su ounded by a diazobenzene and mannose moie ies. In e es ingly, bo h he in e nal hyd ophobic a ea and he diazobenzene co ona o hese micelles can, hos hyd ophobic gues molecules such as cy o oxic o image enhan- cing agen s. 30 As a p elimina y es o show he encapsula ion Fig. 6 Fluo escence s udy o he effec o he spa io empo al a ange- men o mannose on gel-1-lec in in e ac ions. Me hod 1: Image o he cis gel-1 a e incuba ion wi h ConA-Texas Red (a). Me hod 2: Image o he ans gel-1 a e incuba ion wi h ConA-Texas Red (b), image o he ans gel-1-ConA Texas Red a e pho oisome iza ion (c) Pape Jou nal o Ma e ials Chemis y B Open Access A icle. Published on 09 Oc obe 2023. Downloaded on 6/11/2024 11:45:18 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online This jou nal is © The Royal Socie y o Chemis y 2023 J. Ma e . Chem. B, 2023, 11, 10189–10205 | 10197 pe o mance o ManMic-3 micelles, Nile Red was chosen as a model o hyd ophobic d ug and dye. Nile Red is insoluble in wa e (solubili y o1mgmL 1 ), Fig. 7e(i), he luo escence o which inc eases subs an ially in hyd ophobic en i onmen s. The micelles o mula ed wi h Nile Red we e p epa ed simply mixing he p e iously o med ManMic3 micelles wi h Nile Red (Fig. 7e). A e s i ing and hea ing a 50 1C o 24 hou s, il a ion o elimina ion o he non-encapsula ed dye, a pink solu ion wi hou isible p ecipi a ion was ob ained (Fig. 7e(iii)). In compa ison, applying he same p ocedu e in wa e wi hou micelles esul ed in a clea colo less solu ion (Fig. 7e(i )), which also did no show he cha ac e is ic band abso bance o Nile Red (Fig. 7 ). Subsequen ly, we in es iga ed he po en ial o MicMan3 as a nanoca ie o hyd ophobic d ugs, wi h he aim o imp o ing hei wa e solubili y and p o ec ing hem wi hin he hyd ophobic co e. Doce axel (DTX), a d ug widely used in clinical p ac ice bu wi h e y low aqueous solubili y (solubili y 0.3 mgmL 1 a 37 1C), p esen ed a signi ican challenge. In esponse o his challenge, we endea o ed o c ea e inclusion complexes using a sol en - ee me hod in ol ing s i ing and hea ing. This app oach elimina ed he need o o ganic sol en s. The quan i y o inco po a ed DTX was de e mined by measu ing he diffe ence in mass be ween he loaded and emp y micelles, complemen ed by HPLC analysis. The d ug loading con en (DLC) and encapsula ion efficiency (DLE) we e calcula ed using eqn (1) and (2) as ollows: DLC %ðÞ¼ Weigh o loaded DTX W o MicMan3 þW o loaded DTX 100% (1) DLE %ðÞ¼ Weigh o loaded DTX Weigh o inpu DTX 100%(2) The d ug loading con en and encapsula ion efficiency o MicMan3 o DTX we e 2.8% and 11.2%. These p elimina y indings unde sco e he capabili y o MicMan3 o enhance he wa e solubili y o highly insoluble o ganic molecules, which is o u mos signi icance o hei po en ial u ili y as effec i e d ug deli e y sys ems. Conclusions In conclusion, we ha e de eloped a e sa ile design o he syn hesis o mul i unc ional diazobenzene/diace ylene glycoam- phiphiles. By adjus ing he space leng h and he p esence o a iazole ing, we can con ol he hie a chical sel -o ganiza ion o hese molecules. Neoglycolipid 3 o ms sphe ical micelles (MicMan-3) wi h a shel e ed inne co e, capable o solubilizing hyd ophobic molecules like Nile Red and doce axel. On he o he hand, monome 1 o ms a hyd o-alcoholic gel wi hou he need o addi ional p ocessing, consis ingo en angled Fig. 7 Syn hesis and use as nanocon aine o s a ic mannose-coa ed nanomicelles ManMic-3 h ough sonica ion-p omo ed sup amolecula sel - assembly ollowed by in e molecula pho o-polyme iza ion o neoglycolipid 3in wa e (a). TEM images (b) and (c) o he o med micelles and liposomes. CMC and DLS size de e mina ion o MicMan-3 (d). Nile Red encapsula ion s udy (e). Abso p ion spec a o Nile Red in wa e (black line) and in ManMic-3 solu ion (pu ple line) ( ). Raman spec um o he polyme ized ManMic-3 (g). Jou nal o Ma e ials Chemis y B Pape Open Access A icle. Published on 09 Oc obe 2023. Downloaded on 6/11/2024 11:45:18 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online 10204 | J. Ma e . Chem. B, 2023, 11, 10189–10205 This jou nal is © The Royal Socie y o Chemis y 2023 26 J. Y. C. Lim, Q. Lin, K. Xue and X. J. 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