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

This journal is © The Royal Society of Chemistry 2023 J. Mater. Chem. B, 2023, 11, 10189–10205 | 10189 Cite this: J. Mater. Chem. B, 2023, 11, 10189 Divergent approach to nanoscale glycomicelles and photo-responsive supramolecular glycogels. Implications for drug delivery and photoswitching lectin affinity† Elena Romero-Ben, ‡ a M Carmen Castillejos, ‡ a Cristian Rosales-Barrios, a Marı ´a Expo ´sito, a Pilar Ruda, a Paula M. Castillo, a Stefania Nardecchia, b Juan de Vicente b and Noureddine Khiar * a The field of stimuli-responsive supramolecular biomaterials has rapidly advanced in recent years, with potential applications in diverse areas such as cancer theranostics, tissue engineering, and catalysis. However, designing molecular materials that exhibit predetermined hierarchical self-assembly to control the size, morphology, surface chemistry, and responsiveness of the final nanostructures remains a significant challenge. In this study, we present a divergent synthetic approach for the fabrication of spherical micelles and functional 1D-glyconanotube-based photoresponsive gels from structurally related diazobenzene/diacetylene glycolipids. The resulting nanostructures were characterized using NMR, TEM, and SEM, confirming the formation of spherical and tubular nanostructures in both the gel and solution states. Upon UV irradiation, a reversible gel–sol transition was observed, resulting from the photoswitching of the azobenzene unit from the stretched trans form to the compact, metastable cis form. Our gels were shown to enable spatio-temporal control of the adhesion and release of the lectin Concanavalin A, demonstrating potential use as regenerable biomaterials to fight against infections with toxins and pathogens. Additionally, our micelles and gels were evaluated as nanocontainers for loading and controlled release of hydrophobic dyes and antitumoural agents, suggesting their possible use as smart theranostic drug delivery systems. Introduction The development of complex nanoscale systems able to receive and execute commands from external stimuli has received increased attention in recent years due to their potential use in drug delivery, tissue engineering,biomineralization,molecular electronics, and catalysis. 1–7 Of relevance for the design of these systems as cancer theranostics, structure–activity relationship studies have shown that topology and size are key factors for their cellular uptake, circulation time and interaction with specific receptors. 8–12 Indeed, while spherical nanomaterials are well suited to processes involving rapid cellular uptake, 13 1D-rod structuresaremoreappropriateforthoserequiringalonger circulation time. 14 On the other hand, it has been shown that spherical micelles are potent inhibitors of globular receptors, while 1D-nanofibers as well as 3D-gels are more suitable for adhesion and inhibition of bacterial motility. 15–18 Therefore, developing divergent and cost-effective synthetic approaches allowing the synthesis of smart functionalized organic materials and modulating their sizes, topology and functionality from welldesigned molecular monomers is highly desirable, although it remains an outstanding challenge. 19 One family of molecules suitable for the creation of supramolecular diversity are the amphiphiles, whose self-organization can lead to the formation of species with a wide range of sizes and whose functionality can range from ordinary soap to extracellular matrix mimics., 20,21 A paradigmatic example are supramolecular gels, 22,23 obtained from low molecular weight organogelators (LMOGS), 24–26 whose hierarchical self-organization lead to flexible 1D-nanofibers with a high aspect ratio having sizes ranging from submicron to hundreds of micrometers which by entanglement, form the macroscopic supramolecular gels of millimeter size. 27,28 This hierarchy provides an excellent opportunity to design intelligent supramolecular a Asymmetric Synthesis and Functional Nanosystems Group, Instituto de Investigaciones Quı ´micas (IIQ), CSIC-Universidad de Sevilla, Avda. Ame ´rico Vespucio 49, 41092, Seville, Spain. E-mail: [email protected] b Department of Applied Physics and Excellence Research Unit ‘Modeling Nature’ (MNat), Faculty of Sciences, University of Granada, C/Fuentenueva s/n, 18071 – Granada, Spain †Electronic supplementary information (ESI) available. See DOI: https://doi.org/ 10.1039/d3tb01713c ‡These authors contributed equally. Received 28th July 2023, Accepted 7th October 2023 DOI: 10.1039/d3tb01713c rsc.li/materials-b Journal of Materials Chemistry B PAPER Open Access Article. Published on 09 October 2023. Downloaded on 6/11/2024 11:45:18 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue 10190 | J. Mater. Chem. B, 2023, 11, 10189–10205 This journal is © The Royal Society of Chemistry 2023 systems with functional responses by incorporating motifs responsive to external stimuli into the molecular structures of amphiphiles. 29–31 Among the different stimuli, using light offers the advantage of being easily turned on and off, employing adjustable wavelength and intensity, and offering high spatial and temporal control. 29,30,32–36 Moreover, an interesting class of amphiphiles are glycolipids whose hierarchical supramolecular organization leads to complex systems endowed with important biological activities due to the involvement of carbohydrates in vital processes such as pathogen adhesion, transplant rejection, fertilization, and cell proliferation/differentiation. 29,37–40 Based on these premises and within our interest in the development of functional soft glyconanomaterials, 41–44 we report, herein, a new modular synthetic approach of self-associative multifunctional neoglycolipids 1–3 for the divergent nanofabrication of functional spherical micelles, 1D-lipid glyconanotubes, and photoresponsive 3D-gels, Fig. 1. Results and discussion Synthetic design In the design of the self-associative monomers, diazobenzene, linked directly to the biorecognition element mannose, was used as a light-responsive group. 29,34 The design also includes a variable oligoethylene glycol to fine-tune the hydrophobic– hydrophilic balance of the neoglycolipid. Furthermore, as hydrophobic tail a 25 carbon chain bearing a photo-polymerizable diacetylenic function, known for its ability to form nanomaterials with interesting chromatic properties was used. 44–49 The design was validated by synthesizing functional spherical micelles (Fig.1a),and1D-lipidglyconanotubes(Fig.1b),thatentangleto form light responsive 3D-gels (Fig. 1c). Synthesis of the monomers The synthesis of the three compounds (1,2and 3), use tetra-O- acetyl-a-D-mannopyranosyl trichloroacetimidate 4, obtained in two steps from mannose pentaacetate, as starting material, Scheme 1. Monoglycosylation of 4,40-dihydroxyazobenzene 5 acceptor, 50 using donor 4and boron trifluoride as an activator gives the desires compound 6with 86% yield. For the modulation of the hydrophilic–hydrophobic balance, as well as for unravelling the effect of the connecting group between the polar head group and the apolar tail, bifunctional spacers 7–9, derived from tetraethyleneglycol (7,8) or eicosaetyleneglycol (9), were used, Scheme 1. The condensation of alcohol 6with tosylated spacer 7in the presence of K 2 CO 3 , LiCl in acetonitrile gives the corresponding derivative 10 in 60% yield, Scheme 1a. Next, the deprotection of the NHBoc derivative 10 using trifluoroacetic acid in methylene chloride gives the free amine 11. Amidation with pentacosadiinoic acid (PCDA) in the presence of TBTU and DIPEA in DMF gives the corresponding amide 12 with 60% yield and, finally, Zemplen deacetylation gives the desired neoglycolipid 1, Scheme 1a. For the synthesis of the neoglycolipids 2and 3, Scheme 1b, condensation of potassium alcoholate of 6with the tosylated azido spacers derived from tetraethyleneglycol 8or Fig. 1 Divergent synthesis of mannose-coated micelles (a) and photoresponsive mannose-coated gel (b) through supramolecular self-organization of sugar-coated diazobenzene/diacetylene amphiphiles. Amhiphile 3with a large PEG 20 chain self-organize into spherical micelle able to host and solubilize the hydrophobic dye Nile red (a), while amphiphile 1with a short tetraethyleneglycol chain self-organize into 1D-nanotubes (b) which evolve to tridimensional gels, able to establish selective interactions with mannose-specific lectin Texas red s concanavalin A (d), and to perform a reversible gel– sol transition in response to light irradiation (c and e). Paper Journal of Materials Chemistry B Open Access Article. Published on 09 October 2023. Downloaded on 6/11/2024 11:45:18 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online This journal is © The Royal Society of Chemistry 2023 J. Mater. Chem. B, 2023, 11, 10189–10205 | 10191 eicosaethyleneglycol spacer 9, in acetonitrile gives the ether derivatives 13 and 14 in 67% and 70% yields respectively. Cu(I)-catalyzed azide alkyne 1,3-dipolar cycloadditions (CuAAC) between azides 13 or 14 and the N-(2-propynyl)pentacosa-10,12- diynamide alkyne 15, itself obtained in one step from PCDA and propargylamine, in the presence of sodium ascorbate and in a methylene chloride/water mixture, afforded regioselectively the 1,2,3-triazole derivatives 16 and 17 with 60 and 80% yield respectively. Finally, Zemplen deacetylation gives the desired compounds 2and 3with 97 and 99% chemical yield. The structures of the 3 monomers were confirmed by monodimensional and bidimensional NMR as well as by mass spectroscopy (see experimental part and ESI†). Photoisomerization study of the monomers Azobenzene compounds are well-known for their reversible photoisomerization between the trans and cis forms under alternating UV and visible light irradiation. 51 The light-responsive behavior of three neoglycolipids 1–3,Fig.1,wasstudiedby 1 HNMR(Fig.2b and c), UV-Vis (Fig. 2d and e), and HPLC-MS spectroscopies (vide infra). The most significant protons for studying the isomerization kinetics of neoglycolipids 1–3 by 1 H NMR are the diazobenzene protons,theproton5ofthetriazoleandtheanomericprotonof sugar. In the stable trans (E) isomers the signals of protons 3, 5 and 30,5 0ofthediazobenzenemoietyappearastwodoubletsat 6.90 ppm and 7.10 ppm respectively, while protons 2.6 and 20.60 appear together at a lower field (about 7.75 ppm). The triazole proton is observed as a singlet at 7.90 ppm, and the anomeric proton appears either as a broad singlet (in the case of 1-trans and 2-trans) or as a doublet with a small coupling constant (1.34 Hz) in the case of 3-trans, at approximately 5.60 ppm. In the cis (Z) isomers [1-cis,2-cis (see ESI†), and 3-cis], the signals of the 3, 5 and 30,5 0protons undergo a slight shift at 7.00 and 6.90 ppm, while the 2, 6 and 20,6 0protons alpha to the diazo function undergo a pronounced shielding effect, from 8.00 ppm to 6.80 ppm. Although the anomeric proton undergoes a slight chemical shift change (from 5.60 ppm to 5.50 ppm), its location in a position where there are no other signals allows it to be used as an additional control to monitor and quantify the trans–cis isomers. To gain further structural insights, we conducted selective 1D-NOESY and 1H/1H EASY ROESY 2D experiments (see ESI†). In the case of the 2-trans derivative, our NOESY 1D experiments facilitated more precise signal assignments for both aromatic rings (see Fig. S53–S56 in the ESI†). Additionally, 2D 1H/1H EASYROESY experiments, carried out on a predominantly 2-cis configuration, revealed contact NOEs between the H3 and H5 protons of the aromatic ring linked to the sugar and a methylene protons of thePEGchain(seeFig.S60intheESI†). Our study allowed us to quantify the photostationary (Z:E) state of the three neoglycolipids in DMSO. Neoglycolipid 1 exhibited a photostationary state of 75% cis (Z) and 25% trans (E) isomers (Fig. 2b), while neoglycolipid 2displayed a photostationary state of 82% cis (Z) and 18% trans (E) isomers (see ESI†). Remarkably, and contrary to most reported diazobenzene glycolipids, neoglycolipid 3showed a unique behavior as its photoswitching was independent of the solvent and complete even in methanol, with a photostationary state of 100% cis (Z) isomer (Fig. 2c). The complete isomeric conversion of neoglycolipid 3is of great interest in studying the influence of carbohydrate orientation on lectin–carbohydrate interactions. The slow thermal return from pure 3-cis to pure 3-trans isomer, occurring over a period of 96 hours in the dark, further adds to the significance of neoglycolipid 3. Moreover, under blue light irradiation (460 nm), the return from the 3-cis to 3-trans isomer in neoglycolipid 3was significantly accelerated, taking only 2 hours (Fig. 2c). UV/Vis spectroscopic studies were conducted using DMSO as the solvent and under gelation conditions (see details below). Glycolipids exhibit a broad absorption peak around 320–350 nm, primarily attributed to the trans form of the azobenzene group (Fig. 2d and e). Upon irradiation, the photo-induced trans–cis isomerization was observed to be reversible, as the cis form of the azo-surfactants relaxed back to the trans form after exposure to visible light or slowly in the absence of light (Fig. 2e). Furthermore, a series of successive ‘‘on–off’’ switching experiments on the absorption bands of glycolipid 1was carried out. The results of these experiments demonstrated that the process was reversible for at least nine cycles, indicating that sample 1 exhibits good stability (Fig. 2f). Supramolecular self-assembly of amphiphile 1 and characterization of the photoresponsive-glycogel In order to determine the supramolecular self-association capacities of the monomers, we determined first their water and organic solvents solubilities. Although monomers 1and 2are insolubleinmostsolvents,themorehydrophilicmonomer3is Scheme 1 Synthesis of self-associatives neoglycolipid 1(a), 2and 3(b). (i) BF 3 Et 2 O, CH 3 CN, 0 1C; (ii) K 2 CO 3 , LiCl, CH 3 CN; (iii) TFA, CH 2 Cl 2 ; (iv) TBTU, DIPEA, DMF; (v) MeONa, MeOH; (vi) CuSO 4 , AsCNa, CH 2 Cl 2 /H 2 O. Journal of Materials Chemistry B Paper Open Access Article. Published on 09 October 2023. Downloaded on 6/11/2024 11:45:18 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 10192 | J. Mater. Chem. B, 2023, 11, 10189–10205 This journal is © The Royal Society of Chemistry 2023 soluble in various solvents including water (vide infra). The use of mixture of solvents reveals water–ethanol as the best mixture. Indeed, in 1 : 1 or 1 : 2 water–ethanol mixture, monomer 1showed a great ability to form gels. In the same conditions monomer 2 was insoluble while monomer 3gave a clear yellowish solution. Interestingly, while in the 1 : 2 water ethanol mixture, the gelation of 1takes place at a concentration of 1% (w/v), in 50% aqueous ethanol solution it takes place at a concentration as low as 0.1%. Additionally, in the latter case the compound gelificate instantaneously, Fig. 3, at room temperature with no need of heating–cooling or sonication. The structure of the as-formed gel as well as the xerogel formed from monomer 1at 1% was characterized by transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM) (Fig. 3). Interlocked agglomerated tubules with a uniform diameter of 31 nm, a thickness of 9 nm, and micron-scale lengths were regularly found in the glycogel-1 under TEM analysis (Fig. 3a). The observation of xerogel-1 under SEM at the tens of micrometer scale showed interesting intertwined and twisted structures in a bush-form (see Fig. S32, ESI†). Moreover, FESEM analysis allowed observing the detailed fibrillar aggregates forming the bundles shown in the standard SEM images (Fig. 3b). Small-angle X-ray scattering (SAXS) studies allowed us to advance in the exact structural determination of the fibrillar glycogel-1 (Fig. 3c). Maximum peaks corresponding to averaged repeating distances of about 9.5 nm were obtained (Fig. 3c). It should be noted that the starting monomer is approximately 5 nm in length when extended, as determined by Chem3D analysis (Fig. 3f). This suggests that a single bilayer would extend for a maximum of 10 nm, indicating that the tube walls are likely formed by a single amphiphilic bilayer with some interpenetration of the lipid tail (as shown in Fig. 3d–f). A second peak in the SAXS spectrum at 2y=0.51, corresponding to a separation of 16.6 nm, can be assigned to the inner diameter of the lipid nanotube, which is likely influenced by the packing of the amphiphilic bilayer. Under such supramolecular self-organization, the tube in the gel would expose the mannose residue usually used for the adhesion of pathogens to the external phase, whose specific receptor is involved in several biological processes of interest. 51,52 On the other hand, the internal cavity of the tube, larger than 10 nm, could accommodate molecules of interest such as cytotoxic compounds, dyes or couldalsoserveasaconfinedmedium for organic reactions. 53 The mechanical properties of gel-1 were characterized through rheological strain sweep and frequency sweep Fig. 2 Schematic representation of the photoisomerization of neoglycolipids 1–3 (a). 1 HNMR studies of trans–cis and cis–trans photo and thermal isomerization of the neoglycolipid 1in DMSOd 6 (b), and 3in MeOD (c). UV-Vis studies of trans–cis (d) photoisomerization and cis–trans recovery under blue light irradiation (e) of neoglycolipid 1(0,005 wt/v% in DMSO). Cycle of absorption after alternate irradiation with UV (360 nm, green bar) and visible light (460 nm, yellow bar) (f). Paper Journal of Materials Chemistry B Open Access Article. Published on 09 October 2023. Downloaded on 6/11/2024 11:45:18 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online This journal is © The Royal Society of Chemistry 2023 J. Mater. Chem. B, 2023, 11, 10189–10205 | 10193 experiments, revealing its high low-shear viscosity and shearthinning behavior. The gel exhibited a hysteresis loop in the flow curve, indicating thixotropic behavior. Dynamic oscillatory shear tests showed that the gel behaved as a solid-like material with a linear viscoelastic range, followed by non-linear behavior and yielding at higher strain amplitudes. The full study with detailed results can be found in the ESI.† Diacetylenic amphiphiles (DA) possess photoresponsive properties as they can undergo polymerization through a 1,4- addition reaction of diradicals generated by UV or girradiation. This process leads to the formation of smart polydiacetylene (PDA)-nanomaterials, exhibiting intriguing chromatic properties. Prior to investigating the functionality of gel 1, we examined the ability of the self-associated DA monomer 1to photopolymerize in the gel state using Raman spectroscopy. The results of these studies revealed that photopolymerizationoccursonlyinthedrystateand not in the gel state (for detailed information, see the ESI†). Studies on the UV-induced reversible gel–sol transition To investigate the light-responsive behavior of glycogel 1, we, first, conducted UV/Vis spectroscopic studies using dilution solutions in ethanol/water (Fig. 4). The results revealed two distinct absorption peaks at 365 nm and 445 nm, which correspond to the p–p* and an n–p* transitions, respectively. Fig. 3 Characterization of the glycogel formed by supramolecular self-organization of neoglycolipid 1. Large scale high (a) and enlarged (1) TEM images. (1). Large-scale height (b) and enlarge micrograph of xerogel. Small angle X-ray scattering (c). Model of the molecular bilayer nanotube derived from SAXS study (d). Schematic illustration of the molecular packing in the self-assembled state (e). Chem 3D schematic illustration of the size of self-assembled amphiphile 1. Journal of Materials Chemistry B Paper Open Access Article. Published on 09 October 2023. Downloaded on 6/11/2024 11:45:18 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 10194 | J. Mater. Chem. B, 2023, 11, 10189–10205 This journal is © The Royal Society of Chemistry 2023 Upon exposure to UV light, a noticeable decrease in the p–p* transition band was observed, accompanied by a slight increase in the n–p* transition, indicating the occurrence of trans–cis photoisomerization of monomer 1upon UV irradiation (Fig. 4a). After 20 minutes of exposure, the absorption bands stabilized, indicating that the photostationary state had been reached. Furthermore, we conducted High-Performance Liquid Chromatography (HPLC) to separate the two isomers, and the results showed that 1-cis eluted at 11.3 minutes, while 1-trans eluted at 11.7 minutes (Fig. 4c and d). This separation allowed us to quantify the proportions of both isomers at different time points. The analysis revealed that neoglycolipid 1exhibited a Fig. 4 Reversible gel–sol transition and rhodamine release studies. UV-Vis studies of trans–cis (a) photoisomerization and cis–trans recovery under blue light irradiation (b) of 1(0,005% in EtOH/H 2 O). HPLC chromatograms after exposure of 1to UV light (c) and to white light from 0 to 20 minutes (d). Photographs of vials showing the gel–sol transition and color change under UV irradiation (e). Large-scale height (f) and enlarge phase (1) TEM imagesof spherical nanotubes in the solution. Rhodamine release in the dark (g) and under UV irradiation (h). Paper Journal of Materials Chemistry B Open Access Article. Published on 09 October 2023. Downloaded on 6/11/2024 11:45:18 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online This journal is © The Royal Society of Chemistry 2023 J. Mater. Chem. B, 2023, 11, 10189–10205 | 10195 photostationary state with 88% in the cis (Z) isomer and 12% in the trans (E) isomer (Fig. 4c). An important observation for practical applications is that this photo-induced isomerization is reversible. When the glycolipid is in the cis form, it relaxes back to the trans form after exposure to visible light (Fig. 4d) or gradually in the dark (see ESI†for more details). Photoexcitation of glycogel 1 with UV light at 365 nm causes a gel–sol transition accompanied by a color change from yellow to orange, (Fig. 4e). Remarkably, the gel–sol transition takes place faster (after a few minutes, see video in ESI†), in the case of gel formed in a 33% compared to that formed in 50% aqueous ethanol mixture (10– 12 h). The resulting solution was analysed by transmission electron microscopy, which revealedthepresencedispersedlipid nanotubes (Fig. 4f). Interestingly, the diameter of the tubes (about 39 nm) was found to be slightly larger than that of the tube in the gel state. It is believed that the increase in diameter is mainly due to the widening of the inner cavity of the tube, as the walls remained the same size. The precise cause of this change is challenging to elucidate, but it is likely attributed to alterations in the curvature of the lipid layer resulting from the isomerization of the glycogel-1 molecules. The analysis of cis-glycogel-1 through SAXS studies (see ESI†) indicates that the size of the tube walls, formed by a single amphiphilic bilayer, remained unchanged. However, the inner diameter of the lipid nanotubes exhibited an increaseandshowednon-uniformity. Specifically, we observed three distinct peaks corresponding to averaged repeating distances of approximately 9.17 nm, 17.86 nm, and 20.18 nm. These findings suggest that the diameter increment primarily arises from the widening of the inner cavity of the tube, rather than the widening of the tube walls. The gel is also capable to maintain several cycles of formation and disintegration promoted by light stimuli with no noticeable degradation (data not shown). Study of rhodamine storage and controlled release from glycogel-1 Next, in order to investigate the capability of glycogel-1 for small molecules storage, as well as passive and light-controlled release we measured the diffusion of rhodamine B (a common biological dye) from gel-1 formed at 0.5% in the dark and upon UV light irradiation (Fig. 4g and h). For this, freshly prepared Rho/gel-1 was covered with the same hydroalcoholic solution used for its preparation and, at different time intervals, the entire supernatant was removed, replaced, and the absorptions at 550 nm recorded. As deduced from sigmoid curve of Fig. 5d and the reach of a plateau, a total rhodamine release from Rho/gel-1 was attained at 50h(Fig.4g)inthedark.Incontrast,irradiationat365nmledto the release of 100% of the drug within 15 h, Fig. 4h, indicating that the diffusion of rhodamine from the gel-1 in the darkness is at least 3-fold slower than its release under UV light irradiation. Spatiotemporal control of adhesion and release of the lectin concanavalin A Once demonstrated the ability of the gel to entrap and release molecules of interest in a controlled manner, we focused on the additional functionality conferred by spatial orientation of the mannose residue. It is pertinent to recall at this point that spectroscopic and microscopic studies (vide supra), support the proposition of a model according to which glycogel-1 derives from the interconnection of nanotubes whose walls are formed by a single bilayer of the amphiphile. If this model is correct, the external face of the nanotubes will expose mannose residues, providing an excellent opportunity to study the presentation and orientation of carbohydrate moieties in their multivalent interactions with specific receptors for the first time in the gel state. The interaction of the plant lectin concanavalin A (ConA), obtained from jack bean (Concavalia ensiformis), with multivalent mannosylated materials is well studied and provides an excellent tool to validate our model. 54–56 ConA is known to selectively recognizes a-manno- pyranoside, a-glucopyranoside and to a lesser extent a-N- acetylglucosamine. 57–59 As a lectin control we used the Peanut agglutinin lectin (PNA) from Arachis hypogaea, known to selectively recognize b-galactose but not a-mannose epitopes. The studies were conducted by fluorescence spectroscopy using Texas Red s -labelled ConA and FITC-labelled PNA. Considering that PDA derivatives can exhibit autofluorescence, we conducted a lectin-free control assay (Fig. 5a and b), which showed Fig. 5 Fluorescence study of the selective interaction of mannosecoated glycogel-1 with lectins. The images for the detection of the green fluorescence were acquired upon excitation at 480/40 nm with a band pass filter at 527/30 nm. The images for the detection red fluorescence were acquired after excitation at 560/40 nm with a band filter 645/75. Images of the glycogel-1 in the trans form alone (a) and (b), after incubation with PNA-FITC (c), after interaction with ConA-Texas Red (d), after incubation with reversely labelled lectins ConA-FITC (e), and with PNA-Alexa Fluort594 (f). Journal of Materials Chemistry B Paper Open Access Article. Published on 09 October 2023. Downloaded on 6/11/2024 11:45:18 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 10196 | J. Mater. Chem. B, 2023, 11, 10189–10205 This journal is © The Royal Society of Chemistry 2023 that glycogel-1 lacks both green (Fig. 5a) and red fluorescence (Fig. 5b). However, an image showing a gel having a red fluorescence was observed upon treatment of glycogel-1 with ConA-Texas Red s (Fig. 5d), indicating an effective interaction between lectin and mannose on the surface of the fibers. Conversely, no fluorescence was observed when glycogel-1 was treated with PNA-FITC (Fig. 5c), indicating that glycogel-1 is able to establish selective interactions and is resistant to non-specific interactions. As an additional control, the same assay was performed using reverse labelled lectins (ConA-FITC and PNA-AlexaFluort). In this case (Fig. 5e and f), and as expected, glycogel-1 exhibited fluorescence only in green. It is also remarkable that this specific recognition takes place in a very short time (30 s), comparable to the best systems reported in the literature. 60 This efficiency can be explained by the so-called glycoside cluster effect, 61–63 consequence of mannose multivalent exposition on the 1D-glyco- nanotubes in glycogel-1. In this sense we recently reported that mannose-coated single-walled carbon nanotubes are among the most efficient binder to ConA. 31 Taking advantage of the possibility of visualizing sugarlectin interactions by fluorescence microscopy, we conducted a study on the effect of the spatiotemporal arrangement of mannose on these interactions (Fig. 6) in the gel state. For this, we carried out two different tests. The initial one consisted of isomerizing the gel from the stable trans form to the cis form, followed by incubation with lectin. After carrying out successive washes, we analysed the fluorescence of the aggregates obtained (method 1, Fig. 6). No fluorescence was detected in this case (Fig. 6a), highlighting the absence of ConA-mannose interaction. This result indicates for the first time, that after isomerization, the mannose within gel-1 in the cis form is no longer accessible and therefore cannot establish effective interactions with ConA. This observation is in line with previous research conducted by the Jayaraman group, 64 the Hartmann group, 65 and the Lindhorst group. 66,67 These studies have highlighted the critical role of carbohydrate ligand orientation in lectin recognition, mainly in solution. The Jayaraman group pioneered the development of photoswitchable multivalent carbohydrate ligands, showcasing photoinduced variations in binding affinities. 64 Similarly, the Hartmann group reported changes in inhibitory concentrations upon photoswitching, underscoring the importance of ligand structure and orientation. 65 Additionally, the Lindhorst group demonstrated light-dependent differences in bacterial adhesion on immobilized glycosylated azo-benzene derivatives, both on microtiter plates 66 and human cell membranes. 67 Interestingly, in the second test, once the glycogel-1-ConA Texas Red aggregate was obtained (Fig. 6b), UV irradiation (360 nm, 30 min), followed by successive washes, led to the loss of the initial red fluorescence (method 2, Fig. 6c). This test indicates that glycogel-1 is able to release the adherent lectin by changing from the trans form to the cis form. The ability of glycogel-1 to capture and release soluble lectins highlights its potential for the development of regenerable biomaterials to fight against toxins and pathogen infections. Supramolecular self-assembly of the more hydrophilic monomer 3 Finally, we focused on the supramolecular self-organization of monomer 3, which unlike the other two neoglycolipids is water soluble. We were especially interested in knowing if this monomer is capable of forming micelles or liposomes due to their importance as smart drug delivery systems. The amphiphilic neoglycolipid 3showed a critical micellar concentration (CMC) in water of 22 mM as determined by DLS technique using a Zetasizer Nano ZS system. Formation of the nanomicellar system ManMic-3 wascarriedoutbyasimpledispersion of the neoglycolipid 3in water at a concentration above the CMC. The photo-polymeri- zation of the diacetylene function upon ultraviolet irradiation (254 nm) afforded a partially conjugated polydiacetylene backbone of alternating enyne groups (Fig.7a).Thecharacteristic size and morphology of the formed nanosystems were determined by DLS and TEM. The TEM micrograph shows the formation of several entities, being the spherical micelles ManMic-3 with sizes of 15 nm the most abundant (Fig. 7b). TEM analysis also shows the presence of liposome with approximately 150 nm in size (Fig. 7c). DLS analysis confirms the presence of vesicle having an average diameter of 184 nm, uniform in shape and monodisperse as indicated by the PDI of0.05(Fig.7d).However,the micelles detected by TEM, could not be detected by DLS, since the signal is dominated by the powerful scattering of the liposome thus hiding the micelles present. Indeed, being micelles more than one order of magnitude smaller, scatter more than three orders of magnitude less for similar volume phase. As for the gel, we studied the photopolymerization of the starting DA monomers in the micelle by Raman spectroscopy (Fig. 7g). In this case the Raman spectrum shows the presence of the peak at 2268 cm 1 corresponding to the triple bond in the monomer together with the peaks at 2084 cm 1 and 1454 cm 1 corresponding to the conjugated ene-yne system, indicating an incomplete photopolymerization of the DA monomers in the micelles (Fig. 7g). The obtained micelles ManMic-3 present a sheltered hydrophobic inner cavity formed by the hydrocarbon tails, a hydrophilic PEG chain surrounded by a diazobenzene and mannose moieties. Interestingly, both the internal hydrophobic area and the diazobenzene corona of these micelles can, host hydrophobic guest molecules such as cytotoxic or image enhancing agents. 30 As a preliminary test to show the encapsulation Fig. 6 Fluorescence study of the effect of the spatiotemporal arrangement of mannose on gel-1-lectin interactions. Method 1: Image of the cis gel-1 after incubation with ConA-Texas Red (a). Method 2: Image of the trans gel-1 after incubation with ConA-Texas Red (b), image of the trans gel-1-ConA Texas Red after photoisomerization (c) Paper Journal of Materials Chemistry B Open Access Article. Published on 09 October 2023. Downloaded on 6/11/2024 11:45:18 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online This journal is © The Royal Society of Chemistry 2023 J. Mater. Chem. B, 2023, 11, 10189–10205 | 10197 performance of ManMic-3 micelles, Nile Red was chosen as a model of hydrophobic drug and dye. Nile Red is insoluble in water (solubility o1mgmL 1 ), Fig. 7e(i), the fluorescence of which increases substantially in hydrophobic environments. The micelles formulated with Nile Red were prepared simply mixing the previously formed ManMic3 micelles with Nile Red (Fig. 7e). After stirring and heating at 50 1C for 24 hours, filtration for elimination of the non-encapsulated dye, a pink solution without visible precipitation was obtained (Fig. 7e(iii)). In comparison, applying the same procedure in water without micelles resulted in a clear colorless solution (Fig. 7e(iv)), which also did not show the characteristic band absorbance for Nile Red (Fig. 7f). Subsequently, we investigated the potential of MicMan3 as a nanocarrier for hydrophobic drugs, with the aim of improving their water solubility and protecting them within the hydrophobic core. Docetaxel (DTX), a drug widely used in clinical practice but with very low aqueous solubility (solubility 0.3 mgmL 1 at 37 1C), presented a significant challenge. In response to this challenge, we endeavored to create inclusion complexes using a solvent-free method involving stirring and heating. This approach eliminated the need for organic solvents. The quantity of incorporated DTX was determined by measuring the difference in mass between the loaded and empty micelles, complemented by HPLC analysis. The drug loading content (DLC) and encapsulation efficiency (DLE) were calculated using eqn (1) and (2) as follows: DLC %ðÞ¼ Weight of loaded DTX Wt of MicMan3 þWt of loaded DTX 100% (1) DLE %ðÞ¼ Weight of loaded DTX Weight of input DTX 100%(2) The drug loading content and encapsulation efficiency of MicMan3 for DTX were 2.8% and 11.2%. These preliminary findings underscore the capability of MicMan3 to enhance the water solubility of highly insoluble organic molecules, which is of utmost significance for their potential utility as effective drug delivery systems. Conclusions In conclusion, we have developed a versatile design for the synthesis of multifunctional diazobenzene/diacetylene glycoamphiphiles. By adjusting the spacer length and the presence of a triazole ring, we can control the hierarchical self-organization of these molecules. Neoglycolipid 3forms spherical micelles (MicMan-3) with a sheltered inner core, capable of solubilizing hydrophobic molecules like Nile Red and docetaxel. On the other hand, monomer 1forms a hydro-alcoholic gel without the need for additional processing, consistingofentangled Fig. 7 Synthesis and use as nanocontainer of static mannose-coated nanomicelles ManMic-3 through sonication-promoted supramolecular selfassembly followed by intermolecular photo-polymerization of neoglycolipid 3in water (a). TEM images (b) and (c) of the formed micelles and liposomes. CMC and DLS size determination of MicMan-3 (d). Nile Red encapsulation study (e). Absorption spectra of Nile Red in water (black line) and in ManMic-3 solution (purple line) (f). Raman spectrum of the polymerized ManMic-3 (g). Journal of Materials Chemistry B Paper Open Access Article. 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