Pattern-mediated supramolecular assembly of lipids into nanostructures

Abstract

A method for creating lipid supramolecular structures. The method includes preparing an ink made of an aqueous solution of lipid micelles, which is deposited on a polymer pen or an array of polymer pens, for example by electrospraying techniques, to achieve uniform coverage of single isolated micelles. The method further includes transferring the ink to a substrate using polymer pen lithography (PPL). The nanoconfinement of lipid micelles associated with the disclosed method allows the lipid micelles to rearrange, ultimately resulting in a highly ordered and uniform supramolecular lipid structure. Further disclosed are supramolecular assemblies created using the disclosed method and nanoscale delivery systems comprising lipid supramolecular assemblies.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 289935, filed December 15, 2021, which is incorporated by reference in its entirety herein.

[0002] This disclosure relates generally to patterned lipids and methods for making the same, which are based on nanoconfinement as a means to promote the self-assembly of such lipids into various secondary / tertiary structures. This disclosure relates generally to novel quaternary lipid structures such as nanopalms. [Background technology]

[0003] Self-assembly is a fundamental bottom-up process stimulated by nature and used to form higher order structures starting from a liquid disordered phase. While intramolecular self-assembly produces well-defined secondary and tertiary structures from polymers, instability exists in the corresponding lipid structures due to their molecular dynamics, which is often determined by the lipid composition and / or lipid geometry when present in a liquid environment. The ability of lipids to form quaternary structures via intermolecular self-assembly from secondary and tertiary structures via supramolecular assembly may govern the formation of highly stable lipid structures that are increasingly compatible with a wide variety of medical products ranging from organ self-targeting to mRNA delivery and CRISPER-Cas9 in-vivo gene editing to accelerate clinical trials.

[0004] Under appropriate conditions, lipids may be able to self-assemble into a variety of nano- / micro- / meso-scale structures, including micelles, spherical vesicles, nanotubes, nanoribbons (planar, wavy, and helical), spiral, or discotic liquid crystals. In 1975, Papahadjopouloset et al. reported that Ca ions form via electrostatic interactions and their subsequent transformation into large sheets. 2+The spiral shape was first discovered when observing the large fusion and structural rearrangement of unilamellar negative phosphatidylserine vesicles after the addition of glycerol. To minimize interactions with surrounding water molecules, the sheets could fold into a spiral to form a tertiary structure known as a lipid cochleate. In addition to sheets, ribbons are another secondary / tertiary structure that could undergo curling, adhesion, or fusion to form a spiral due to their large energetically unfavorable surface. Discotic liquid crystals consist of various multi-chain lipids such as triglycerides that can self-assemble into Y-structures with fully splayed chains (120° apart) upon increasing the temperature or addition of non-polar solvents. These splayed chains are loosely associated to form flexible columns / cylindrical rods in disk-like structures that self-assemble via "interdigitating" stacking. At first glance, the molecular self-assembly of lipids is fairly well understood; yet a significant portion of the formation of supramolecular lipid structures remains unexplained.

[0005] Patterning lipids on solid substrates in a spatiotemporally ordered manner could provide an ideal opportunity to study quaternary lipid structures. Polymer pen lithography (PPL) has been successfully used to create patterned features made of hard materials such as metals or soft materials including proteins, carbohydrates, oligonucleotides, and lipids, making it potentially useful in advancing the field of supramolecular chemistry. A typical polymer pen array contains thousands of pyramidal elastomeric pens made of polydimethylsiloxane (PDMS) to deliver different inks to desired substrates and allows for high throughput scanning cm with sub-100 nm resolution. 2It facilitates the development of superior patterning capabilities such as multiplexing over area. Nanometer and micrometer length scale features can be fabricated thanks to time- and force-dependent ink transport, ensuring uniform pattern development when in contact with the substrate after automated electrical leveling, extending the PPL tunability within the same pen array. Combinatorial libraries with tunable gradients of feature sizes can also be fabricated with individual pens by varying the residence time or relative z-piezo extension, or by deliberately tilting the pen array with respect to the substrate. PPL has proven to be a powerful strategy for synthesizing complex multi-element nanoparticles by patterning polymeric features, so-called nanoreactors, that are filled with metal precursors and converted into rigid structures of metal nanoparticles by post-patterning gas-heat treatment. However, the generation of soft nanomaterials via PPL-defined nanoconfinement remains unexplored. Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need for improved methods and associated systems for generating soft nanomaterials via PPL-defined nanoconfinement. The present method, which describes the effect of patterned lipid nanoconfinement on promoting self-assembly into various secondary / tertiary structures when brought into close proximity, is directed to overcoming one or more of the problems described above and / or other problems of the prior art. [Means for solving the problem]

[0007] A method for forming lipid supramolecular structures is described. In one embodiment, the method includes preparing an ink containing an aqueous solution of lipid micelles that form the primary lipid structures, and performing polymer pen lithography (PPL) by electrospraying the lipid micelles uniformly onto one or more pens, or even onto an array of pens. In one embodiment, electrospraying is performed with optimized parameters including spray distance, flow rate, and spray voltage to ensure micelle uniformity along with uniform coverage of the pen array. In one embodiment, PPL is performed in attoliter volumes via a nanoscale tip to achieve nanoconfinement of lipid micelles and rearrangement of lipid micelles as they are printed onto a substrate. Rearrangement of lipid micelles forms patterned lipid secondary structures.

[0008] In one embodiment, the method described further comprises performing at least one post-printing treatment on the patterned lipid secondary structures to form quaternary structures as they pass through the intermediate patterned lipid tertiary structures. For example, a process is described in which the patterned lipid secondary structures are chemically treated with a solvent. In one embodiment, the solvent is a mixture, such as chloroform (CHCl 3 ) / ethanol (EtOH) / double distilled water (DDW) (3:1:1) v / v%.

[0009] In one embodiment, following the chemical treatment step, the patterned lipid secondary structures are subjected to ultrasonication and mechanical agitation to form the lipid tertiary structures via intramolecular self-assembly of the patterned lipids. The method further comprises subjecting the intramolecularly self-assembled lipid tertiary structures to continuous stirring and simultaneous sonication with a solvent, such as CHCl. 3 and performing at least one treatment step by complete evaporation of EtOH to form a patterned lipid quaternary structure via intermolecular self-assembly of lipids.

[0010] In one embodiment, the supramolecular assembly of lipids produced by the disclosed method is disclosed. For example, the supramolecular assembly can be in the form of nanostructures such as nanoperms, discotic liquid crystals, vortexes, or combinations thereof.

[0011] In another embodiment, a nanoscale delivery system is disclosed that includes the lipid supramolecular assembly disclosed herein. The disclosed nanoscale delivery system can be configured to deliver pharmaceutical ingredients, proteins, genes or portions thereof, chemicals, nutrients, foods, pesticides, or combinations thereof.

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate disclosed embodiments and, together with the description, serve to explain the disclosed embodiments. [Brief description of the drawings]

[0013] [Figure 1] Figures 1(a) and 1(b) show a preparation scheme according to one embodiment involving a gold (Au) substrate primed with 1-pentanethiol (PTT) and an inked pen array. Figures 1(c) and 1(d) show scanning electron microscope (SEM) characterization of a pen array inked via electrospray (Figure 1(c)) and a pen array inked after wet-out (Figure 1(d)). [Diagram 2] 2(a)-2(d) show polymer pen lithography (PPL) writing schemes and their corresponding patterned lipid structures at different forces for embodiments disclosed herein. [Diagram 3] 3(a)-3(c) are scanning electron microscope (SEM) images of supramolecular lipid structures prepared according to the present disclosure. [Figure 4]Figure 4(a) shows SEM micrographs of nanoperm and its components [sheet cochleates (SCs) and ribbons], Figure 4(b) shows a close-up view showing the SCs (annotated with white circles), ribbons (annotated with white stars), and nanoperm (annotated with white arrows), while Figure 4(c) shows a counts vs. structure histogram verifying the high structural uniformity of nanoperm. [Diagram 5] Figures 5(a)-5(c) show the characterization of an embodiment of lipid nanoperm by transmission electron microscopy (TEM) [Figures 5(a) and 5(b)] and scanning electron microscopy (SEM) [Figure 5(c)]. [Figure 6] Figures 6(a)-6(c) show scanning electron microscopy (SEM) characterization of lipid ribbons printed using PPL on a gold (Au) substrate primed with 1-pentanethiol (PTT) [Figures 6(a) and 6(b)] and in solution after emulsification of the printed lipid ribbons [Figure 6(c)]. [Figure 7] Figure 7(a) is a schematic diagram showing a proposed mechanism for lipid nanoperm formation according to the present disclosure, and Figures 7(b)-7(g) are scanning electron microscope (SEM) micrographs of embodiments of nanoperm suspensions according to the present disclosure. [Figure 8] Figures 8(a)–8(d) show the transmission electron microscopy (TEM) characterization of the sheet [Figure 8(a)], ribbon [Figure 8(b)], SC [Figure 8(c)], and ribbon wrapping the SC to form nanoperms [Figure 8(d)]. [Figure 9] 9(a)-9(f) are scanning electron microscope (SEM) micrographs showing nanoperm suspensions according to the present disclosure. [Figure 10] FIG. 10 is a schematic diagram showing force-mediated fabrication of patterned disks, sheets, and sheets and disks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] definition As used herein, "patterning-mediated self-assembly" refers to self-assembly between molecules within patterned nanoscale features upon printing via nanolithography.

[0015] "Inking" as used herein refers to the uniform deposition of single, isolated droplet particles of submicron size upon application of an electric field to a liquid passing through a narrow capillary. Inking using electrospray is influenced by several process parameters including applied voltage, flow rate, injector-to-collector distance, and spray time.

[0016] As used herein, "nanocontainment" refers to the physical restriction of molecules within patterned attoliter features (nanoreactors) of the ink defined by the nanoscale tip size of the polymer pen. In one embodiment, the nanoreactors described herein cause nanoconfinement of molecules by restricting their movement.

[0017] As used herein, an "attoliter" is 10 -18 A unit of fluid measurement equal to the liter.

[0018] As used herein, "stability" refers to the preservation of certain properties, including shape, size, and structural composition.

[0019] As used herein, "highly ordered" refers to the production of uniform morphology as a result of rearrangement of primary, secondary, or tertiary structures in a hierarchy.

[0020] As used herein, "homogeneous" or "homogeneity" refers to the physical continuity of a substance with respect to structural similarity (i.e., uniform shape, uniform size, uniform composition).

[0021] Molecular self-assembly has provided a prominent means for the fabrication of well-defined and individual nano / micro structures through programmed arrangements of amphiphilic molecules driven primarily by electrostatic attraction and influenced by molecular chirality, elasticity of orientational order, or natural curvature. Hydrophobic interactions, H-bonding, and van der Waals attractions could also drive the self-assembly of amphiphilic molecules when they are dispersed in an aqueous environment at concentrations above their critical micelle concentration (CMC). Despite extensive studies of supramolecular self-assembly by others, understanding the rules governing this phenomenon remains elusive. A dynamic transition from one self-assembled structure to another occurs under certain conditions until a hierarchical supramolecular structure of enhanced stability is reached.

[0022] Various embodiments of the present disclosure are described herein with reference to the figures. The disclosed polymer pen lithography approach allowed the assembly of lipids under nanoconfinement. Figure 1(a) and Figure 1(b) show a preparation scheme according to an embodiment, including a gold (Au) substrate primed with 1-pentanethiol (PTT) and an inked pen array. In this embodiment, the scheme for Figure 1(a) shows an Au substrate primed with PTT, and Figure 1(b) shows a PDMS pen array inked with a lipid micelle ink. Figure 1(c) further shows SEM characterization of the pen array inked by electrospraying, with scale bars at 100 μm (left) and 10 μm (right). Figure 1(d) shows the pen array inked after 2 hours of humidification, with scale bars at 100 μm (left) and 10 μm (right).

[0023] Upon adhesion of the printed lipid micelles to the PTT-primed substrate and following their rupture due to hydrophobic interactions, when they were brought into close proximity, a nano-confinement effect promoted the self-assembly of lipid molecules into disks. Figures 2(a)-2(d) show PPL writing schemes and their corresponding patterned lipid structures at different forces for embodiments disclosed herein. In particular, Figure 2(a) is a disk, Figure 2(b) is a sheet, and Figure 2(c) is a sheet & disk. Scale bars are 50 μm (left image) and 10 μm (right image). Figure 2(d) shows the area (μm) of the patterned lipid structures as a function of printing force (mN). 2 ) is a plot showing

[0024] These patterned disks [Figure 2(a)] were transformed into various secondary / tertiary structures such as sheets [Figure 2(b)], sheets & disks [Figure 2(c)], or ribbons [Figures 6(a and 6(b)] through intramolecular self-assembly mediated primarily by force and moisture in the confined space. The structures were then transformed into the desired structures using a solvent mixture (CHCl) via sonication and mechanical agitation (shaking). 3 After their transfer into 1000 μm aqueous solutions of 100 μm to 1000 μm aqueous solutions of 100 μm to 15 ...

[0025] Figures 3(a)-3(c) are SEM micrographs of supramolecular lipid structures prepared according to the present disclosure. In particular, the following structures are shown at various scales; 100 μm-500 nm; the scale bar in the inset is 2 μm. 3Continuous stirring together with complete evaporation of EtOH triggered the formation of various supramolecular structures, including vortexes [Figure 3(a)], discotic liquid crystals [Figure 3(b)], and nanoperms [Figure 3(c)]. Hierarchical growth during controlled self-assembly resulted in novel highly ordered supramolecular structures through the explicit manipulation of the lipid structures printed via PPL. Indeed, supramolecular chemistry could open up new perspectives towards the realm of supramolecular materials in materials science.

[0026] Figure 4(a) is an SEM micrograph showing 7 SCs, 8 ribbons, and 90 nanoperms, which are clearly distinguished in Figure 4(b). Figure 4(c) is a count versus structure histogram presenting strong evidence of the high structural uniformity of the nanoperms, originally resulting from PPL-mediated lipid nanoconfinement.

[0027] 5(a) and 5(b) are TEM micrographs, while FIG. 5(c) is an SEM micrograph, of an embodiment of a nanoperm according to the present disclosure.

[0028] Figures 6(a) and 6(b) show lipid ribbons printed using PPL on a 1-PTT primed Au substrate. Figure 6(c) shows the lipid ribbons printed using a solvent mixture (CHCl 3 Figure 1 shows random stacking of printed lipid ribbons after emulsification in PBS / EtOH / DDW (3:1:1) v / v%).

[0029] FIG. 7(a) is a schematic diagram showing a proposed mechanism of lipid nanoperm formation according to the present disclosure. FIGs. 7(b)-7(g) are scanning electron microscope (SEM) micrographs of embodiments of nanoperm suspensions according to the present disclosure. For example, FIG. 7(b) shows a printed "sheet & disk" structure, while FIG. 7(c) shows fully transformed nanoperm into sheets and ribbons after high speed vortexing of a 3-week-old nanoperm suspension. FIGs. 7(d), 7(e), and 7(f) show partially transformed nanoperm, while FIG. 6(g) shows intact nanoperm according to the present disclosure.

[0030] Figure 8 shows transmission electron microscope (TEM) micrographs of an embodiment of a 3-week-old nanoperm suspension after high speed vortexing. For example, Figure 8(a) shows a fully deformed sheet of nanoperm, while Figure 8(b) shows a ribbon. Figure 8(c) shows the SC, while Figure 8(d) shows the ribbon encasing the SC to form nanoperm according to the present disclosure.

[0031] 9(a)-9(f) are scanning electron microscope (SEM) micrographs showing nanoperm suspensions according to the present disclosure. These figures show the characterization of nanoperm suspensions aged for 120 days. FIG. 9(a) shows intact nanoperm, FIG. 9(b) shows fully deformed (square area), partially deformed (*) and intact nanoperm (→), FIG. 9(c) shows a close-up of fully deformed nanoperm showing stable sheets and ribbons, FIG. 9(d) shows folded sheets, FIG. 9(e) shows unfolded sheets and FIG. 9(f) shows stacked ribbons. Analysis of the aged suspensions of nanoperm revealed the high stability of the resulting assemblies (sheets & ribbons) even after 120 days in relation to the same assemblies formed in solution due to cationic crosslinking. Favorable intramolecular interactions within the confined nanoreactors could stabilize the resulting self-assembled structures. This undergoes intuitive intermolecular self-assembly upon mechanochemical processing to generate a highly stable quaternary structure; nanoperms. However, in the absence of confinement, structural degradation may occur due to the metastability of the lipid structures formed.

[0032] Reported supramolecular lipid structures such as whorls and tubules derived from unrestrained self-assembly in solution suffer from heterogeneity or high polydispersity. This lack of homogeneity could be overcome by PPLs, which could facilitate patterning-mediated self-assembly by generating secondary / tertiary structures of high uniformity. Indeed, an engineering variable / design principle for generating uniform supramolecular lipid structures is proposed herein. It is known that molecules in confined spaces behave differently than in bulk. Nanoconfinement of lipid molecules thereby determines the structural shape, size, and stability by affecting their self-assembly spatially and temporally.

[0033] Other variables include controlled environmental parameters such as temperature and humidity, and instrumental parameters including force and printing time, which are primarily influenced by dwell time, pattern size, and z-piezo contact position. Such parameters govern the volume of delivered ink and its diffusivity, which affects the shape, area, and uniformity of the resulting patterned structures. Post-patterning processing is another principle of intense influence on the final supramolecular nanostructures. In one non-limiting embodiment, solvent, heat, and agitation all contributed to the folding, scrolling, and formation of highly uniform nanoperms. These cooperative and synergistic principles result in the patterning-mediated self-assembly of well-defined nanoperms. Indeed, the PPL-controlled generation of nanoperms via self-assembly provides a very powerful alternative to nanofabrication and nanomanipulation, circumventing the implementation of conventional procedures and providing a novel route to nanoscience and nanotechnology.

[0034] In one embodiment, the present disclosure describes the effect of nano-confinement of patterned lipids on promoting their self-assembly into various secondary / tertiary structures when brought into close proximity. Thus, a PPL approach was developed to self-assemble novel quaternary lipid structures; sheets and ribbons, termed "nanoperms", each predicted to be the supramolecular product of self-assembled secondary / tertiary structures. In one embodiment, controlling the printing force results in the creation of patterns of various structures, including disks, sheets, and sheets & disks, when lipid micelle inks are delivered to PTT-primed Au substrates under appropriate conditions, as shown in FIG. 10. Subsequent mechanical agitation and solvent evaporation of the developed "sheet & disk" structures together with sonochemical processing allowed their self-assembly into lipid nanoperms. EXAMPLES

[0035] The following non-limiting examples, which are intended to be illustrative, further clarify the present disclosure.

[0036] As shown in Figure 1(a), the Au substrate was plasma cleaned to remove organic contaminants and primed with an ethanolic solution of PTT for 24 h under static vacuum at ambient conditions. The PTT molecules could self-assemble on the Au substrate to form a monolayer arranged in a trans configuration with a tilt angle of approximately 20°–30°, known as n-alkanethiols self-assembled on the Au surface.

[0037] PTT modification gives the Au substrate hydrophobic properties, which affects the spreading behavior of the transferred ink on the substrate. Since the lipid concentrations chosen were above their CMC, lipid micellar inks were formed in aqueous solutions. Concurrently, the PDMS pen array was plasma cleaned to promote ink wetting prior to electrospraying of the micellar ink solution [Fig. 1(b)]. In one embodiment, electrospraying was the inking strategy used over spin-coating and drop-casting, as it resulted in uniform ink coverage [Fig. 1(c)]. In a typical experiment, the pen array was inked via electrospraying using a voltage of 8.4 kV, a flow rate of 1 mL / hr, a syringe-to-collector distance of 10 cm, and a duration of 10 min. This step is crucial to ensure the uniform coverage of the pen array along with the micelle uniformity. The inked pen array was then mounted on the instrument and then incubated for 2 h at a moderate temperature of approximately 32 ± 2 °C and 92 ± 2% relative humidity (RH) prior to the actual printing process. This step is necessary to facilitate the transport of the ink to the substrate by allowing hydration, spreading, and diffusion.

[0038] Figure 1(d) shows the synergistic effect of temperature and humidity on the ink spreading with pen coating. The phase transition temperatures (Tm) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are -17°C and -16°C, respectively. Hence, their mixture with cholesterol will certainly be in Lα liquid state at 32±2°C. DOPC is widely used as phospholipid ink in dip pen lithography or PPL due to its controllable spreading with RH ranging from 40 to 95% under ambient conditions. At RH higher than 70%, the phospholipid ink becomes fluid enough to coat the tip easily. Hence, temperature and humidity are parameters to be considered when controlling the ink mobility in a uniform manner.

[0039] Nanolithography Printing Design Printing was performed using 40,000 pen-to-pen 100 μm tip-spacing PDMS pen arrays at various printing forces (150, 230, or 420 mN) and pattern sizes (30×30 or 40×40 dot matrix per pen) with fixed applied voltage (5 V), current threshold (0.20 mA), dwell time (3 s), step size (0.7 μm), and other instrument parameters affecting the printing process including approach (100 μm / s) and retract speeds (30 μm / s). A temperature of 32±2° C. and a RH of 92±2% were used during the entire printing process.

[0040] Upon adhesion to the PTT-primed Au substrate, the printed lipid micelles tend to invert due to the hydrophobic nature of the modified Au substrate (Figure 10). Micelle deformation and rupture can then occur as a result of hydrophobic interactions between the inverted micelles and the substrate, followed by self-assembly of the lipid amphiphiles, a mechanism known as lipid vesicle dynamics on hydrophobic surfaces. Similar to molecular ink spreading behavior, the phospholipid ink can spread isotropically, adhere to the substrate, and then self-assemble when brought into close proximity, resulting in round printed features (discs) at a printing force of 150 mN, as shown in Figure 2(a). These results are consistent with previously reported data showing round features of printed phospholipids. These features tended to stack three-dimensionally on the substrate due to the slow spreading kinetics of phospholipids; nevertheless, they could spread laterally on hydrophobic surfaces to form thin homogeneous layers when printing was performed at high humidity (≥90%) and moderate temperature (26 ± 2 °C) for extended periods of time (≥1 h).

[0041] Increasing the printing force to 230 mN (b) and 420 mN (c) increased the tip-substrate contact area as well as the amount of ink delivered, allowing the lipid disks to fuse together to form other structures; sheets (pattern size 30 × 30) and sheets & disks (pattern size 40 × 40), respectively. Clearly, the implementation of diverse parameters within the same overall assembly program results in different self-assembly processes and hence different lipid assembly structures such as disks, sheets, or sheets & disks. Further SEM characterization of the patterned lipid structures can be seen in Figure 2(c). Being in close proximity thanks to the containment effect together with the applied force and humidity, the printed disks tend to come together and their outer bilayer leaflets can then coalesce together with their inner contents to enable their intramolecular self-assembly. Within the force threshold, a linear relationship can be observed between the printing force and the area of ​​the lipid structures at a fixed dwell time of 3 seconds and RH 92 ± 2%, as shown in Figure 2(d).

[0042] Apparently, generating structures of various areas and shapes is greatly influenced by force-dependent ink transport parallel to the force and moisture-mediated feature self-assembly. When using a pattern size of 30×30, the area of ​​the patterned sheet printed at 230 mN was larger than the patterned disk printed at 150 mN. Utilizing a pattern size of 40×40 and increasing the printing force to 420 mN, the areas of the patterned sheet and disk were even larger.

[0043] Another means of obtaining structures of different sizes and shapes is by adjusting the total time required for printing, which is related to various parameters including dwell time, pattern size, and best contact position (precise z-coordinate of piezo xyz motors at obtaining electrical contact of array and substrate after automated electrical leveling). By increasing the pattern size and fixing the dwell time with longer time per point (related to approach and retract speeds and set z-piezo contact position), the total printing time could be greatly increased, which affects the obtained shape and size. Indeed, it was possible to generate structures of different sizes and shapes by simply controlling the printing force and time. Table 1 summarizes the various experimental and instrumental parameters used for lipid ink printing.

[0044] [Table 1]

[0045] Another interesting lipid structure consists of disks fused to ribbons. As shown in Figures 2(a) and 2(b), these structures were obtained with some modifications to the instrument parameters used to obtain the sheets and disks, including the printing force (450 mN) and the z-piezo contact position (64 μm). A slightly shorter z-piezo contact position greatly influenced the total printing time (3.25 h) and allowed lateral fusion of the disks to ribbons in a highly humid environment (92 ± 2%). Indeed, the total printing time in combination with high humidity could drastically change the final shape of the obtained lipid structures. Thus, PPL offers excellent control over the spatial and temporal features of lipids.

[0046] Emulsions of assembled lipid structures CHCl with sonication (10 min) and mechanical agitation (20 min) 3Chemical treatment of the patterned lipid structures in a solvent mixture of CHCl / EtOH / DDW (3:1:1) v / v% promoted their transfer into the liquid phase and possible fragmentation into smaller structures while preserving their original shape. EtOH could easily penetrate the lipid bilayer and H-bond with the ester oxygens to reduce the interfacial tension of the lipid bilayer. Similarly, CHCl 3 could partition into different parts of the lipid bilayer and alter their interleaflet interactions. The combined effect of such solvent mixtures may cause structural curvature and compaction. To the same extent, sonication could alter bilayer curvature and disrupt large lipid structures by inducing local stresses via stable cavitation that creates a shear field. This mechanism is consistent with the effect of a shear field on generating smaller liposomes. CHCl under continuous stirring at 900 rpm at 50±5°C was 3 and fast evaporation of EtOH produced different supramolecular outputs for emulsified lipid structures. Well-known lipid structures such as the spiral or discotic liquid crystals shown in Figure 3(a) or (b) could be fabricated thanks to hierarchical self-organization via intermolecular self-assembly of disks or sheets, respectively. Similarly, CHCl in conjunction with emulsification of sheet & disk composite structures could be fabricated using the hierarchical self-organization technique. 3and slow evaporation of EtOH (48 h under continuous stirring at 900 rpm at 35 ± 5 °C) produced a novel uniform supramolecular structure, the shape of which resembles that of Adonidia Merrillii (also known as Christmas palm), shown in Figure 3(c), hence the term "lipid nanoperm". Each nanoperm is presumed to consist of numerous ribbons formed upon disk fusion that continue to wrap around the SC to form a tightly wrapped lower part while continuing to wrap around to form a loosely wrapped upper part. Figures 4(a) and 4(b) show the structural characterization of the nanoperm and its constituents, while Figure 4(c) represents a quantitative determination of the high structural uniformity of the nanoperm. The average outer diameters of the tightly and loosely wrapped parts of the nanoperm were 206 ± 20 nm and 267 ± 25 nm, respectively, while its total length was approximately 3 ± 1 μm. Further SEM and TEM characterization of the lipid nanoperm can be seen in Figures 5(a)-5(c). At first glance, the described PPL method relies heavily on printing high consistency disks with their continuous secondary / tertiary structures to produce different supramolecular entities with high uniformity. In contrast, the ribbon structures shown in Figures 6(a) and 6(b) when emulsified give rise to random stacking as shown in Figure 6(c), mainly due to the lack of chirality in the phospholipids used, which is a common driving force for ribbon scrolling.

[0047] The role of nanocontainment The disclosed findings suggest the ability to pattern various structures including lipid disks, sheets, sheets and disks, or ribbons when lipid molecules are brought into close proximity. This facilitated the fabrication of well-defined morphologies that may not exist in practice such as whorls, discotic liquid crystals, and nanoperms. The novelty of nanoperms directed the research direction to investigate their formation mechanism proposed in Figure 7(a). Emulsification and sonication of the sheet & disk structures shown in Figure 7(b) followed by complete solvent evaporation initiated the fusion of the disks into ribbons and sheets that folded into SCs, followed by the ribbons wrapping around the SCs while continuing to wind up to finally form nanoperms. The intermolecular self-assembly between these two secondary / tertiary structures, sheets and ribbons, was mostly mediated by hydrophobic interactions and H-bonds. To shed more light on the mechanism of nanoperms formation, a 3-week-old suspension was analyzed after vigorous vortexing, which forcefully deformed the nanoperms due to high shear stress. The fully deformed nanoperms shown in Figure 7(c) generated their secondary / tertiary structures; sheets (folded or unfolded) and ribbons (wrapped or unwrapped). Some nanoperms were partially deformed [Figures 7(d)-7(f)] to show ribbons torn along their direction of rotation, exposing the inner SC, whereas others remained fully intact, see Figure 7(g). These results together with the TEM images shown in Figures 8(a)-8(d) substantiate the proposed mechanism of nanoperms formation. Apparently, the SC formed enhances the wrapping of ribbons along their folding direction, since chirality, the usual stimulus for ribbon scrolling, is absent in the phospholipids used.

[0048] The aged suspensions were further examined after 120 days to determine the stability of the various lipid structures. Stable quaternary structures of nanoperms [Figure 9(a)] were observed along with their constituents; sheets & ribbons [Figure 9(b) and Figure 9(c)]. Folded [Figure 9(d)] or unfolded sheets [Figure 9(e)] and ribbon stacks [Figure 9(f)] could also be detected. Nanoconfinement apparently stabilizes the dynamic assemblies via strengthening intramolecular bonds in a humid environment. Indeed, the high stability of the formed lipid structures gives rise to excellent control over engineering principles adapted to the supramolecular assemblies of extremely stable nanomaterials. [Industrial Applicability]

[0049] Methods are disclosed for forming lipid supramolecular structures that can be used as nanoscale delivery systems. Indeed, a wide variety of products may be compatible with this invention. Non-limiting examples of such products include: Medical applications ranging from organ self-targeting to accelerate clinical trials, to delivery of imaging agents for diagnostic purposes, and drug delivery, including CRISPER-Cas9 for mRNA-based therapies and in vivo gene editing; · Delivery systems used in the petroleum industry for Enhanced Oil Recovery (EOR); Cosmetic formulations for improved topical delivery of cosmetic products; · Processing, packaging and development of functional foods to enhance homogenization techniques, protect food from physical damage, increase its shelf life and improve the nutritional value of food; · insecticide formulations to increase the efficacy and durability of insecticides while reducing the amount of active ingredient that needs to be used; · The coatings industry, reducing the cost of coatings while enhancing properties such as mechanical strength, smoothness and cleanliness; Thermal coatings to improve insulating properties; and · Packaging sector to enhance the quality of shock absorbers.

[0050] To describe such use, a method of creating lipid supramolecular structures is first described. In one embodiment, the method includes preparing an ink containing an aqueous solution of lipid micelles that form primary lipid structures, and electrospraying the lipid micelles to uniformly deposit them on one or more polymer pens, or an array of polymer pens, to ensure the formation of isolated and unique micelles that homogeneously cover the array, facilitating uniform deposition on a substrate using an array of polymer pens, for example, comprising one or more elastomeric pens made of polydimethylsiloxane (PDMS), by polymer pen lithography (PPL). As used herein, "array of polymer pens" refers to an array that comprises at least 10 pens, for example, at least 100 pens, or even at least 1000 pens, or more than 10,000 pens, or even 100,000 pens. Any of these values ​​can be used as endpoints to define the size of the array of polymer pens. For example, as used herein, an "array of polymer pens" refers to an array that includes at least 10 pens, such as at least 100 pens, or even at least 1000 pens, or more than 10,000 pens, or even 100,000 pens. In one embodiment, the array of polymer pens contains between 100 and 100,000 polymer pens, such as between 10,000 and 50,000 polymer pens.

[0051] PPL can be performed in attoliter volumes via nanoscale tips to achieve nanocontainment of lipid micelles. In some embodiments, the polymer pen array can be cleaned with oxygen plasma prior to electrospraying of the ink. In some embodiments, lipid micelles rearrange as they are printed onto the substrate to form patterned lipid secondary structures.

[0052] Non-limiting examples of secondary structures include disks, sheets, or combinations thereof. In one embodiment, the printing force of the PPL can be varied, for example, within the range of 150-450 mN, or even 200-400 mN, to change the shape of the secondary structure from a disk to a sheet. In one embodiment, the secondary structure includes a ribbon, a sheet, a disk, or combinations thereof, with the proviso that when the secondary structure includes a sheet or disk, they are larger in size than the primary structure.

[0053] The method further includes performing at least one post-printing treatment on the patterned lipid secondary structure. For example, the post-printing treatment can include sonochemical treatment, mechanical agitation, or a combination. In addition, after printing, the patterned lipid secondary structure can be treated with a solvent-containing mixture, such as CHCl. 3 / EtOH / DDW. In some embodiments, at least one post-printing treatment step on the patterned lipid secondary structures forms the patterned lipid tertiary structures via intramolecular self-assembly of said lipids.

[0054] Non-limiting examples of tertiary structures include wrapped ribbons, rolled sheets, or combinations thereof, provided that when said tertiary structures include wrapped ribbons or rolled sheets, they are smaller in size than the secondary structures.

[0055] The method described herein may further comprise performing at least one treatment step on the intramolecularly self-assembled lipid tertiary structure to form a patterned lipid quaternary structure via lipid intermolecular self-assembly. At least one treatment step may be performed on the secondary / tertiary structure to form a quaternary structure including solvent evaporation. In one embodiment, the rate of solvent evaporation may be adjusted to control the shape of the quaternary structure, such as nanoperm, discotic liquid crystal, vortex, or combinations thereof.

[0056] In one embodiment, electrospray may be performed using a voltage of 7.0-9.0 kV, a flow rate ranging from 0.5 mL / hr to 1.5 mL / hr, an injector-to-collector distance of 5-15 cm, and a duration of 5-15 minutes.

[0057] Prior to deposition, the aqueous solution of lipid micelles may be held at a constant temperature and humidity for a time sufficient to impart to the ink viscosity properties that allow it to uniformly coat the polymer pen array. In one embodiment, printing is performed at a temperature in the range of 30-34° C. and a relative humidity (RH) in the range of 90-94% RH.

[0058] As described herein, intramolecular self-assembly of secondary lipid structures can be mediated by nanoconfinement resulting from the physical restriction of molecules within patterned attoliter features of the ink defined by the nanoscale tip of a polymer pen.

[0059] As mentioned, the self-assembled lipids produced by the disclosed method are highly ordered and uniform supramolecular lipid structures that typically have a stability of up to 120 days.As a result, excellent nanoscale delivery systems are produced.Non-limiting examples of nanoscale delivery systems that can be produced from supramolecular assemblies are those that can be configured to deliver proteins, genes, or parts thereof, such as mRNA, DNA, or CRISPER-Cas9, pharmaceutical ingredients such as chemicals, nutrients, food, pesticides, or combinations thereof, to biological organs.

[0060] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure.

[0061] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated in the following claims.

Claims

1. 1. A method for forming a lipid supramolecular structure, comprising: providing an ink comprising an aqueous solution of lipids, wherein the concentration of lipids in the aqueous solution is above a critical micelle concentration (CMC) for forming a micellar ink of the lipids in said aqueous solution; depositing a micellar ink of lipids in an aqueous solution onto one or more polymer pens; printing a lipid micelle ink onto a substrate with one or more polymer pens using polymer pen lithography (PPL), where the printed lipid micelles rearrange during printing onto the substrate to form patterned lipid secondary structures; performing at least one post-printing treatment on the patterned lipid secondary structure, the at least one post-printing treatment step on the patterned lipid secondary structure forming a patterned lipid tertiary structure by self-assembly of said lipids; and performing at least one treatment step on the self-assembled lipid tertiary structure to form a patterned lipid quaternary structure by self-assembly of said lipids; A method comprising:

2. The method of claim 1 , wherein the depositing is performed using electrospray, and the polymer pen or pens are plasma cleaned with oxygen gas prior to electrospraying.

3. 3. The method of claim 2, wherein the electrospraying is performed with a voltage in the range of 7.0 to 9.0 kV, a flow rate in the range of 0.5 mL / hr to 1.5 mL / hr, and a duration of 5 to 15 minutes, and the injector to collector distance is in the range of 5 to 15 cm.

4. The method of claim 1 , wherein the post-printing treatment comprises chemically treating the secondary structure of the patterned lipids with a solvent.

5. The method of claim 1 , wherein the secondary structure of the patterned lipid is selected from a disk, a sheet, or a combination thereof.

6. 6. The method of claim 5, wherein the polymer pen lithography is performed with an array of polymer pens with a printing force sufficient to change the shape of the secondary structures from a disk to a sheet.

7. The method of claim 6, wherein the printing force is in the range of 150 to 450 mN.

8. 2. The method of claim 1, wherein the printing is carried out at a temperature in the range of 30-34° C. and a relative humidity (RH) in the range of 90-94% RH.

9. The method of claim 1 , wherein at least one post-printing treatment comprises sonochemical treatment, mechanical agitation, or a combination thereof.

10. The method of claim 1, wherein the secondary structures comprise ribbons, sheets, disks, or combinations thereof, and when the secondary structures of the patterned lipids comprise sheets or disks, they are larger in size than the primary structures.

11. The method of claim 1 , wherein the tertiary structures include wrapped ribbons, rolled sheets, or combinations thereof, and when the tertiary structures include wrapped ribbons or rolled sheets, they are smaller in size than the secondary structures.

12. 2. The method of claim 1, wherein at least one treatment step performed on the secondary / tertiary structures to form the quaternary structures comprises solvent evaporation.

13. 13. The method of claim 12, wherein the rate of solvent evaporation is adjusted to alter the shape of the quaternary structure, the shape being selected from nanoperm, discotic liquid crystal, vortex or combinations thereof.

14. The solvent is CHCl ３ 2. The method of claim 1, comprising a mixture of EtOH / DDW.

15. The method of claim 1 , wherein the self-assembly of the secondary lipid structures is intramolecular and mediated by nanoconfinement.

16. 16. The method of claim 15, wherein the one or more polymer pens have nanoscale tips that cause nanoconfinement of secondary lipid structures.

17. 10. The method of claim 1, wherein prior to deposition, the aqueous solution of lipid micelles is kept at constant temperature and humidity for a time sufficient to impart the desired viscosity characteristics to the ink.

18. The method of claim 1 , wherein the polymer pen comprises one or more elastomeric pens made of polydimethylsiloxane (PDMS).

19. 2. The method of claim 1, wherein the quaternary structure comprises an ordered, uniform supramolecular lipid structure having a stability of up to 120 days.

20. 10. The method of claim 1, wherein the polymer pen lithography is performed with an array of polymer pens comprising 500 to 50,000 pens in the array.

21. A supramolecular assembly of lipids prepared by the method of claim 1.

22. 21. The supramolecular assembly of claim 20, which is in the form of nanoperms, discotic liquid crystals, vortices, or combinations thereof.

23. A nanoscale delivery system comprising the supramolecular lipid assembly of claim 20.

24. 24. The nanoscale delivery system of claim 23, configured to deliver a pharmaceutical ingredient, a protein, a gene or portion thereof, a chemical, a nutrient, a cosmetic, a food, a paint, a pesticide, or a combination thereof.

25. 25. The nanoscale delivery system of claim 24, wherein the pharmaceutical ingredient is delivered to a biological organ.

26. The nanoscale delivery system of claim 24, wherein the protein, gene or portion thereof comprises mRNA, DNA, or CRISPER-Cas9.

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