β-Cyclodextrin-based geometrically frustrated amphiphiles as one-component, cell-specific and organ-specific nucleic acid delivery systems

β-Cyclodextrin-based geometrically frustrated amphiphiles as one-component, cell-specific and organ-specific nucleic acid delivery systems Gonzalo Rivero-Barbarroja a , Jos´ e L´ opez-Fern´ andez b , Inmaculada Ju´ arez-Gonz´ alvez c , Carlos Fern´ andez-Clavero d , Christophe Di Giorgio e , Itziar V´ elaz f , María J. Garrido c , Juan M. Benito b , Carmen Ortiz Mellet a,* , Francisco Mendicuti d,* , Conchita Tros de Ilarduya c,* , Jos´ e M. García Fern´ andez b,* a Department of Organic Chemistry, Faculty of Chemistry, University of Seville, 41012 Sevilla, Spain b Instituto de Investigaciones Químicas (IIQ), CSIC −Universidad de Sevilla, 41092 Sevilla, Spain c Department of Pharmaceutical Sciences, School of Pharmacy and Nutrition, University of Navarra, 31080 Pamplona, Spain d Departamento de Química Analítica, Química Física e Ingeniería Química and Instituto de Investigaci´ on Química “Andr´ es del Rio”, Universidad de Alcal´ a, Spain e Institut de Chimie Nice, UMR 7272, Universit´ e Cˆ ote d’Azur, F-06108 Nice, France f Department of Chemistry, School of Sciences, University of Navarra, 31080 Pamplona, Spain ARTICLE INFO Keywords: Cyclodextrins Non-viral gene delivery Self-assembling Molecular nanoparticles Precision macromolecular synthesis ABSTRACT We introduce an innovative β-cyclodextrin (βCD)-prototype for delivering nucleic acids: “geometrically frustrated amphiphiles (GFAs).” GFAs are designed with cationic centers evenly distributed across the primary O6 and secondary O2 positions of the βCD scaffold, while hydrophobic tails are anchored at the seven O3 positions. Such distribution of functional elements differs from Janus-type architectures and enlarges the capacity for accessing strictly monodisperse variants. Changes at the molecular level can then be correlated with preferred self-assembly and plasmid DNA (pDNA) co-assembly behaviors. Specifically, GFAs undergo pH-dependent transition between bilayered to monolayered vesicles or individual molecules. GFA-pDNA nanocomplexes exhibit topological and internal order characteristics that are also a function of the GFA molecular architecture. Notably, adjusting the pK a of the cationic heads and the hydrophilic-hydrophobic balance, pupa-like arrangements implying axial alignments of GFA units flanked by quasi-parallel pDNA segments are preferred. In vitro cell transfection studies revealed remarkable differences in relative performances, which corresponded to distinct organ targeting outcomes in vivo. This allowed for preferential delivery to the liver and lung, kidney or spleen. The results collectively highlight cyclodextrin-based GFAs as a promising class of molecular vectors capable of finely tuning cell and organ transfection selectivity. 1. Introduction The introduction of nucleic acids into target cells offers a pathway for precise gene expression control, unlocking opportunities for personalized medicine and innovative therapeutic options (Dunbar et al., 2018). These prospects hinge critically on the efficient and safe delivery of nucleic acids to the intentional point of intervention (Sung & Kim, 2019). While viral vectors have demonstrated high efficiency and site specificity (Bulcha et al., 2021; Manche˜ no-Corvo & Martín-Duque, 2006) they pose risks such as severe immune responses, cellular toxicity, and genotoxicity (Kaiser, 2018; Othman et al., 2021). Non-viral vectors, emerging as promising alternatives, offer improved safety profiles, ease of manufacturing, and potential for large-scale production (Dong et al., 2024; Lostal´ e-Seijo & Montenegro, 2018; Mendes et al., 2022; Zu & Gao, 2021). Among them, four-component lipid nanoparticles (LNPs), incorporating ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-conjugated lipids, have garnered attention as leading systems for nucleic acid delivery (Cullis & Hope, 2017; Hou * Corresponding authors. E-mail addresses: [email protected] (C. Ortiz Mellet), [email protected] (F. Mendicuti), [email protected] (C. Tros de Ilarduya), [email protected] (J.M. García Fern´ andez). Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol https://doi.org/10.1016/j.carbpol.2024.122776 Received 3 July 2024; Received in revised form 12 September 2024; Accepted 18 September 2024 Carbohydrate Polymers 347 (2025) 122776 Available online 24 September 2024 0144-8617/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). et al., 2021; Kulkarni et al., 2019), notably contributing to the success of mRNA-based COVID-19 vaccines (Chung et al., 2020; Rana et al., 2023; Rueda-Fern´ andez et al., 2022; Verbeke et al., 2021). Notwithstanding, optimization of LNPs for broader clinical translation is hampered by the unknown distribution of their four components within the nanoparticle structure. The segregation of the neutral ionizable lipid as an oil phase in the LNP core hinders cytosolic release and significantly contributes to their low transfection efficiency (1–2 %) (Kulkarni et al., 2018; Ramezanpour et al., 2019). Moreover, LNPs are prone to stability issues that undermine both their integrity and efficacy (Schoenmaker et al., 2021; Blenke et al., 2023). Additionally, the use of PEG as a stealth coating on LNPs introduces the “PEG dilemma”: while extending LNP circulation time in the bloodstream, it concurrently diminishes gene expression by up to four orders of magnitude, primarily by reducing intracellular trafficking and impeding cellular uptake and endosomal escape (Hatakeyama et al., 2011; Whitfield et al., 2021). Additionally, PEG raises concerns regarding immunogenicity and potential off-target effects (Bigini et al., 2021; de Vrieze, 2021), underscoring the pressing need for targeted approaches to enhance therapeutic selectivity (Dilliard & Siegwart, 2023). Addressing the limitations of LNPs and advancing nucleic acid therapeutics requires exploring novel strategies, focusing on precision chemistry, molecular engineering, and innovative design principles (Mitchell et al., 2021). In this context, molecular nanoparticle (MNP)- based systems, characterized by persistent shape and volume (Jim´ enez Blanco et al., 2017; Yin et al., 2017), have emerged as a highly promising class of nonviral vectors (Xu & Zhang, 2018). MNPs, such as polyhedral oligomeric silsesquioxanes (POSS) (Wang et al., 2018), fullerenes (C 60 ) (Kazemzadeh & Mozafari, 2019; Wang et al., 2017) polyoxometalates (POM) (Bijelic et al., 2019), and folded proteins (Moreno-Gutierrez et al., 2023) possess tunable structures and particle-like characteristics. This category also includes macrocyclic oligomers with limited flexibility composed of aromatic ((e.g. calixarenes (Sansone et al., 2006; Bagnacani et al., 2013; Barr´ an-Berd´ on et al., 2015; Liu, Zhang, et al., 2021) or pillarenes (Xiao et al., 2019; Zyryanov et al., 2023)), heterocyclic ((e.g. cucurbit[n]uryls (Chernikova & Berdnikova, 2020; Yuan et al., 2020)), monosaccharide ((e.g. cyclodextrins (Haley et al., 2020; Kali et al., 2024; Rivero-Barbarroja et al., 2020; Sehgal et al., 2024) or cyclotrehalans (Carbajo-Gordillo et al., 2020; Carbajo-Gordillo et al., 2022; Jim´ enez Blanco et al., 2016; Manzanares et al., 2017)) or amino acid building blocks ((e.g. regioselectively addressable functionalized template cyclopeptides - RAFTs (Bartolami et al., 2015)). Importantly, the distinctive geometries of MNPs and their ability to undergo regioselective elaboration can be leveraged to achieve efficient nanocomplexation and protection of nucleic acids. This opens up possibilities for the development of sequence-defined multifunctional prototypes, which can be incorporated into one-component vector formulations. By eliminating the need for complex lipid mixtures, these systems provide greater control over the delivery process, facilitating optimization for specific applications (Rhan et al., 2024). The pursuit of MNP candidates represents a constantly sought-after objective in the field of gene therapy. However, progress in this domain has been somewhat constrained, predominantly within the scope of macrocyclic MNPs (Gallego-Yerga et al., 2015; Geng et al., 2019). Among them, cyclodextrins (CDs), with their unique toroidal structure, anisotropic distribution of hydroxyl groups and tunable hostguest interactions serve as privileged scaffolds (Mousazadeh et al., 2022; Ooi et al., 2024; Taharabaru et al., 2024). The precise molecular accuracy regarding composition, spatial relationships, and functionality renders these macromolecules exceptional for exploring the interplay between diverse molecular parameters in the hierarchical assembly processes that culminate in the formation of CD-nucleic acid nanocomplexes (CDplexes) (Bienvenu et al., 2012; Díaz-Moscoso et al., 2009; Gallego-Yerga et al., 2014). Through precision chemistry, cyclodextrinbased amphiphiles can be also functionalized with ligands or targeting moieties, enabling selective interaction with specific cells or tissues, thereby enhancing the therapeutic specificity (Aranda et al., 2013; Gooding et al., 2015; Guo et al., 2012; McMahon et al., 2012; M´ endezArdoy et al., 2015; Sun et al., 2023; Symens et al., 2012). However, the search for diastereomeric purity in conjunction with the incorporation of biorecognition modules entails relatively costly synthetic efforts (Díaz-Moscoso et al., 2011). An appealing alternative that presents opportunities for liganddevoid targeted delivery of nucleic acids consists in controlling factors such as size, shape, internal order, and surface charge of the nanocomplexes (Jia et al., 2024). Recent research underscores the tangled relationship between the primary structure of lipid components in LNPs (Cheng et al., 2020; Liu, Cheng, et al., 2021) or amphiphilic Janus dendrimers-mRNA dendriplexes (Zhang, Atochina-Vasserman, Maurya, Huang, et al., 2021; Zhang, Atochina-Vasserman, Maurya, Liu, et al., 2021; Zhang et al., 2022; Percec & Sahoo, 2024) and their fate within the body. Similarly, our previous findings revealed that subtle modifications in the architecture of trehalose-based vectors can lead to diverse assembly behaviors, resulting in distinct nanocomplex topologies and specific selectivities towards certain organs and cells (Carbajo-Gordillo et al., 2019; Carbajo-Gordillo et al., 2021; Ortega-Caballero et al., 2024). In the case of CDs, the neat face distinction allows for efficient synthesis of Janus-type vectors with separate cationic and lipophilic domains located either at the primary and secondary rim (Fig. 1A) (Ortiz Mellet et al., 2011), or vice versa (Fig. 1B) (Ortega-Caballero et al., 2008). Upon co-assembly with nucleic acids, spherical nanoparticles featuring onionlike multilamellar arrangements of CD bilayers and DNA chains are obtained (Fig. 1C) (Villari et al., 2013), which show a broad range of cell transfection capabilities in vitro and a preferential tropism to the liver in vivo. Alternative self-assembling patterns have been attained by breaking the symmetry of the macrocycle, achieved through dimerization (Gallego-Yerga et al., 2018) or the incorporation of aromatic “clips” at specific locations (Neva et al., 2020). While distinct cell and organ selectivities were successfully attained through these approaches, the associated synthetic challenges are significantly heightened. Here we proposed a new βCD-based nucleic acid delivery platform that surmounts the former archetype in its capacity to encode topological information. These vectors display cationic centers homogeneously distributed at both the primary O6 and the secondary O2 positions. The hydrophobic tails are then anchored at the seven O3 positions (Fig. 1D). Such distribution of functional elements frustrates the two-face sharp segregation of cationic and lipophilic domains characteristic of Janus MNPs. In the scientific literature, the term “frustrated amphiphiles” has been coined to refer to amphiphilic molecules for which the divergent alignment of the hydrophilic head and the lipid tail is hindered due to unfavorable hydrogen-bonding (Rutkauskaite et al., 2022) or electrostatic interactions (K¨ ostereli & Severin, 2012). By analogy, we use here “geometrically frustrated amphiphiles” (GFAs) to refer to amphiphilic MNPs for which the typical opposite orientation of hydrophilic and lipophilic moieties is disfavored by architectural constrains. The goal of this publication is to present the design, synthesis and supramolecular properties of βCD-based cationizable GFAs, using principles built upon insights gained from Janus MNPs. In aqueous environments, these GFAs exhibit self-assembly into bilayer vesicles, with their stability significantly influenced by pH variations within the physiological range. Additionally, they demonstrate co-assembly with plasmid DNA (pDNA), leading to the formation of CDplexes upon a simple mixing process, thereby functioning as one-component delivery systems. Screening experiments with a library of 12 GFAs provide evidence regarding the significance of the vector’s primary structure in determining the physical characteristics of the resulting nanocomplexes. This encompasses the cationic heads and the hydrophobic part. Computational simulations (molecular mechanics and molecular dynamics; MM and MD), dynamic and static light scattering (DLS and SLS, respectively), and transmission electron microscopy (TEM) were utilized to assess the preferred interaction modes and the ensuing arrangements. In vitro and in vivo transfection experiments demonstrate the proof of G. Rivero-Barbarroja et al. Carbohydrate Polymers 347 (2025) 122776 2 concept of βCD-based ionizable GFAs, highlighting their potential utility as models to elucidate fundamental aspects of nonviral delivery systems across multiple scales and their potential applications in the formulation of celland organ-selective nucleic acid therapeutics. 2. Materials and methods 2.1. Materials Ethylenediaminetetraacetic (EDTA) acid and DMSO Hibry-Max were supplied from Sigma. Sodium dodecyl sulfate (SDS) and NaCl (Roig Farma, Barcelona, Spain) were used to release DNA from the complexes. Alamar blue dye was purchased from Accumed International, Inc. (Westlake, OH, USA). The plasmid pCMV-Luc VR1216 (6934 bp) encoding luciferase was obtained from Clontech Laboratories, Inc. (Palo Alto, CA, USA). The plasmid pCMV100-IL-12 (5500 bp) encoding IL-12 was kindly provided by Dr. Chen Qian (University of Navarra, Spain). Cytokine levels were obtained using the kit BD OptEIA ELISA Set (BD Biosciences Pharmingen, San Diego, CA, USA) for IL-12 p40 following the manufacturer’s instructions. Heptakis(2,6-di-O-allyl)cyclomaltoheptaose (1) was synthesized according to a previously reported procedure (Eskandani et al., 2011), with modifications to eliminate the need for column chromatography. Details of the synthesis are provided in the Supplementary data. The absence of mixtures of positional isomers was confirmed by the presence of a single spin system in the 1 H NMR spectrum (Fig. S62), consistent with the C 7 -molecular symmetry of the compound. Sample homogeneity was further supported by MS (Fig. S63) and combustion analysis, ruling out the presence of underor over-allylated byproducts. Purity and homogeneity were systematically verified for all prepared batches of compound 1. 2-(tert-Butoxycarbonylamino)ethyl isothiocyanate (5) (Benito et al., 2004), 2-(N-tert-butoxycarbonyl-N-methylamino)ethyl isothiocyanate (6) (Kim et al., 2015) and 2-[N,N-bis(2-(tert-butoxycarbonylamino) ethyl)amino]ethyl isothiocyanate (7) (Díaz-Moscoso et al., 2009) were prepared according to reported procedures. N-tert-butoxycarbonyl-2aminoethanethiol (4) and 2-(4-morpholino)ethyl isothiocyanate (8) were purchased from BLD Pharmatech Ltd. (Reinbek, Germany). 2-(4Hydroxypiperidino)ethyl isothiocyanate was prepared from 2-(4-aminopiperidino)ethylamine (BLD Pharmatech Ltd., Reinbek, Germany) by isothiocyanation reaction with thiophosgene as described in the Supplementary data. Silica gel column chromatography was performed on silica gel 60 (E. Merck, 230–400 mesh) using 50 g of silica per 1 g of mixture. The crude product was loaded dissolved in the minimum amount of the less polar solvent component of the eluent. For Sephadex LH-20 size-exclusion chromatography purification, a ratio of 100 g of stationary phase per 1 g of crude material was used. The product was loaded dissolved in the minimum amount of MeOH. 2.2. Synthesis 2.2.1. βCD-based GFA synthesis As depicted in Schemes 1 and 2, the synthesis of β-cyclodextrincentered geometrically frustrated amphiphiles entailed: (a) per-(O3)- alkylation (hexylation or dodecylation) of the tetradecaallylated derivative 1 (→2 and 3, respectively); (b) multiple thiol-ene coupling with Boc-protected cysteamine 4 (→10a,b); (c) carbamate hydrolysis by treatment with aqueous TFA (→11a,b); and (d) subsequent thioureaforming reaction with either the Boc-protected aminoethyl isothiocyanates 5–7 (→12a,b-14a,b), followed by a final deprotection step (→15a,b-17a,b), or with the tertiary amine-equipped isothiocyanates 8 and 9 (→18a,b and 19a,b, respectively). Full characterization data are provided in the Supplementary data. Heptakis(2,6-di-O-allyl-3-O-hexyl)cyclomaltoheptaose (2). To a chilled (0 ◦C, ice-water bath) suspension of NaH (90 % purity, 220 mg, 8.26 mmol, 5 eq) in dry DMF (3 mL) under Ar atmosphere, a solution of heptakis(2,6-di-O-allyl)cyclomaltoheptaose 1 (400 mg, 0.236 mmol) in dry DMF (9 mL) was added. The ice-water bath was removed and the mixture was stirred for 1 h, allowing it to reach rt. 1-Bromohexane (1.16 mL, 8.26 mmol, 5 eq) was added, and stirring was continued for 16 h. The excess of NaH was quenched by dropwise addition of a small amount of MeOH, and the mixture was evaporated to dryness. A solution of the residue in DCM (10 mL) was washed with H 2 O (3 ×5 mL) and brine (1 ×5 mL), dried (MgSO 4 ), evaporated and purified by column chromatography (1:10 → 1:6 → 1:2 EtOAc-cyclohexane). Yield 270 mg (50 %). Heptakis(2,6-di-O-allyl-3-O-dodecyl)cyclomaltoheptaose (3). To a chilled (0 ◦C, ice-water bath) suspension of NaH (90 % purity, 385 mg, 14.4 mmol, 5 eq) in dry DMF (5 mL) under Ar atmosphere, a solution of 1 (0.70 g, 0.413 mmol) in dry DMF (15 mL) was added. The ice-water bath was removed and the mixture was stirred for 1 h, allowing it to reach rt. 1-Bromododecane (3.5 mL, 14.4 mmol, 5 eq) was then added, and the mixture was heating at 60 ◦C for 16 h. The excess of NaH was quenched by dropwise addition of a small amount of MeOH, and the mixture was evaporated to dryness. A solution of the residue in DCM (15 mL) was washed with H 2 O (3 ×10 mL) and brine (1 ×10 mL), dried (MgSO 4 ), evaporated and purified by column chromatography (1:10 → 1:2 EtOAccyclohexane). Yield: 1.08 g (91 %). Heptakis[2,6-di-O-(3-(2-(N-tert-butoxycarbonylamino)ethylthio)propyl)-3-O-hexyl]cyclomaltoheptaose (10a). To a solution of 2 (270 mg, 0.118 mmol) and N-tert-butoxycarbonyl-2-aminoethanethiol 4 (558 μ L, 3.31 mmol, 2 eq) in toluene (6 mL), 2,2-dimethoxy-2-phenylacetophenone (DMPA; 27 mg, 0.105 mmol) in toluene (0.5 mL) was added and the mixture was irradiated with UV light (λ =365 nm) at −50 ◦C under N 2 atmosphere for 1 h. The solvent was evaporated and the resulting Fig. 1. A) and B) Schematic representation of Janus-type βCD-based cationizable amphiphiles showing the two possible relative dispositions of the cationic heads (red circles; X is generally oxygen or sulfur) and lipid tails (purple swinging lines). C) Typical lamellar arrangement in CDplexes co-assembled from Janus-type CD vectors and nucleic acids; the characteristic lipid bilayer organization of the amphiphilic CD component is shown. D) βCD-based geometrically frustrated amphiphiles proposed in this work. G. Rivero-Barbarroja et al. Carbohydrate Polymers 347 (2025) 122776 3 residue was purified by column chromatography (15:1 DCM-acetone → 25:1 DCM-MeOH). Yield: 326 mg (58 %). Heptakis[2,6-di-O-(3-(2-(N-tert-butoxycarbonylamino)ethylthio)propyl)-3-O-dodecyl]cyclomaltoheptaose (10b). To a solution of 3 (500 mg, 0.174 mmol) and 4 (823 μ L, 4.87 mmol, 2.0 eq) in toluene (3 mL), DMPA (50 mg, 0.194 mmol) in toluene (0.5 mL) was added and the mixture was irradiated with UV light (λ =365 nm) at −50 ◦C under N 2 atmosphere for 1 h. The solvent was evaporated and the resulting residue was purified by column chromatography (50:1 → 40:1 DCM-MeOH). Yield: 876 mg (94 %). Heptakis[2,6-di-O-(3-(2-aminoethylthio)propyl)-3-O-hexyl]cyclomaltoheptaose tetradecahydrochloride (11a). Compound 10a (0.49 g, 0.104 mmol) was dissolved in a 1:1 TFA-H 2 O mixture (10 mL) and stirred at rt. for 1 h. The solvents were removed under reduced pressure and the traces of TFA were coevaporated with toluene (4 ×5 mL). The residue was dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 403 mg (quant). Heptakis[2,6-di-O-(3-(2-aminoethylthio)propyl)-3-O-dodecyl]cyclomaltoheptaose tetrahydrochloride (11b). Compound 10b (0.55 g, 0.103 mmol) was dissolved in a 1:1 TFA-H 2 O mixture (10 mL) and stirred at rt. for 2 h. The solvents were removed under reduced pressure and the traces of TFA were coevaporated with toluene (4 ×5 mL). The residue was dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 459 mg (quant). Heptakis[2,6-di-O-(3-(2-(2-(N-tert-butoxycarbonylamino)ethylthioureido)ethylthio)propyl)-3-O-hexyl]cyclomaltoheptaose (12a). To a solution of compound 11a (115 mg, 29.7 μ mol) in DMF (4 mL), Et 3 N (75 μ L, 0.54 mmol, 1.3 eq) and 2-(N-tert-butoxycarbonylamino)ethyl Scheme 1. Structures of the building blocks and reagents used in the synthesis of the βCD-based ionizable GFAs prepared in this work. Scheme 2. Synthesis of βCD-scaffolded GFA library. G. Rivero-Barbarroja et al. Carbohydrate Polymers 347 (2025) 122776 4 isothiocyanate (5, 101 mg, 0.50 mmol, 1.2 eq) were sequentially added. The resulting clear solution was stirred overnight at room temperature without the formation of any precipitate. Afterward, the solvent was evaporated under reduced pressure. The remaining residue was then purified by Sephadex LH20 column using MeOH as eluent. Yield: 92 mg (50 %). Heptakis[2,6-di-O-(3-(2-(2-N-tert-butoxycarbonylamino)ethylthioureido)ethylthio)propyl)-3-O-dodecyl]cyclomaltoheptaose (12b). To a solution of compound 11b (101 mg, 22.4 μ mol) in DMF (3.5 mL), Et 3 N (56 μ L, 0.407 mmol, 1.3 eq) and 5 (76 mg, 0.376 mmol, 1.2 eq) were sequentially added. The reaction mixture was stirred overnight at rt. and then the solvent was removed under reduced pressure. The resulting residue was purified by Sephadex LH20 column using MeOH as eluent. Yield: 129 mg (85 %). Heptakis[2,6-di-O-(3-(2-(2-(N-tert-butoxycarbonyl-N-methylamino)ethylthioureido)ethylthio)propyl)-3-O-hexyl]cyclomaltoheptaose (13a). To a solution of compound 11a (205 mg, 52.9 μ mol) in DMF (10 mL), Et 3 N (205 μ L, 1.48 mmol, 2 eq) and 2-(N-tert-butoxycarbonyl-N-methylamino)ethyl isothiocyanate (6, 319 mg, 1.48 mmol, 2 eq) were added sequentially. The resulting clear solution was stirred overnight at room temperature without the formation of any precipitate. Afterward, the solvent was evaporated under reduced pressure. The remaining residue was then purified by column chromatography (40:1 DCM-MeOH). Yield: 210 mg (62 %). Heptakis[2,6-di-O-(3-(2-(2-(N-tert-butoxycarbonyl-N-methylamino)ethylthioureido)ethylthio)propyl)-3-O-dodecyl]cyclomaltoheptaose (13b). To a solution of compound 11b (69 mg, 15.4 μ mol) in DMF (5 mL), Et 3 N (37 μ L, 0.27 mmol, 1.25 eq) and 6 (65 mg, 0.30 mmol, 1.4 eq) were added sequentially. The resulting clear solution was stirred overnight at room temperature without the formation of any precipitate. Afterward, the solvent was evaporated under reduced pressure. The remaining residue was then purified by column chromatography (25:1 DCM-MeOH). Yield: 79 mg (73 %). Heptakis[2,6-di-O-(3-(2-(2-(N,N-bis(2-N-tert-butoxycarbonylaminoethyl)amino)ethylthioureido)ethylthio)propyl)-3-O-hexyl] cyclomaltoheptaose (14a). To a solution of compound 11a (80 mg, 20.6 μ mol) in DMF (3.5 mL), Et 3 N (52 μ L, 0.376 mmol, 1.3 eq) and 2-[N,N-bis (2-(tert-butoxycarbonylamino)ethyl)amino]ethyl isothiocyanate (7, 135 mg, 0.347 mmol, 1.2 eq) were sequentially added. The reaction mixture was stirred overnight at rt. and then the solvent was removed under reduced pressure. The resulting residue was purified by Sephadex LH20 column using MeOH as eluent. Yield: 81.8 mg (45 %). Heptakis[2,6-di-O-(3-(2-(2-(N,N-bis(2-N-tert-butoxycarbonylaminoethyl)amino)ethylthioureido)ethylthio)propyl)-3-Ododecyl]cyclomaltoheptaose (14b). To a solution of compound 11b (112 mg, 25.1 μ mol) in DMF (4 mL), Et 3 N (63 μ L, 0.45 mmol, 1.3 eq) and 7 (163 mg, 0.42 mmol, 1.2 eq) were sequentially added. The reaction mixture was stirred overnight at rt. and then the solvent was removed under reduced pressure. The resulting residue was purified by Sephadex LH20 column using MeOH as eluent. Yield: 162.6 mg (69 %). Heptakis[2,6-di-O-(3-(2-(2-aminoethylthioureido)ethylthio)propyl)-3O-hexyl]cyclomaltoheptaose tetrahydrochloride (15a). Compound 12a (49.3 mg, 7.96 μ mol) was dissolved in a 1:1 TFA-H 2 O mixture (4 mL) and stirred at rt. for 1 h. The solvents were removed under reduced pressure and the traces of TFA were coevaporated with toluene (4 ×5 mL). The residue was dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 42 mg (quant). Heptakis[2,6-di-O-(3-(2-(2-aminoethylthioureido)ethylthio)propyl)-3O-dodecyl]cyclomaltoheptaose tetradecahydrochloride (15b). Compound 12b (55 mg, 8.1 μ mol) was dissolved in a 1:1 TFA-H 2 O mixture (4 mL) and stirred at rt. for 2 h. The solvents were removed under reduced pressure and the traces of TFA were coevaporated with toluene (4 ×5 mL). The residue was dissolved 0.1 M aqueous HCl and freeze-dried. Yield: 48 mg (quant). Heptakis[2,6-di-O-(3-(2-(2-(N-methylamino)ethylthioureido)ethylthio) propyl)-3-O-hexyl]cyclomaltoheptaose tetradecahydrochloride (16a). Compound 13a (193 mg, 30.2 μ mol) was dissolved in a 1:1 TFA-H 2 O mixture (10 mL) and stirred at rt. for 2 h. The solvents were removed under reduced pressure and the traces of TFA were coevaporated with toluene (4 ×10 mL). The residue was dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 166 mg (quant). Heptakis[2,6-di-O-(3-(2-(2-(N-methylamino)ethylthioureido)ethylthio) propyl)-3-O-dodecyl]cyclomaltoheptaose tetradecahydrochloride (16b). Compound 13b (57 mg, 8.16 μ mol) was dissolved in a 1:1 TFA-H 2 O mixture (10 mL) and stirred at rt. for 1 h. The solvents were removed under reduced pressure and the traces of TFA were coevaporated with toluene (4 ×10 mL). The residue was dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 49 mg (quant). Heptakis[2,6-di-O-(3-(2-(2-(N,N-bis(2-aminoethyl)amino)ethylthioureido)ethylthio)propyl)-3-O-hexyl]cyclomaltoheptaose (17a) dotetracontahydrochloride. Compound 14a (60.6 mg, 6.9 μ mol) was dissolved in a 1:1 TFA-H 2 O mixture (3 mL) and stirred at rt. for 2 h. The solvents were removed under reduced pressure and the traces of TFA were coevaporated with toluene (4 ×5 mL). The residue was dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 48 mg (quant). Heptakis[2,6-di-O-(3-(2-(2-(N,N-bis(2-aminoethyl)amino)ethylthioureido)ethylthio)propyl)-3-O-dodecyl]cyclomaltoheptaose (17b) dotetracontahydrochloride. Compound 14b (60.2 mg, 6.4 μ mol) was dissolved in a 1:1 TFA-H 2 O mixture (4 mL) and stirred at rt. for 2 h. The solvents were removed under reduced pressure and the traces of TFA were coevaporated with toluene (4 ×5 mL). The residue was dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 49 mg (quant). Heptakis[2,6-di-O-(3-(2-(2-(4-morpholino)ethylthioureido)ethylthio) propyl)-3-O-hexyl]cyclomaltoheptaose tetradecahydrochloride (18a). To a solution of compound 11a (150 mg, 38.7 μ mol) in DMF (5 mL), Et 3 N (106 μ L, 0.76 mmol, 1.4 eq) and 2-(4-morpholino)ethyl isothiocyanate (8, 115 mg, 0.66 mmol, 1.25 eq) were sequentially added. The reaction mixture was stirred overnight at rt. and then the solvent was removed at reduced pressure and the resulting residue was purified by Sephadex LH20 column using MeOH as eluent. The product-containing fraction was concentrated to dryness, dissolved in 0.1 M aqueous HCl and freezedried. Yield: 170 mg (71 %). Heptakis[2,6-di-O-(3-(2-(2-(4-morpholino)ethylthioureido)ethylthio) propyl)-3-O-dodecyl]cyclomaltoheptaose tetradecahydrochloride (18b). To a solution of compound 11b (138 mg, 30.9 μ mol) in DMF (5 mL), Et 3 N (78 μ L, 0.56 mmol, 1.4 eq) and 8 (84 mg, 0.49 mmol, 1.25 eq) were sequentially added. The reaction mixture was stirred overnight at rt. and then the solvent was removed under reduced pressure. The resulting residue was purified by Sephadex LH20 column using MeOH as eluent. The product-containing fraction was concentrated to dryness, dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 167 mg (78 %). Heptakis[2,6-di-O-(3-(2-(2-(4-hydroxypiperidino)ethylthioureido)ethylthio)propyl)-3-O-hexyl]cyclomaltoheptaose tetradecahydrochloride(19a). To a solution of compound 11a (150 mg, 38.7 μ mol) in 10:1 DMF-H 2 O mixture (5.5 mL), Et 3 N (106 μ L, 0.76 mmol, 1.4 eq) and 1-(2-isothiocyanatoethyl)piperidin-4-ol 9 (124 mg, 0.66 mmol, 1.25 eq) were sequentially added. The reaction mixture was stirred overnight at rt. and then the solvents were removed under vacuum, the resulting residue was purified by Sephadex LH20 column using MeOH as eluent. The productcontaining fraction was concentrated to dryness, dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 175 mg (71 %). Heptakis[2,6-di-O-(3-(2-(2-(4-hydroxypiperidino)ethylthioureido)ethylthio)propyl)-3-O-dodecyl]cyclomaltoheptaose tetradecahydrochloride(19b). To a solution of compound 11b (125 mg, 28 μ mol) in 10:1 DMF-H 2 O (5.5 mL), Et 3 N (78 μ L, 0.56 mmol, 1.4 eq) and 9 (89 mg, 0.48 mmol, 1.3 eq) were added consecutively and the reaction mixture was stirred overnight at rt. The solvents were then removed under reduced pressure and the resulting residue was purified by Sephadex LH20 column using MeOH as eluent. The product-containing fraction was concentrated to dryness, dissolved in 0.1 M aqueous HCl and freeze-dried. Yield: 180 mg (90 %). G. Rivero-Barbarroja et al. Carbohydrate Polymers 347 (2025) 122776 5 2.3. Formulation of pDNA-GFA nanocomplexes The stock solution of nucleic acid was prepared in ddH 2 O at 1.0 mg/ mL concentration. The stock solution of the GFA vector was prepared in 1:1 DMSO-ddH 2 O at 5–10 mM concentration (depending on the cationic density), selected to achieve the desired N/P ratio of the final CDplexes upon formulation. Each CDplex formulation was prepared by mixing two purposely-prepared phases in 9:1 (v:v) ratio: (i) the nucleic acid phase in HEPES buffer (10 mM, pH 7.4) and (ii) the vector phase in 1:1 DMSO-ddH 2 O. The nucleic acid phase was prepared from the stock solution by diluting 10-fold with HEPES, so that the final concentration was set up at 0.1 mg/mL ([phosphate] =0.30 mM). The vector phase was prepared by diluting the corresponding vector stock with 1:1 DMSO-ddH 2 O at the concentration required to achieve the desired N/P ratio, considering the number of cationizable groups in the vector molecular structure. Mixing was followed by gentle sample homogenization and storage at 4 ◦C until CDplexes were used. Particle size for each formulation was measured (DLS) immediately after preparation and monitored upon storage at 4 ◦C during several weeks without noticing relevant variations in size and polydispersity. Such formulation procedure ensures that the nucleic acid concentration, pH and DMSO ratio (5 % v:v) are identical throughout all formulations. For in vitro and in vivo assays, the formulations were diluted with HEPES buffer in order to reach a DMSO proportion below the 1 % threshold. Control experiments confirmed that the dilution process does not affect CDplex size and polydispersity. As an illustrative example, the preparation of CDplexes from pDNA and 16b at N/P 10 is detailed. Calculations were made for the preparation of 100 μ L of the final pDNA-16b formulation, containing 0.1 mg/ mL pDNA. a. Nucleic acid phase (100 μ L). Prepared by diluting 11 μ L of the 1.0 mg/ mL ([phosphate] 3.0 mM) pDNA stock solution in the appropriate volume of HEPES buffer (10 mM, pH 7.4). b. Vector phase (100 μ L). The concentration of the vector phase was calculated on the basis of the number of cationizable groups per molecule of the vector (14 in the case of 16b), the desired N/P ratio (10 in the present example), and the 9:1 phase mixing ratio, according to the formula: [vector phase] × (n◦cationic groups) = [phosphate]formulation ×N/Pratio)× (9:1 mixing ratio) For an N/P 10 the required concentration of 16b is 2.16 mM. It was achieved by diluting 43 μ L of a 5 mM stock solution of 16b with 1:1 DMSO-ddH 2 O to a final 100 μ L volume. c. Finally, the nucleic acid (90 μ L) and vector (10 μ L) phases were gently mixed and stored at 4 ◦C. Detailed calculations for all formulations prepared in this work are collected in the Supplementary data, Tables S1 and S2. The amount of GFA or control vector (bPEI) used in each formulation was calculated according to the desired pDNA concentration, the N/P ratio, the molecular weight, and the number of protonable nitrogen atoms in the corresponding cationic derivative. Typically, the plasmid pCMV-Luc VR1216 was diluted in HEPES (10 mM, pH 7.4) and then the required amount of GFA derivative or control vector was dispersed in this solution according the above from a stock solution in DMSO (typically 1–10 mM). The resulting mixture (with a final DMSO content below 1 % in all cases) was instantly vortexed thoroughly, and the complexes were incubated for 1 h at rt. prior to subjecting them to characterization or transfection experiments. 2.4. Procedure for determination of in vitro transfection activity Cells (COS-7, HepG2, RAW264.7 or NPTr) were seeded in medium in 48-well plates (Iwaki Microplate, Japan) and incubated for 24 h at 37 ◦C in 5 % CO 2 . The medium was removed, and 0.3 mL of complete medium (activated FBS) and 0.2 mL of CDplexes (containing 1 μ g of pDNA) were added to each well. After 4 h incubation the medium was replaced for complete medium, and the cells were further incubated for 48 h. Cells were washed with phosphate-buffered saline (PBS) and lysed with 100 μ L of Reporter Lysis Buffer (Promega, Madison, WI) at rt. for 10 min, followed by a freeze−thaw cycle. A 20 μ L aliquot of the supernatant was assayed for total luciferase activity by using the luciferase assay reagent (Promega), according to the manufacturer’s protocol. A luminometer (Sirius-2, Berthold Detection Systems, Innogenetics, Diagn´ ostica y Terap´ eutica, Barcelona, Spain) was used to measure luciferase activity. The protein content of the lysates was measured by de DC protein Assay Reagent (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard. The data were expressed as picograms of luciferase (based on a standard curve for luciferase activity) per milligram of protein. Samples were analyzed in a plate spectrophotometer Power Wave XS and a data processor KC junior, BioTek. 2.5. Cell viability, Alamar blue assay Cell viability was quantified by a modified AlamarBlue assay (Invitrogen). Briefly, 1 mL of 10 % (v/v) Alamar blue dye in complete medium was added to each well 24 h post-transfection. After 2.5 h of incubation at 37 ◦C, 200 μ L of the supernatant was assayed by measuring the absorbance at 570 and 600 nm. Cell viability (as percentage of control cells) was calculated according to the formula (A 570 −A 600 ) of treated cells ×100/(A 570 −A 600 ) of control cells. 2.6. In vivo transfection activity Balb-c mice (6–8 weeks of age, 20–25 g weight) were purchased from Harlan Ib´ erica Laboratories. All animals were studied in accordance with guidelines established by Directive 86/609/EEC and with the approval of the Committee on Animal Research at the University of Navarra-CIMA (id ES/31-2010-000132; accreditation number CEEA 017-19). Individual mice in groups of eight (four males and four females) were injected via the tail vein with 200 μ L of CDplexes containing 50 μ g of pCMV-Luc VR1216 plasmid DNA at N/P 5. Naked DNA was injected as control. Twenty-four hours after injection the mice were sacrificed. The liver, heart, kidneys, lungs, and spleen were collected and washed with cold PBS. We homogenized the organs with 1 mL of lysis buffer using a homogenizer at 5000 rpm (Mini-Beadbeater; BioSpec Products, Inc., Bartlesville, OK) and centrifuged at 10000 rpm for 3 min. A 20 μ L aliquot of the supernatant was analyzed for luciferase activity following the same procedure as for in vitro assays. Cytokine levels were obtained using the kit BD OptEIA ELISA Set (Pharmingen, San Diego, CA, USA) for IL-12 p70 following the manufacturer’s instructions. Values were calculated based on a standard curve. Samples were analyzed in a plate spectrophotometer Power Wave XS and a data processor KC junior, BioTek®. 2.7. Statistical analysis Statistical analyses were performed using SPSS software from SPSS Inc. (Chicago, IL). The analysis of the transfection efficiency of CDplexes was performed with a two-tailed unpaired Student’s t-test. P <0.05 was considered statistically significant. 2.8. Molecular mechanics (MM) and molecular dynamics (MD) calculations Molecular Mechanics (MM) and Molecular Dynamics (MD) G. Rivero-Barbarroja et al. Carbohydrate Polymers 347 (2025) 122776 6 calculations were performed to study the stability of a fully protonated GFA dimer in the presence of two B-DNA helical fragments in explicit water. Compound 15b was selected for this purpose. The Sybyl X-2.0 (SYBYL-X 2.0; Tripos Associates; St. Louis, MO, 2012) and the Tripos Force Field were used for all calculations (Clark et al., 1989). B-DNA fragments contained twelve nucleotides with a CGCGAATTCGCG sequence each. Charges for 15b were obtained by MOPAC (AM1) using the Gaussian program (Frisch et al., 2009), to provide a total net charge of +14 (esu) in the absence of chloride ions. DNA fragment charges were obtained by using the Gasteiger and Marsili method (Gasteiger & Marsili, 1980; Gasteiger & Marsili, 1987). The Molecular Silverware algorithm (MS) and periodic boundary conditions (PBC) were used for solvation (Blanco, 1991). A relative permittivity ε =1.0 was used for electrostatic contributions in explicit water. Nonbonded cutoff distances were set at 12 Å. Optimizations were performed with the simplex algorithm, and the conjugate gradient was used as a termination method with gradients of 3.0 kcal/(mol Å) for the MM calculations (Brunel et al., 1975; Press et al., 2007). Initially, a neutralized (uncharged) 15b dimer formation and its stability in water was studied by MM. In the neutral structure, the N⋅⋅⋅Cl virtual bonds lengths located at the end of the hydrophilic chains were constrained to keep them near the length for the minimum of the potential energy. For this purpose, a harmonic penalty function equation for the length of the bonds between the N and Cl atoms was added to the force field. This energy function was written as E =k (l-l e ) 2 , where k = 200 kcal/molÅ 2 , and l e and l are the most favorable N⋅⋅⋅Cl length and the variable distance during the MD trajectory, respectively. The optimized (gradient 0.5 kcal/molÅ) minima binding energy (MBE) structures for the neutral dimer structures were used as the initial conformations for the 1.0 ns MD simulations in the presence of water. During MD, the bonds where H atoms were involved were constrained from vibrating, but the rest of the conformational parameters were variable. Trajectories were performed starting from 1 K, and the temperature was increased by 20 K intervals equilibrating the system at each intermediate temperature for 500 fs up to reaching the temperature of interest of 300 K. Once at this temperature, an additional equilibration period of up to 25 ps was used. The whole heating/equilibration period was discarded from the analysis. From this point on, the rest of the trajectory time (ns) was simulated at 2 fs integration time steps. The velocities were rescaled at 10 fs intervals. Structures obtained from the analysis of MD trajectories were saved every 500 fs, yielding n snapshots [n =time (ns) ×10 6 /500] for subsequent analysis. The (15b) 2 -(DNA) 2 complex formation and stability in water was also studied by MM and MD. MM calculations for the most favorable 15b dimer structure in the presence of two DNA helix fragments were performed. Two possible orientations of the dimer, relative to the DNA helix, were considered: (a) either the dimer was placed along the y-axis and perpendicular to the two DNA fragments or (b) parallel to them (along the z-axis. The proposed approach of the DNA fragments to the dimer was always through the major groove. Optimized MBE structures for the (15b) 2 -(DNA) 2 (gradient 0.5 kcal/molÅ) were used as the starting conformations for MD simulations. To maintain a regular helical structure for the pair of DNA chains and to avoid the fraying of the end portions of the short DNA helix during MD, the simulations were performed on MBE structures where the N⋅⋅⋅HN hydrogen bond distances for each pair of DNA complementary bases were constrained to keep them constant. For this purpose, a harmonic penalty function was added to the force field for those atoms which were involved in the constraint. This energy function is written as E =k (r-r i ) 2 , where k =200 kcal/ molÅ 2 , and r i and r are the initial distance and the variable distance during the MD trajectory, respectively. A full description of the computational methods and results is provided in the Supplementary data. 3. Results and discussion 3.1. GFA design criteria, synthesis and properties Cyclodextrins exemplify how molecular architecture plays a defining role in the chemical reactivity of MNPs. Within the torus structure, the narrower and wider rims are flanked by primary and secondary hydroxyls, respectively. The narrower rim primary OH6 hydroxyls exhibit higher nucleophilicity due to reduced steric hindrance. On the other hand, the OH2 groups, owing to the intramolecular hydrogen bonding network, display increased acidity and can be readily deprotonated (pK a of 12.2). Accessing the OH3 position poses a relatively greater challenge. These fundamental principles provide a conceptual framework for achieving regioselective alkylation concurrently at positions O2 and O6. Allylation (→1) is particularly advantageous in this context (Eskandani et al., 2011). First, it enables modification of the remaining OH3 hydroxyls for the incorporation of the lipid tails by a next alkylation reaction. We used hexyl (→2) and dodecyl bromide (→3) for this purpose. Second, the allyl groups in the compound keeping the free OH3 groups or in the related hepta-(O3)-hexyl and hepta-(O3)-dodecyl ethers can be engaged in multiple thiol-ene “click” reaction (Sinha & Equbal, 2019) with tert-butoxycarbonyl (Boc)-protected cysteamine (4). The methodology implemented to generate molecular diversity at the cationic domain then relies upon design principles originally developed for the synthesis of polycationic amphiphilic Janus CDs, but refined and expedited. It involves the isothiocyanates 5–9 as crucial precursors (Scheme 1). Photochemical addition of thiol 4 to the tetradeca-O-allyl βCD derivatives 2 and 3 proceeded smoothly at −50 ◦C in toluene to give the corresponding thioethers 10a,b in 58–94 % yield. Acid-promoted hydrolysis of the carbamate groups (→11a,b) then exposes 14 peripheral primary amino groups. Note that this step already unmasks the GFA feature in just four sequential reactions from commercial βCD. Each of the two pivotal tetradecacysteaminyl βCD thioethers 2 or 3 was coupled with five aminoethyl-type isothiocyanates (5–9). The reaction is highly efficient without need of catalyst and insensitive to moisture, meeting all the criteria of click chemistry (Barner-Kowollik et al., 2011). Potential oxidation of the amine reagent by molecular oxygen can be prevented by generating the free base in situ from the corresponding hydrochloride salt, avoiding prolonged storage. Moreover, the resulting thiourea segments provide anchoring centers for hydrogen bonding, which are known to interact with phosphate groups in biology and in synthetic supramolecular chemistry and have been previously found to promote reversible complexation of nucleic acids (Breton et al., 2011). Final hydrolysis of the Boc-protected primary or secondary amino groups in the respective adducts was monitored using 1 H and 13 C NMR after converting the products into their respective perhydrochloride salts. The characteristic signals for the methyl (δ H 1.5, δ C 29 ppm) and carbonyl groups (δ C 157 ppm) of the tert-butoxycarbonyl residues completely disappeared, indicating successful transformation. In this manner, a set of twelve sequence-defined GFAs combining amino, N ′ -(2-aminoethyl) thioureido, N ′ -(2-(N-methylamino)ethyl)thioureido, N ′ -(2-(N,N-bis(2aminoethyl)amino)ethyl)thioureido, N ′ -(2-(4-morpholin-1-yl)ethyl)thioureido or N ′ -(2-(4-hydroxypiperidin-1-yl)ethyl)thioureido moieties linked at positions O2 and O6 on the βCD platform through 3-thiopropyl segments and hexyl (10a–15a) or docecyl tails attached at positions O3 (10b–15b) was gathered (Scheme 2). Assessing structural integrity through NMR was challenging due to significant line broadening caused by slow rotation around the N – C ( – – S) thiourea bonds (Jim´ enez Blanco et al., 1999). The substantial reduction in motion within the central core region of the GFA macromolecules also leads to an extended relaxation time for the associated carbon atoms, resulting in considerably lower intensities of their signals in the 13 C NMR spectra compared to the carbon resonances of the external chains. It is important to note that such spectroscopic features do not indicate structural defects. Nonetheless, electrospray ionization G. Rivero-Barbarroja et al. Carbohydrate Polymers 347 (2025) 122776 7 (ESI) mass spectrometry confirmed the expected molecular weights through pseudomolecular ions with exceptional molecular homogeneity considering the double click coupling synthetic strategy should quantitatively take place at 14 positions. Specifically, no pseudomolecular ions for N-dealkylation products were detected, which would be expected if N-oxidation of secondary or tertiary amino groups had occurred. Combustion analysis data were also compatible with sample homogeneity. Although these techniques cannot detect diastereomeric mixtures, the combined results strongly indicate a high level of purity in the target compounds. 3.2. Self-assembly of βCD-based GFAs The spontaneous formation of self-assembled structures from amphiphilic (macro)molecules consisting of ionic and hydrophobic domains in aqueous environments intricately balances attractive and repulsive forces. Hydrophobic attraction, hydrogen bonding, and steric or electrostatic repulsion all play significant roles in this delicate equilibrium, often resulting in a diverse array of nanostructures that exhibit pH-responsive behavior. In our study, the cationizable groups on compounds 11a,b and to 15a,b–19a,b were strategically chosen to cover a range of pK a values spanning approximately from 6 to 10. In principle, groups with pK a values 5–7 will be predominantly neutral at pH around 7.4, the extracellular pH, while they will be mainly reprotonated at pH 4.5, the pH at the lysosome. This is expected to deeply impact the stability and the characteristics of their aggregates (Mixich et al., 2024). Conversely, cationizable groups with pK a values over 8 will be essentially protonated at any physiological pH and thus less pH-sensitive in the biological environment. For compounds 11a and 11b, the pK a value of the isolated cationizable heads may closely approach that of S-methyl cysteamine (20, pK a 9.7). However, the intrinsic polarization of the thiourea segment in compounds 15a,b to 19a,b is predicted to decrease basicity compared to the corresponding alkylamine (García Fern´ andez & Ortiz Mellet, 2000). To determine the intrinsic pK a values, methylthiourea adducts, namely compounds 21–25, were prepared and titrated. The corresponding data are presented in Fig. 2 (see the Supplementary data for experimental details). It must be emphasized, however, that any assumptions made regarding the intrinsic pK a of the isolated heads should be approached cautiously when dealing with aggregates. The locally intense electrostatic potential at charged surfaces hinders the extent of protonation, typically resulting in an effective pK a downshift of several pH units compared to the intrinsic pK a and widen pH protonation-deprotonation range (Borisov et al., 2011). To evaluate the pH sensitivity of the self-assembly properties of the compounds, we initially investigated the size and topology of their aggregates in pure water across a 0.2–500 μ M concentration range. This range corresponds to a pH change of approximately 2 units based on the intrinsic pK a of the amine-type heads. The assessment involved a combination of dynamic and static light scattering (DLS and SLS), fluorescence spectroscopy measurements in the presence of a fixed concentration of pyrene (50 μ M), and transmission electron microscopy (TEM). DLS provides information on the hydrodynamic diameter (D h ) of the aggregates, offering insights into rearrangements or dissociation into individual amphiphile molecules, whereas SLS provides the particle molecular weight (MW). On the other hand, the fluorescence excitation spectrum of pyrene experiences a significant shift when transitioning from bulk water to a hydrophobic environment. This phenomenon is commonly utilized to detect the formation of micelles with a hydrophobic core, thereby establishing the critical micellar concentration (CMC) of conventional amphiphiles (Hofmann et al., 2011). Notably, pyrene is also capable of entering the βCD cavity through the wider rim, forming an inclusion complex, making it a useful probe for judging the accessibility of βCD cavities within the aggregates (Udachin & Ripmeester, 1998). This dual role of pyrene enables the evaluation of surface properties and internal order of the aggregates. DLS/SLS experiments revealed consistent patterns in the series of βCD-based GFAs 11b and 15a,b to 19a,b. At the lower concentrations (higher pH values), objects with a diameter (D h ) of 37–65 nm were observed, which transformed into much smaller entities with a D h of approximately 4 nm at higher concentrations (lower pH values). The estimated molecular weights (MW SLS ) of the latter matched those calculated from their molecular formula within the experimental error, indicating a dissociation process (Table 1). The fluorescence of pyrene from its excitation spectrum, with the emission fixed at λ ex 372 nm, remained constant within the concentration range where only aggregates were present in the solution. Subsequently, it experienced a linear increase with concentration. The intersection point of these two linear regimes aligns with the concentration at which the disruption of the aggregates occurs according to DLS (transition concentration, C t ; Supplementary data, Figs. S32-S43 and Table 1). This suggests that while individual βCD-based GFA molecules can form complexes with pyrene, the aggregates lack a hydrophobic interior capable of accommodating pyrene molecules. Additionally, the βCD cavity is inaccessible both within the internal core and at the external corona of the particles. The determination of the pH of the solutions used in the previous experiments provided the corresponding transition pH (pH t ) value in each case, defined as the pH at which a shift between distinct mesophases occurs. Ensuing DLS/SLS experiments conducted at fixed GFA concentrations (5 and 50 μ M) and solution pH both below and above the pH t values (pH 3 and 7, respectively), conclusively verified than the shift from vesicles to individual molecules is a pH-driven process independent of the concentration. The experimental data can be concealed assuming that at pH >pH t values only a fraction of the amine groups is protonated. This allows for intermolecular hydrophobic interactions between the lipophilic tails at the secondary face to be reinforced by additional hydrophobic contacts involving the linker moieties connecting the ionizable heads. Furthermore, hydrogen bonding interactions involving NH/C=S acceptor/donor thiourea groups may contribute to the stabilization of a bilayer vesicle architecture. As the pH decreases, coulombic repulsions exert a dominant influence, leading to the dissociation of the vesicles and enabling the formation of pyrene inclusion complexes. TEM micrographs recorded at concentrations above the respective pH t confirmed the existence of the anticipated vesicles. Beyond the pH t , the vesicles are no longer observable (Fig. 3). The exception to the above scenario is the hexylated GFA 11a, lacking the peripheral aminoethylthiourea segments. Whereas in the pH >pH t regime the behavior was comparable to the other GFAs, a transition to larger vesicular aggregates of approximately 150 nm occurred as the protonation degree gradually increased (Table 1 and Fig. 3). This restructuring concurred with a notable increase in pyrene fluorescence excitation spectrum within the solution, strongly suggesting that the broader rim of the βCD platform became exposed and accessible to pyrene molecules, aligning with a monolayer organization. It is important to note that the central region of the βCD-scaffolded GFA, which separates the two polycationic domains, consists primarily of hydrophobic thioalkyl spacers attached to both βCD faces. By rearranging into monolayer vesicles, the exposure of hydrophobic surfaces to bulk water is minimized, akin to classical bolaamphiphiles. In contrast, the Fig. 2. Structure of the amines 20–25 used in titration experiments to assess the intrinsic pK a values (in parenthesis) of the cationic heads displayed in the βCD GFAs prepared in this work. G. Rivero-Barbarroja et al. Carbohydrate Polymers 347 (2025) 122776 8 dodecylated counterpart 11b would presumably expose the O3-linked longer lipid tails to the aqueous solvent, a thermodynamically unfavorable outcome. The higher hydrophilicity of protonated aminoethylthiourea segments further promotes dissociation versus monolayer vesicles in the case of 15a,b–19a,b. This collective information provides a logical explanation for the experimental observations. The cumulative findings support the prevalence of GFA bilayer configurations under the nearly neutral conditions applied in CDplex formulation. This dominance is expected to bolster the nucleic acid nanocondensation process by actively fostering desolvation through hydrophobic effects, subsequent to the initial electrostatic interactions. Transitioning to monolayer arrangements (11a) or single molecules (11b and 15a,b–19a,b) typically occurred within the pH range of 3.1 to 5.0 in the specified experimental conditions. In the context of GFAnucleic acid co-assemblies, we anticipate that this shift would manifest at higher pH values, taking into account the influence of the anionization state of the polyphosphate chain on bilayer stability, suggesting the potential for distinct intralysosomal behaviors. Table 1 Transition concentration (C t ), transition pH (pH t ), average hydrodynamic diameter (D h ), polydispersity index (PDI), theoretical molecular weight (MW) and experimental molecular weight determined by SLS (MW SLS ) for aqueous solutions of compounds 11a,b and 15a,b–19a,b. Comp. C t ( μ M) pH t D h above pH t (nm) PDI D h below pH t (nm) PDI MW MW SLS 11a 6.0 3.4 53 ±2 0.01 149 ±18 0.16 3286.34 – 11b 4.5 3.6 54 ±2 0.11 4.1 ±0.3 0.19 4366.42 4485 ±120 15a 5.1 3.9 52 ±4 0.05 3.3 ±0.4 0.23 5305.64 5384 ±114 15b 10.1 3.8 64 ±5 0.28 3.5 ±0.3 0.16 5894.78 5906 ±65 16a 4.2 4.7 37 ±3 0.13 3.8 ±0.4 0.2 5502.02 5617 ±115 16b 28.2 4.5 45 ±4 0.1 4.2 ±0.4 0.14 6091.16 6135 ±83 17a 44.4 3.4 65 ±6 0.21 4.0 ±0.3 0.06 7021.99 7120 ±115 17b 54.1 3.1 43 ±5 0.28 4.9 ±0.4 0.15 7611.12 7802 ±195 18a 11.7 3.6 55 ±3 0.09 3.5 ±0.3 0.18 6286.92 6318 ±76 18b 46.1 3.4 62 +4 0.23 4.9 ±0.3 0.13 6876.05 7003 ±158 19a 18.7 4.7 40 ±2 0.19 3.4 ±0.2 0.1 6483.30 6512 ±47 19b 4.8 5.0 63 ±4 0.22 3.3 ±0.3 0.12 7072.43 7201 ±130 Fig. 3. Upper panel: Representative TEM micrographs recorded from aqueous solutions of 11a and 11b at pH above and below the pH t . Middle panel: cartoons of the corresponding bilayer vesicles (blue), monolayer vesicles (orange) or individual molecules (green); the accessibility of the βCD secondary rim to pyrene inclusion in the two later cases, resulting in enhanced fluorescence emission, is indicated. Lower panel: DLS profiles recorded from the same aqueous solutions of 11a and 11b. G. Rivero-Barbarroja et al. Carbohydrate Polymers 347 (2025) 122776 9 pDNA, nanocomplexes with varied topologies were formed. Notable differences in their internal order were also evidenced by TEM. Short range order preferentially involves interactions of individual molecules with the oligonucleotide chain. Conversely, long-range order, particularly pupa-like arrangements, can be rationalized in terms of pDNAtemplated longitudinal alignment of face-to-face GFA dimers, which can be considered the basic constituent of lipid bilayers. In vitro and in vivo evaluation of the transfection efficiency revealed striking cell and organ selectivity patterns that can be correlated with specific nanocomplex topologies. Remarkably, pupa-like nanocomplexes exhibited a clear preference for RAW264.7 macrophages, considered to be rather refractory to transfection, that correlated with a marked tropism to the spleen. The intriguing overlap of cell linages and tissue targets strongly suggests that receptor-mediated mechanisms, issued from preferential adsorption of distinct serum proteins on the particle surface, might operate to a certain extent. Nevertheless, the contribution of nanocomplex topology and protein corona composition to the in vitro and in vivo transfection mechanisms of GFA-based nanoparticles needs experimental validation, and it is essential to be cautious about making any generalizations. Despite this, the results collectively highlight cyclodextrin-based GFAs as a promising class of molecular vectors capable of finely tuning cell and organ transfection selectivity through precise chemical engineering. Abbreviations CD cyclodextrin DLS dynamic light scattering EDTA ethylenediaminetetraacetic acid TFA trifluoroacetic acid GFA geometrically frustrated amphiphile HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid bPEI branched poly-(ethylenimine) MBE minima binding energy MD molecular dynamics MM molecular mechanics MNP molecular nanoparticle pDNA plasmid DNA SD standard deviation SDS sodium dodecyl sulfate SLS static light scattering TEM transmission electron microscopy CRediT authorship contribution statement Gonzalo Rivero-Barbarroja: Writing – review & editing, Methodology, Investigation, Formal analysis. Jos´ e L´ opez-Fern´ andez: Writing – review & editing, Methodology, Investigation. Inmaculada Ju´ arezGonz´ alvez: Writing – review & editing, Methodology, Investigation, Formal analysis. Carlos Fern´ andez-Clavero: Writing – review & editing, Methodology, Investigation, Formal analysis. Christophe Di Giorgio: Writing – review & editing, Validation, Methodology, Investigation, Formal analysis. Itziar V´ elaz: Writing – review & editing, Validation, Supervision, Investigation. María J. Garrido: Writing – review & editing, Validation, Supervision, Investigation. Juan M. Benito: Writing – review & editing, Validation, Supervision, Investigation, Funding acquisition. Carmen Ortiz Mellet: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization. Francisco Mendicuti: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization. Conchita Tros de Ilarduya: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization. Jos´ e M. García Fern´ andez: Writing – review & editing, Writing – original draft, Supervision, Resources, Funding acquisition, Conceptualization. Declaration of competing interest The authors declare no competing financial interest. Acknowledgements We acknowledge the Ministerio de Ciencia, Innovaci´ on y Universidades and the Agencia Estatal de Investigaci´ on, AEI/10.13039/ 501100011033 and “ERDF A way of making Europe” (PID2020118384GB-I00, PID2021-124247OB-C21, PID2021-124247OB-C22 and PID2022-141034OB-C21) and the CAM (CM/JIN/2021-022). We also acknowledge funding by the European Union’s Horizon Europe research and innovation programme under the Marie Skłodowska-Curie grant agreement 101130235 – Bicyclos and the PTI+Global Health (CSIC). The CITIUS (Univ. Seville) is additionally acknowledged for technical support. G.R.-B and J. L.-F. (Grant numbers FPU18/02922 and PRE2019-088271, respectively). C.F.-C. thanks the UAH collaboration grant awarded. Appendix A. 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