Engineering amphiphilic alkenyl lipids for self-assembly in functional hybrid nanostructures

Engineering amphiphilic alkenyl lipids for self-assembly in functional hybrid nanostructures Raúl Gimeno-Ferrero1, Victoria Valdivia1, Inmaculada Fernández1, María Luisa García-Martín2,3,4 & Manuel Pernia Leal 1 The development of biocompatible hybrid nanosystems for advanced functional applications presents significant challenges to the research community. Key obstacles include the poor solubility of these nanosystems in water and the difficulty of precisely controlling their nanostructure dimensions and composition. A promising approach to overcoming these challenges is the self-assembly of surfactant-based building blocks into well-ordered hybrid nanostructures. In this study, we explore the relationship between structure and self-assembly in novel low molecular weight amphiphilic molecules to produce stable and biocompatible hybrid nanostructures. We investigated the self-assembly behavior of two families of amphiphiles derived from alkenyl lipids with one or two double bonds, leading to distinct hybrid supramolecular structures facilitated by the incorporation of hydrophobic iron oxide nanoparticles (IONPs) as templates. The presence of double bonds in the lipid tail and the morphology of the amphiphile influence the arrangement on the hydrophobic NPs. Amphiphiles with a single double bond in the lipid tail form highly water-soluble, well-ordered micellar-like structures on the IONP surfaces, while those with two double bonds create disordered lipid nanoparticles. Furthermore, these amphiphilic molecules can self-organize into higher-order hybrid supramolecular structures, such as vesicles, with potential applications in magnetic resonance imaging (MRI). Self-assembly of building blocks for the construction of functional materials has received significant interest in many fields, including nanotechnology, materials sciences, and biomedicine, in recent decades1–8. The diversity in the design and nature of the building blocks allows researchers to expand and enhance the library of resulting materials, offering excellent optical, electronic, and magnetic properties9–12. Many of these selfassembled materials are based on inorganic nanoparticles (NPs), which act as building blocks to form highly ordered superstructures ranging from 0D to 3D through hydrophobic alkane-based surfactants interparticle interactions. Nevertheless, the poor solubility in physiological media and the challenges in controlling the dispersity, size, geometrical dimensions, and homogenous composition of these self-organized nanostructures complicate their use in biological applications such as drug delivery systems, biosensors, and therapeutic agents13–18. To overcome these issues, one of the most elegant and cost-effective methods is the surfactantassisted cooperative self-assembly of hydrophobic inorganic NPs using amphiphilic molecules19,20. The selforganization of these components leads to the formation of well-defined and ordered nanostructures. These hybrid complex architectures are formed through non-covalent interactions, such as van der Waals forces, π-π stacking, or hydrogen bonding, between the hydrophobic capped surfactants from the inorganic NPs and the hydrophobic counterpart of the amphiphilic molecules21–24. Micelles and vesicles are among the most studied surfactant-assisted cooperative self-assemblies due to the versatility and ease of synthesis of the amphiphilic molecules4,25–27. Typical amphiphilic building blocks used in the formation of these supramolecular structures range from low molecular weight (MW) amphiphilic molecules or small surfactants to polymeric lipids28–30. Both types of surfactants present several crucial points to be considered ideal building blocks for self-assembly with inorganic NPs, such as the concentration of surfactants above the critical micelle concentration (CMC), the use of hydrophobic inorganic NPs, and the application of energy, such as heating, to evaporate the apolar solvents from the hydrophobic NP solution. Therefore, these key factors, combined with the design of the amphiphilic molecule, lead to their self-organization in micelles or vesicles, passivating the hydrophobic inorganic NPs for 1Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de Sevilla, c/ Profesor García González, 2, Sevilla 41012, Spain. 2Biomedical Magnetic Resonance Laboratory-BMRL, Andalusian Public Foundation Progress and Health-FPS, Seville, Spain. 3Instituto de Investigación Biomédica de Málaga y Plataforma en Nanomedicina-IBIMA Plataforma BIONAND, C/ Severo Ochoa, 35, Málaga 29590, Spain. 4Biomedical Research Networking Center in Bioengineering, Biomaterials &Nanomedicine (CIBER-BBN), Madrid, Spain. email: [email protected] OPEN Scientific Reports | (2024) 14:28887 1 | https://doi.org/10.1038/s41598-024-79917-8 www.nature.com/scientificreports the construction of hybrid building blocks. In addition, the stability of these self-assemblies in aqueous media remains a challenge for both types of surfactants. In the case of the low MW surfactants, due to their nature, they self-assemble dynamically around the inorganic NPs, interdigitating their hydrophobic counterparts with the hydrophobic capping ligands of the inorganic NPs. The resulting nanostructures are highly dependent on the surfactant concentration due to the short residence time of the amphiphilic surfactants16. On the contrary, polymeric surfactants, such as block copolymers, exhibit a high number of hydrophobic chains in the polymer that increase the residence time, enhancing the efficacy and capacity of the micellar architectures31–33. However, amphiphilic polymers present some challenges, such as more precise and versatile synthetic methods, and also require a deeper understanding of the assembly of polymer surfactants with inorganic NPs to yield wellordered hybrid building blocks capable of forming hierarchical superstructures34. In fact, controlling the layer composition and assembly of surfactants in supramolecular structures incorporating inorganic NPs remains a quest in materials science research. In this work, we report the synthesis of two families of low MW surfactants based on lipid chains with one and two double bonds. These surfactants are capable of self-assembling into various water-soluble, stable and well-ordered hybrid supramolecular structures driven by the addition of hydrophobic iron oxide NPs as templates. The presence of double bonds in the lipid chain, and the morphology of the surfactant, induce the type of arrangement of the amphiphilic molecules on the hydrophobic NPs, as well as the maximal capacity of acceptance and embedding of hydrophobic NPs. Moreover, these hybrid supramolecular structures exhibited good characteristics as MRI contrast agents depending on the arrangement of the amphiphiles onto the IONPs. Experimental section Materials All reagents and solvents were obtained from commercial suppliers Merck and Fisher Scientific and used without further purification. Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 analytical plates. The chemicals synthesis was monitored by a handheld UV lamp (254nm) and revealed with a solution of 5% phosphomolybdic acid in EtOH. Silica gel 60 (0.04–0.063mm) from Merck was used for flash chromatography carried out with technical grade solvents. Methods – Nuclear magnetic resonance (NMR) spectra were recorded on a BRUKER AMX-500 apparatus. Deuterated chloroform and methanol were used and indicated in parentheses for each compound. The chemical shift values (δ) were referred to tetramethylsilane used as an internal reference. – High resolution mass spectrometry (HRMS) was recorded on a Q Exactive Hybrid Quadrupole-Orbitrap apparatus from ThermoFisher Scientific. – Transmission electron microscopy (TEM) images were obtained on an HR Fei Talos 200X microscope operated at an accelerating voltage of 100kV. TEM samples were prepared by dropping a solution of the corresponding nanosystem onto a carbon-coated copper grid without staining. The mean sizes were calculated on an average of 100 nanoparticles measured. Stained TEM grid was performed by adding a drop of a solution of 2% uranyl acetate previously filtered through a 0.2μm pore size syringe filter (membrane material: acrylic copolymer housing cellulose acetate/surfactant-free membrane) to a drop of the nanoparticle sample. The drop was left 2min, and then, the grid was dried using paper filter to eliminate uranyl acetate excess. – The intensity distribution of the nanosystems was measured on a Zetasizer Nano ZS90 from Malvern Panalytical. The dynamic light scattering (DLS) measurements were performed on a cell type: ZEN0040 disposable cuvette, setting a refractive index of 2.3 for iron oxide NPs and 1.40 for the micelles without IONPs. The measurement duration was set as automatic, and the number of measurements was three. The general purpose, normal resolution, was chosen as analysis model. The nanosystems were suspended in the corresponding solvent at a concentration of 1mg/L of micelles and/or IONPs. – Small Angle X-ray scattering (SAXS) was measured using a Bruker D8 DISCOVER diffractometer. The sample is located in a borosilicate capillary of 1mm of diameter with a concentration of 1mg/mL. The two-di- mensional area detector (Eiger2 500K) allows for fast measurements with high angular resolution and a large dynamic range. Additionally, a Goebel mirror with a 0.3mm collimator is used to reduce the divergence of the incident beam. The mirror and the detector are used in combination with additional vacuum-enclosures to measure the SAXS powder samples in transmission geometry. Results and discussion Design and synthesis of alkenyl-based amphiphilic molecules To determine the influence of the alkenyl-based lipid counterpart on the self-assembly of amphiphilic molecules, two families of low MW surfactants were designed. One family was based on a hydrophobic chain with a single double bond (cis alkene, as oleic acid), while the other family had a hydrophobic chain with two double bonds (cis-cis alkene, as linoleic acid). In both families, three types of amphiphiles were synthesized with different hydrophobic/hydrophilic balance and structural morphology (I-VI). These types included a single hydrophilic polar head with a single hydrophobic tail (I and IV), two hydrophilic polar heads with a single hydrophobic tail (II and V), and a single hydrophilic polar head with two hydrophobic tails (III and VI). The chosen polar head was a tetraethylene glycol (TEG) chain with an amino as terminal group (Fig.1). Amphiphilic molecules I and IV, with a single hydrophilic polar head and a mono alkenyl-based lipid, were synthesized through a two-step reaction, as shown in Scheme 1. The alkyne-based lipids 1 and 2 were combined with the hydrophilic chain, amino-TEG-azide, via a Cu(I)-catalyzed [3 + 2] cycloaddition (known as click reaction), yielding the amphiphilic molecules I and IV. The Y-shape surfactants II, III, V and VI were Scientific Reports | (2024) 14:28887 2 | https://doi.org/10.1038/s41598-024-79917-8 www.nature.com/scientificreports/ prepared using dopamine as the starting building block material (Scheme 1). The choice of dopamine was based on its great versatility derived from the different reaction sites it possesses, allowing for orthogonal reactions to construct a variety of complex molecular scaffolds. The amidation of the alkenyl-based lipid acids, oleic acid, and linoleic acid, with dopamine, resulted in the amide 3 and 6, respectively. Following the incorporation of two terminal alkynes into the dopamine derivatives and subsequent double click reactions with two molecules of aminoBOC-TEG-azide, the corresponding triazoles 5 and 8 were obtained, with excellent yield for the oleyl derivative and moderate yield for the linoleyl derivative. Subsequent deprotection of aminoBOC in the presence of trifluoroacetic acid and dichloromethane gave the desired Y-shaped amphiphiles II and V, with two polar heads and a mono alkenyl-based lipid. The other Y-shaped amphiphiles III and VI, with a single hydrophilic polar head and two alkenyl-based lipids, were also synthesized via a four-step reaction, as shown in Scheme 1. Unfortunately, the bis-linoleyl amphiphilic molecule VI could not be synthesized. The incorporation of two linoleic acids into the dopamine-derived compound resulted in mainly traces of the bis-linoleyl product, as determined by HRMS. This poor reactivity was likely due to the high steric hindrance of the first attached linoleyl lipid, which hindered the incorporation of the second one. Therefore, further study of the bis-linoleyl-derived surfactant was not pursued. This steric hindrance was also observed in all reactions involving the linoleyl-based lipid, which exhibited low yields compared to their oleyl analogues. All these low molecular weight alkenylbased amphiphilic molecules were characterized using1H and13C NMR spectroscopies and high mass resolution spectrometry (Figures S1-S16). Self-assembly behaviour of alkenyl-based amphiphilic molecules The capacity of the synthesized alkenyl-based amphiphilic derivatives to self-assemble into micelles was determined by measuring the critical micelle concentration (CMC). The CMC values were obtained using the pyrene method in water35,36, resulting excellent values from 0.049 to 0.074 mM for the amphiphilic molecules I, II and III (Fig.2 and Figure S17). These values represent an improvement over the CMC values of low MW surfactants and fall within the same range as those reported for amphiphilic polymers in the literature37,38. In fact, the CMC values of our oleyl amphiphiles decrease with an increase in the polar head of the surfactant, particularly in the amphiphilic molecule II, which presents two hydrophilic chains. On the contrary, the presence of bis-oleyl lipid in the surfactant III resulted in the highest CMC value. Dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM) analysis of the selfassemblies I, II and III (hereafter referred to as Mc1, Mc2, and Mc3, respectively) confirmed the formation of micelles without the presence of larger aggregates, PDI values ranging from 0.24 to 0.37 (Fig.2A-C). In fact, these analyses exhibited a similar trend to the CMC values. The surfactant with the higher hydrophilic/ hydrophobic balance, amphiphile II, which possesses a double TEG chain, formed micelles Mc2 with the smallest hydrodynamic size in water, 5.9 ± 0.4nm. A slight increase in the HD size to 7.8 ± 0.5nm was observed for Mc1 derived from amphiphile I with a single TEG chain, while a more than 2-fold increase to 13.7 ± 0.8nm was noted for Mc3 derived from the bis-oleyl lipid-based surfactant III. Indeed, a change in the hydrophilic/ hydrophobic balance of the amphiphilic molecule, achieved by the introduction of two oleyl lipid chains (Y-shaped amphiphile), led to an expansion of the hydrophobic inner core of the self-assemblies, resulting in an increase in size and the number of surfactants molecules required to self-assemble in micelles (CMC value). As shown in the TEM images in Fig.2D and E, the incorporation of two double bonds (cis, cis) in the lipid chain of the low MW surfactants resulted in different self-assemblies, named as lipid nanoparticles (LNP), compared to those observed for derivatives with a single double bond (cis) in the alkenyl chain. LNPs derived from mono- TEG linoleyl IV (LNP1) and di-TEG Y-shaped V (LNP2) exhibited much larger HD sizes than their oleyl analogues, measuring 461 ± 10nm and 410 ± 29nm, respectively (Fig.2D-E). These results clearly indicated a different mode of self-organization compared to micelles of the cis, cis linoleyl lipid amphiphilic molecules. The presence of the second double bond in the lipid chain restricts the movement of the amphiphilic molecule to Fig. 1. Mono- and bis-alkenyl based amphiphilic molecules studied in this work. Scientific Reports | (2024) 14:28887 3 | https://doi.org/10.1038/s41598-024-79917-8 www.nature.com/scientificreports/ adopt the most energetically favourable arrangement, resulting in increased rigidity and volume. This induces the formation of non-ordered structures such as LNPs. Exploring the NP-driven assembly of low MW surfactants As mentioned above, the synthesis of well-ordered hybrid building blocks is crucial for developing new materials for a wide range of applications, particularly in biomedical fields. Therefore, it urges the necessity to develop water-soluble hybrid building blocks that can be used for this purpose. In this sense, we focused our attention on iron oxide nanoparticles (IONPs) as templates for preparing hybrid building blocks. The prepared IONPs were stabilized by oleic acid molecules, which could interact with the lipid tails of the low MW surfactants described above, leading to stable and well-ordered nanostructures. The initial experiments were performed similarly to the formation of micelles Mc1 by adding the amphiphilic molecule I at a concentration above their CMC in the presence of a fixed concentration of IONPs re-suspended in toluene. These experiments were carried out at Scheme 1. Synthesis of mono- and bis-alkenyl based amphiphilic molecules. Scientific Reports | (2024) 14:28887 4 | https://doi.org/10.1038/s41598-024-79917-8 www.nature.com/scientificreports/ different sonication times ranging from 1 to 30min to optimize the resulting ensembles Mc1-IONP. As shown in Fig.3A, the system required a minimum of 10min to reach equilibrium in terms of size, measuring 125.1nm, likely due to the evaporation time of the toluene from the IONPs. Hybrid systems formed with less than 10min of sonication exhibited significant variations in size, with the appearance of some aggregates after several months. Thus, the self-assembly process of the amphiphiles onto the IONPs is driven by the evaporation of the organic solvent, resulting in the embedding of the lipid tails in the oleic acid layer of the NP, leading to highly stable colloidal structures. Therefore, the speed of solvent evaporation plays an important role in the kinetics of the formation of well-ordered supramolecular structures4,13,20. To demonstrate this effect, toluene was substituted for a low boiling point apolar solvent such as hexane in the self-assembly process. Consequently, the 30min Fig. 3. (A) Size vs. sonication time graph of the Mc1-IONPs in water obtained from toluene as apolar solvent. (B) Summary table of the Mc-IONPs, obtained from toluene and hexane as starting apolar solvents, containing the sizes and PDI values determined by DLS in water. Proposed self-assembly models of Mc1-IONPs from (C) toluene and (D) hexane as apolar solvents. Representative TEM images of (E) Mc1-IONPs, (F) Mc2-IONPs and (G) Mc3-IONPs obtained from hexane as starting apolar solvent. Scale bar corresponds to 100nm for low magnification images and 25nm for the insets. (H) TEM image of Mc2-IONPs, obtained from hexane as starting apolar solvent, stained with uranyl acetate showing the organic coating and inorganic nucleus. Scale bar corresponds to 200nm. Fig. 2. Representative TEM images, CMC values and DLS data of (A) Mc1, (B) Mc2 and (C) Mc3. Scale bar in A corresponds to 20nm, in B and C corresponds to 100nm. Representative TEM images and DLS data of (D) LNP1 and (E) LNP2. Scale bars correspond to 1μm for low magnification images and 200nm for the insets. Scientific Reports | (2024) 14:28887 5 | https://doi.org/10.1038/s41598-024-79917-8 www.nature.com/scientificreports/ sonication of amphiphile I in the presence of the IONPs re-suspended in hexane resulted in larger structures with a HD size of 173.6nm (Fig.3B). This increase of ca. 50nm in size of the supramolecular structures demonstrates the effect of the solvent evaporation. The fast evaporation of the organic solvent increases the entropy of the hybrid system, resulting in the formation of kinetic supramolecular assembly, meaning a more disordered system with poor interlinking of the hydrophobic chains in the oleic acid layer on the IONPs. On the contrary, slow evaporation of the organic solvent leads to the formation of thermodynamic supramolecular assemblies through the insertion of the hydrophobic chains of the amphiphiles into the hydrophobic layer of the IONPs. This effect induces a compression and a more stable system arising from multiple van der Waals interactions at the NP surface, resulting in the formation of smaller supramolecular assemblies (Fig.3C-D). The Y-shaped oleyl amphiphiles II and III showed disparate results compared to amphiphilic molecule I. The bis-oleyl amphiphile III exhibited the same trend as the mono oleyl amphiphile I. The use of toluene as the apolar solvent in the selfassembly process led to a well-ordered hybrid system, Mc3-IONP, with a size of 148nm. In the case of hexane, the hybrid system reached a size of 280nm (Fig.3B). As mentioned above, rapid solvent evaporation resulted in increased entropy, leading to a more disordered supramolecular structure with larger sizes. In fact, with the Y-shaped amphiphilic molecule III, the size of the system increased ca. 100% due to the presence of the bis-oleyl chains in the amphiphile, which confer more rigidity and volume to the system Mc3-IONP. However, the other Y-shaped amphiphilic molecule II, with the double TEG polar head, exhibited a decrease in the size of the hybrid system Mc2-IONP from 122nm in toluene to 94nm in hexane (Fig.3B). A possible explanation is that in the assembly process with toluene, the bis-TEG polar head of the Y-shaped amphiphiles is completely stretched towards the aqueous phase since the oleyl chains are embedded within the oleic acid cover of the NP. In hexane, the oleyl chains from the Y-shaped amphiphiles mainly interact without embedding into the hydrophobic layer of the NPs, meaning that the lipid tails are deposited parallel to the surface of the NP with the bis-TEG polar head positioned perpendicular, reducing the size of the supramolecular structure (Figure S18). TEM analysis of the three different Mc-IONPs showed the presence of monodispersed hybrid NPs as mesuared by DLS (Fig.3E- G). In fact, the complete observation of the hybrid supramolecular structure, showing the organic coating and inorganic nucleus, was obtained by staining with uranyl acetate (Fig.3H). Small-angle X-ray scattering (SAXS) studies were carried out to confirm and determine the structural organization of the different assemblies (Fig.4 and Figure S19). The pair distribution function plot against distance (r) typically showed two main populations. In the smaller r region, the main population corresponds to the scattering of the inorganic IONP core, similar to the scattering of the oleic acid-capped IONPs (Figure S20). In the larger r region, the maximal value corresponds to the size of the hybrid supramolecular assembly, which is slightly smaller than the HD size determined by DLS. This difference arises due to the suppression of the contribution of the hydration layer at the surface of the system in the SAXS analysis (Fig.3B)39. Moreover, the other main population at larger r indicates the thickness of the organic shell of the nanosystems. This distribution displays the radius of the organic shell and the inorganic NP core (Figure S21). Therefore, the size of the organic layer for Mc1-IONPs, Mc2-IONPs and Mc3-IONPs in toluene was determined to be 54nm, 54nm and 72nm, Fig. 4. Normalized pair distribution of the SAXS measurements vs. distance graphs of Mc1-IONP, Mc2- IONP and Mc3-IONP obtained from toluene ((A), (C) and (E), respectively) and hexane ((B), (D) and (F), respectively) as starting apolar solvents. Scientific Reports | (2024) 14:28887 6 | https://doi.org/10.1038/s41598-024-79917-8 www.nature.com/scientificreports/ respectively. Larger sizes were obtained in hexane, in accordance with the DLS results, 77nm and 114nm for Mc1-IONPs and Mc3-IONPs, respectively. In the case of Mc3-IONPs, the pair distribution plot exhibits a third population that directly reflects the size of the organic layer without the presence of the NPs. For Mc2-IONPs, the thickness of the organic layer could not be determined due to the overlapping of the two populations. Optimization of the supramolecular assembly of the oleyl-based amphiphilic molecules on the NPs So far, we have analyzed the structure-self-assembly relationship of two families of amphiphilic molecules with alkenyl-based lipids. Additionally, we have studied the template effect in the self-assembly process with the presence of IONPs and oleyl-based surfactants. However, it remains a challenge to demonstrate the limits and capacity of the supramolecular systems based on these new amphiphilic molecules driven by the presence of NPs. Therefore, we carried out more detailed experiments to comprehensively understand the behaviour of the supramolecular assemblies by increasing the number of NPs. The addition of an increased number of IONPs in the self-assembly process of oleyl-based amphiphiles I, II and III exhibited three different areas in the size vs. concentration of NPs representations (Fig.5A-C). The first area corresponds to the presence of supramolecular assemblies with and without IONPs. The observed sizes represent the average between these two formed systems. The addition of more NPs generated the emergence of a second area that exhibits similar sizes of the formed assemblies (coloured area in Fig.5A-C). This area represents the section of the complete assembly of the amphiphiles with the IONPs (Mc1-IONPs, Mc2-IONPs, and Mc3-IONPs). It means that there are practically no supramolecular structures without IONPs. The size of the assemblies in this area remains practically unaltered upon the addition of more NPs. Once the amphiphiles are occupied forming the hybrid assemblies Mc-IONPs, the extra added IONPs precipitated during the process without affecting the nanostructure (Figure S22). This area is named as stability area, which corresponds to the presence of homogeneous hybrid systems independently of the number of IONPs. The supramolecular assembly Mc1-IONPs from amphiphilic molecule I exhibited a wider stability area compared to the assemblies Mc2-IONPs and Mc3-IONPs of Y-shape amphiphilic molecules II and III, respectively. It is also important to highlight the assembly based on the Y-shape amphiphile II with a single lipid tail, Mc2-IONPs, which showed the maximum incorporation of NP in that area. However, the addition of more IONPs to the systems drives the formation of aggregates. The re-organization of the assemblies that embed more IONPs leads to the formation of unstable systems that precipitate (insets Fig.5B and Figure S23). Fig. 5. Size vs. [IONPs]hexane graphs of (A) Mc1-IONPs, (B) Mc2-IONPs and (C) Mc3-IONPs. Stable areas in colour. PDI values vs. [IONPs]hexane graphs of (D) Mc1-IONPs, (E) Mc2-IONPs and (F) Mc3-IONPs. The vertical line shows the maximal acceptance (MA) point. Embedded IONPs plots of (G) Mc1-IONPs, (H) Mc2- IONPs and (I) Mc3-IONPs. Scientific Reports | (2024) 14:28887 7 | https://doi.org/10.1038/s41598-024-79917-8 www.nature.com/scientificreports/ An exhaustive analysis of the stability area of different amphiphile-based assemblies also provides key information about the concentration of NPs required to build the maximum number of well-ordered hybrid supramolecular structures. The polydispersity index (PDI) of the Mc-IONPs vs. concentration of NPs graphs show a minimum PDI value, referred to as the maximal acceptance NP (MA) point, corresponding to the complete embedding of the added IONPs in the medium (Fig.5D-F). These hybrid supramolecular structures with the minimum PDI value could be considered optimal building blocks for constructing advanced functional materials. Therefore, within the stability area, adding fewer NPs than the MA point results in higher PDI values (left side of the MA point) due to the presence of supramolecular assemblies without IONPs. Conversely, adding a higher number of NPs also leads to higher PDI values (right side), indicating the onset of aggregation, forming a third area in the assembly process consisting of aggregates and/or precipitates. The three oleyl-based assemblies exhibited similar MA points, 27.2 × 1012 IONPs per mL, 24.4 × 1012 IONPs per mL, and 26.1 × 1012 IONPs per mL for the assemblies Mc1-IONPs, Mc2-IONPs, and Mc3-IONPs, respectively (Fig.5D-F). A more detailed analysis of the stability area data also showed the maximal concentration (MC) of IONP for the different supramolecular assemblies (Fig.5G-I). This value, named as MC, indicates the maximal capacity of the amphiphilic molecules to embed and water-stabilize IONPs in the assemblies before reaching the aggregation area. The system Mc2-IONPs exhibited the highest maximal capacity for embedding IONPs, 18.1 × 1012 IONPs per mL, followed by the Mc1-IONPs and Mc3-IONPs with 13 × 1012 IONPs per mL and 7.5 × 1012 IONPs per mL, respectively. These results are consistent with the CMC values obtained for the oleylbased amphiphilic molecules, indicating that the bis-TEG polar head amphiphile II exhibits the best capacity for self-organization. Additionally, quantifying the embedded IONPs in the assemblies at the MA point allows us to calculate the efficiency of the amphiphilic molecules in the self-assembling well-ordered hybrid nanostructures. Amphiphile II showed the highest efficiency at 70%, followed by amphiphilic molecules I and III with 53% and 31%, respectively. The efficiency was calculated as shown in the Supporting Information. From the data above, we calculated the number of amphiphilic molecules per NP and per nm2 of NP. The supramolecular assemblies at maximal NP concentration exhibited 10 and 5 amphiphiles per nm2 for the Y-shape amphiphiles based Mc3-IONPs and Mc2-IONPs, respectively, and 10 amphiphiles per nm2 for the Mc1-IONPs. The stability of these hybrid supramolecular structures Mc-IONPs, obtained through the self-assembly methods discussed above, was measured over time at Camphiphiles < CMC. It was found that the HD sizes of the hybrid assemblies I and II remained unaltered for two months (Figure S24). However, the nanosystem Mc3- IONPs assembled by amphiphile III exhibited larger sizes after that time, forming small aggregates due to a poor hydrophilic/hydrophobic balance of the amphiphilic molecule (Fig.6). Interestingly, these results clearly demonstrate the formation of stable hybrid nanostructures through strong interdigitation of the amphiphiles into the IONP surfaces. Although the linoleyl-derived amphiphilic molecules IV and V are unable to self-organize into micelles, they form lipid nanoparticles. Studies were also performed on the NP-driven assembly of this family with a double alkene (cis, cis) in the alkenyl-based lipid tail (Fig.7). The hybrid systems LNP1-IONPs and LNP2-IONPs, based on the amphiphilic molecules IV and V, respectively, exhibited a stability area similar to that observed in the studies with their mono alkene amphiphilic analogues I and II. Interestingly, the HD sizes of 150nm and 220nm for LNP1-IONPs and LNP2-IONPs, respectively, were smaller than the resulting LNPs without the presence of IONPs. This indicates that the presence of IONPs during the self-assembly process serves as a template for the arrangement of these amphiphiles over the IONPs in small assemblies. On the other hand, the maximal acceptance NP point, referred to as the MA point, was determined by observing the internalization of IONPs in the assemblies within the stability area (highlighted in the coloured region in Fig.7A-B). As commented above, in the family of mono-alkene-based amphiphilic molecules, these MA points were determined by the minimum PDI value exhibited by the hybrid assemblies Mc-IONPs in the stability area. Since the double alkene (cis, cis) lipid-based amphiphilic molecules are not able to self-organize into well-ordered structures but form small aggregates as LNPs, the PDI values are not useful as an aggregation parameter. Therefore, the MA points of the hybrid LNPs were determined by the maximal embedding of IONPs in the stability area (Fig.7C-D). In both LNPs-IONPs, the amphiphilic molecules IV and V exhibited MA points of 48 × 1012 IONPs per mL and 34 × 1012 IONPs per mL for the assemblies LNP1-IONPs and LNP2-IONPs respectively, slightly higher values than their mono-alkene based amphiphilic analogues. Regarding the MC of NP, the hybrid nanosystems Mc- IONPs presented higher values, 175-fold greater than their linoleyl analogues, 7.4 × 1010 IONPs per mL and 4.3 × 1011 IONPs per mL for the LNP1-IONPs and LNP2-IONPs respectively. This significant difference is due to the greater rigidity of the double alkene (cis, cis) lipid tail, which limits and complicates their arrangement into more ordered and stable supramolecular structures. Moreover, TEM analysis of the assemblies LNP-IONPs within the stability area demonstrated the presence of small aggregates of IONPs, which indicates the formation of these structures (Fig.7E-F). Self-assembly in liposomes of amphiphilic molecules with mono alkenyl-based amphiphilic molecules The oleyl-derived amphiphilic molecules I, II and III also demonstrated the ability to self-organize into larger supramolecular structures, namely Lipo1, Lipo2 and Lipo3, respectively, compared to those commented above. In all cases, they exhibited similar HD sizes, between 336 and 368nm by DLS, which is likely due to the slight variation in composition used in the liposome formation process, specifically a low proportion of the amphiphile compared to oleic acid (at a ratio 5:95) (Fig.8). The presence of IONPs in the self-assembly process affected poorly to the size and morphology of the resulting liposomes Lipo-IONPs (Figure S25). The small size of the IONPs allowed them to be incorporated into the hydrophobic layer of the liposomes without significantly altering their size and shape. TEM analysis of the supramolecular structures, based on the amphiphile II, demonstrated the Scientific Reports | (2024) 14:28887 8 | https://doi.org/10.1038/s41598-024-79917-8 www.nature.com/scientificreports/ formation of liposomes without and with the presence of the IONPs (Fig.8A-C). These hybrid structures Lipo- IONPs exhibited high stability over time, with no observation of aggregates. Application of these hybrid supramolecular structures as MRI contrast agents Although the primary goal of this work is the construction of ordered hybrid supramolecular structures, we analyzed the capacity of these systems as MRI contrast agents. The effectiveness of an MRI contrast agent in decreasing the T2 relaxation time of surrounding water protons is quantified by a parameter known as transverse relaxivity, r2, expressed in mM− 1 s− 1. The hybrid systems Mc-IONPs and Lipo-IONPs, based on mono-alkene lipids, were chosen due to their better size arrangement and controlled morphology. Thus, the transverse relaxivity, r2, of the supramolecular structures formed through a micellar process was measured. The r2 values of the oleyl family of supramolecular structures Mc-IONPs exhibited a trend dependent on the thickness of the assemblies of the amphiphilic molecules over the IONPs. These results showed that the hybrid assembly Mc2- IONPs exhibited the highest r2 value for this type of structures, 6.2 mM− 1·s− 1 (Table1). This assembly showed the smallest size within the family, with the thinnest thickness of amphiphiles over the IONPs. However, the hybrid nanostructures Mc1-IONPs and Mc3-IONPs resulted in r2 values of 4.6 and 3.8 mM− 1·s− 1, respectively, exhibiting lower r2 values due to the thicker hydrophobic layer40. On the other hand, the hybrid liposomes Lipo-IONPs exhibited higher r2 values compared to the ordered hybrid structures Mc-IONPs commented above (Table1). This effect is likely attributed to the thinner thickness of the hydrophobic layer in which the IONPs are embedded. In fact, the Lipo1-IONPs and Lipo2-IONPs, based on mono-lipid amphiphiles, exhibited 1.5-fold and 2.6-fold higher r2 values, respectively. However, the Lipo3- IONPs, based on the bis-lipid amphiphile, collapsed during the measurements. Conclusions We have demonstrated that the presence of double bonds in the lipid tail of low MW amphiphilic molecules, with different morphologies in their design, can lead to highly water-soluble, stable and well-ordered hybrid Fig. 6. Stability studies after two months for the hybrid nanostructures Mc-IONPs. (A) Size vs. sonication time graph of the Mc1-IONPs in water obtained from toluene as apolar solvent. Size vs. [IONP]hexane graphs of (B) Mc1-IONPs, (C) Mc2-IONPs and (D) Mc3-IONPs. Initial measurements, t = 0 (in black) and after two months (in red). (E) Summary table of the Mc-IONPs, obtained from toluene as starting apolar solvent, containing the sizes determined by DLS in water. Scientific Reports | (2024) 14:28887 9 | https://doi.org/10.1038/s41598-024-79917-8 www.nature.com/scientificreports/