Protein Nanotubes as Advanced Material Platforms and Delivery Systems

Protein nanotubes (PNTs) as state‐of‐the‐art nanocarriers are promising for various potential applications both in the food and pharmaceutical industries. Derived from edible starting sources like α‐lactalbumin, lysozyme, and ovalbumin, PNTs bear properties of biocompatibility and biodegradability. Their large specific surface area and hydrophobic core facilitate chemical modification and loading of bioactive substances, respectively. Moreover, their enhanced permeability and penetration ability across biological barriers such as intestinal mucus, extracellular matrix, and thrombus clot, make it promising platforms for health‐related applications. Most importantly, their simple preparation processes enable large‐scale production, supporting applications in the biomedical and nanotechnological fields. Understanding the self‐assembly principles is crucial for controlling their morphology, size, and shape, and thus provides the ground to a multitude of applications. Here, the current state‐of‐the‐art of PNTs including their building materials, physicochemical properties, and self‐assembly mechanisms are comprehensively reviewed. The advantages and limitations, as well as challenges and prospects for their successful applications in biomaterial and pharmaceutical sectors are then discussed and highlighted. Potential cytotoxicity of PNTs and the need of regulations as critical factors for enabling in vivo applications are also highlighted. In the end, a brief summary and future prospects for PNTs as advanced platforms and delivery systems are included.


Introduction
Nanocarriers refer to nanostructures used to encapsulate/load bioactive compounds or functional ingredients, increasing the solubility, dispersity, and stability of targeted molecules, achieving the purpose of controlled and sustained release, as well as targeted delivery of these bioactive agents. [1]ince most bioactive molecules or functional ingredients are sensitive to pH, temperature, and light, degradation and denaturation can affect their efficacy. [2]Therefore, research on nanocarriers has always been a hotspot for scientific research.Naturally formed nanotubes in bacteria, such as Bacillus subtilis and Escherichia coli, [3] and viruses like the tobacco mosaic virus, bacteriophage M13, T4 component proteins, and bacteriophage tail sheath protein, [4] along with microtubules formed through / tubulin heterodimer polymerization, [5] have been extensively investigated.Over time, various nanocarriers, including nanotubes, nanofibrils, nanogels, etc., have been developed. [6]Among these, nanocarriers with high specific surface areas, such as nanotubes, have attracted a great deal of Figure 1.Schematic overview of key topics discussed in the present review.The self-assembly process: Reproduced with permission. [14]Copyright 2022, Elsevier Ltd.The LbL deposition process: Reproduced with permission. [15]Copyright 2008, WILEY-VCH .Image of drug delivery systems: Reproduced with permission. [16]Copyright 2023, Wiley-VCH .7a] Copyright 2023, Elsevier Ltd.
attention due to their hollow cores, which provide abundant space and protective layers for bioactive substances. [7]Moreover, most proteins, including -lactalbumin (-lac), lysozyme, ovalbumin, and bovine serum albumin (BSA) have been shown to be able to form nanotubes under mild conditions. [8]8c,d] Nanotubes have also been produced from synthetic peptides, [10] though natural proteins possess several advantages compared to synthetic peptides including sustainability, biocompatibility, and biodegradability, as well as the possibility of practical use in the biomedical and pharmaceutical sectors.
8a,c-e,9a,11] For example, peptides were self-assembled into nanotubes by electrostatic interactions, hydrogen bonding, and - stacking. [12]Another approach of template-based LbL technique was also used for the fabrication of PNTs.In this method, a porous membrane is applied as a template for the alternating deposition of polycation and polyanion layers. [13]Ultimately, PNTs feature advantages such as: i) they can be formed by self-assembly processes under mild conditions, that is, neutral pH, relatively low temperature, and atmospheric pressure; ii) their building blocks can be constituted by natural dairy by-products, which are cheap, accessible, biocompatible, and sustainable; iii) they can be prepared at a large-scale and relatively low cost; iv) smart functional groups can be added at desired positions through chemical modifications.
The main topics discussed in this review are outlined in Figure 1.Specifically, we will comprehensively review PNTs including their building materials, preparation and characterization techniques, self-assembly mechanisms, and physicochemical properties.We will collectively review current applications, state-of-the-art PNTs, and recent progress in the field.The safety of PNTs will also be critically discussed, as this is central to the design of PNTs with specific properties as nanocarrier systems.This review will end with a summary and outlook on some of the potential applications of PNTs, such as biomaterials, delivery systems, and nano-devices.

Building Materials for PNTs
In this section, the building materials of PNTs will be discussed focusing on the versatile natural molecular bricks of proteins and their derived peptides.9a] Copyright 2006, Elsevier Ltd.C) The fabrication of long nanotubes (LNT), short nanotubes (SNT), and crosslinked short nanotubes (CSNT) in the presence of Ca 2+ via different conditions.6a] Copyright 2020, American Chemical Society.
of nanostructures, including nanotubes, [7a] nanofibrils, [18] and nano/microspheres. [19]Here, we will summarize the sources, functions, and advantages of proteins and their derived peptides, with a particular focus on those derived from milk, egg, plant, and other sources.Table 1 provides a comprehensive summary of the sources and types of raw materials used in the assembly of PNTs, including information on diameters, synthesis methods, and the main forces that govern the construction of PNTs.These building blocks offer many advantages, such as biocompatibility, biodegradability, and easy functionalization.Additionally, the use of natural materials can reduce the environmental impact of PNT productions compared to synthetic materials.

Milk Proteins
Milk proteins are a diverse group of proteins that are essential for human nutrition due to their varying molecular structures and functions. [38]They can be classified into two main categories: caseins and whey proteins.Caseins form highly hydrated micellar assemblies known as casein micelles due to their hydrophobic nature, [39] while whey proteins, which make up about 20% of milk proteins, have been extensively studied for their potential use in protein-based encapsulation and delivery systems.Among whey proteins, -lac and BSA have been found to form nanotubes with unique properties that make them suitable for various applications in drug delivery and biomedicine. [40]-Lactalbumin: -Lac is a globular protein derived from whey protein and the second most abundant whey protein.With a molecular weight of 14.3 kDa and containing 123 amino acids, [41] -lac is known for its ability to bind calcium ions and other metal ions, such as Zn 2+ , Mn 2+ , and Pb 2+ .Additionally, it plays a crucial role in lactose synthesis and has been reported to selectively induce apoptosis in certain tumor cells.[42] Notably, the partial hydrolysis of bovine -lac using the protease from BLP has been found to result in the formation of PNTs that coordinate with Ca 2+ ions (Figure 2A,B).[8a,9a] The length and rigidity of PNTs can be controlled by adjusting the formation conditions, resulting in the formation of long nanotubes (LNT) at a 30 mg mL −1 -lac concentration, short nanotubes (SNT) through sonication, Table 1.Summary of the types and sources of the primary building blocks of PNTs, along with the corresponding nanotube diameters, synthesis methods, and the key forces responsible for their assembly.

Self-assembly Hydrogen bonding [10e]
6a] Furthermore, -lac PNTs can also be self-assembled using the V8 enzyme and a high concentration of Ca 2+ at neutral pH. [43]These methods demonstrate the tunability of -lac PNT formation to obtain PNTs with specific properties suitable for diverse applications.
Bovine and Human Serum Albumin (BSA, HSA): BSA and HSA are two commonly studied serum albumins.BSA is a watersoluble globular protein with a molecular weight of 66.7 kDa and a radius of gyration (Rg) of about 3.05 nm. [44]Its structure is mainly determined by its 583 amino acids and 17 disulfide bridges between cysteines. [45]On the other hand, HSA is the most abundant protein in human blood plasma, with a molecular weight of 66.5 kDa. [46]It plays a crucial role in regulating osmotic pressure, maintaining pH balance, and transporting various ligands, such as fatty acids, bilirubin, and drugs, throughout the body. [47]The LbL deposition was used to fabricate nanotubes, by a technique involving the sequential adsorption of alternating layers of positively and negatively charged materials onto a porous template membrane. [48]This process relies on electrostatic interactions between the oppositely charged layers, leading to the formation of multilayered tubular structures. [49]Researchers have explored using BSA and HSA as building blocks for nanotube formation.For example, Maldonado and Kokini developed edible polyelectrolyte complex nanotubes using the LbL deposition method, in which BSA and sodium alginate (ALG) act as the positively and negatively charged biopolymers at pH = 3-4, respectively.8d] In addition, Qu et al. also synthesized various PNTs comprised of an alternate LbL assembly using a polycation as an electrostatic glue.They deposited positively charged poly-l-arginine (PLA) or polyethylenimine (PEI) and negatively charged proteins (such as HSA, ferritin, or myoglobin (Mb)) alternatively into a porous polycarbonate (PC) membrane to form PNTs. The template was then dissolved with CH 2 Cl 2 , resulting in the formation of PNTs (Figure 3B). [15]These PNTs have potential applications in drug delivery and biomedical imaging, among others.

Egg Proteins
Eggs are a highly nutritious food source that is widely consumed all over the world.Egg white is mainly composed of water, protein, and minerals, while egg yolk is rich in fat, cholesterol, and vitamins.Egg white proteins, which make up the majority of the protein content in an egg, have several unique properties that make them useful in various applications. [50]To the best of our knowledge, only lysozyme and ovalbumin were reported to form nanotubes under appropriate conditions.
Lysozyme: Lysozyme is a small protein found in egg white that has antimicrobial properties and is commonly used as a natural preservative in the food industry.Hen egg white lysozyme protein (14.5 kDa), which has 129 amino acids, is homologous to human lysozyme (although the primary sequence is significantly different) and the content of -helix secondary structure is 30−40%.8b,10b] ILQINS hexapeptide, which was identified in lysozyme left-handed helical ribbons and nanotubes, can  [8d] Copyright 2017, Elsevier Ltd.B) PNTs comprised of PLA or PEI with different proteins containing HSA, ferritin, and myoglobin.Reproduced with permission. [15]Copyright 2008, WILEY-VCH.
10b] Ovalbumin: Ovalbumin, another protein found in egg white, has several uses due to its versatility, including its ability to froth and form gels when heated. [52]8c] It is worth noting that the potential of other egg proteins to form nanotubes is yet to be fully explored, and further researches in this area could lead to the discovery of new and exciting applications for egg proteins.

Protein-Derived Peptides
Peptides, as smaller and less complex macromolecules compared to proteins, are typically composed of fewer amino acid residues, whose simplicity allows for more precise control over the design and synthesis of peptide-based self-assembled biomaterials.The smaller size also facilitates the study of structure-function relationships and the identification of key motifs responsible for self-assembly.Some peptides fabricated by hydrolysis of the native proteins can possess an intrinsic propensity to self-assemble into tubular nanostructures, which has vast potential in diverse domains including drug delivery, biosensor, and biocompatible electronics.
-Amyloid (A)-Derived Peptides: A peptides have gained increasing attention in recent years owing to their association with Alzheimer's disease.They have the ability to self-assemble into nanofibrils at various pH conditions. [54]The self-assembly of heptapeptides comprising the A(16-20) sequence KLVFF from A has been investigated as a model system. [55]A modified end-capped heptapeptide called CH 3 CONH-AAKLVFF-CONH 2 (CapFF) (Figure 4A) was used to investigate morphological transitions.10a,e,21] These findings are of significance for understanding the structural and functional properties of A peptides-formed nanotubes.Copyright 2011, WILEY-VCH.B) Schematic illustration of mechanism for fiber ribbon twisted into nanotubes upon screening the surface charge on AAKLVFF -sheets.55c] Copyright 2010, American Chemical Society.C) Cryo-TEM images of 1 wt% (14.5 mm) solutions of CapFF in 50 mm NaCl.Reproduced with permission. [21]Copyright 2011, American Chemical Society.D) TEM image of nanotubes stained by uranyl acetate.55a] Copyright 2008, American Chemical Society.

Synthetic Peptides
Synthetic peptides, also known as designed or engineered peptides, offer several advantages over natural peptides for various applications, such as design flexibility, enhanced bioactivity, and alike.As two main categories of synthetic peptides, oligopeptides and cyclic peptides representing diverse classes of self-assembled building blocks for the construction of nanotubes are reviewed and discussed here.
Oligopeptides: Dipeptides (Leu-Leu, Phe-Phe, Leu-Phe, and Phe-Leu) are first noted to form nanotubes via hydrophobic interactions. [56]10f,22,57] For example, Reches and Gazit demonstrated that a Phe-Phe dipeptide can form stable nanotubes that can be diffusion-filled by silver ions, casting silver wires inside. [22]The dipeptides with different chiral amino acids (D-Phe-D-Phe) can also form nanotubes at appropriate concentrations. [58]The solvent conditions of diphenylalanine play an important role in controlling assembly behavior to form nanotubes or nanowires.High freewater content caused by low ionic strength or a low concentration of solutes can enhance the formation of more energet-ically favored nanotubes rather than nanowires. [59]10f] Diphenylalanine with a noncoded, achiral, ,-dehydrophenylalanine residue can also form nanotubes. [60] Moreover, a new type of dipeptide with -alanine (-Ala-l-Xaa, Xaa = Val, Ile, Phe) can self-assemble into nanotubes. [25]Aromatic dipeptide nanotubes represent another class of highly organized nanostructures that can be selfassembled using vapor deposition techniques. [23]Terminally protected acyclic tripeptides (tert-butoxycarbonyl (Boc)-Tyr-Val-Tyr-OMe and Boc-Tyr-Ile-Tyr-OMe) can self-assemble into nanotubes in the crystal state. [61]Two series of bolaform hexapeptides are synthesized with different branching and hydrophobicity to form micrometer-long nanotubes with different diameters from 5 to 230 nm. [62]10e] In addition, the structural transition from nanotubes to ribbons in the A n K peptide system (A = alanine, K = lysine) was investigated by a simple thermodynamic model.The morphological difference between ribbons and tubes in various self-assembling peptide systems has been shown to be dependent on solution pH, solvent polarity, or peptide sequence. [64]Several surfactant-like peptides (A 6 D, V 6 D, V 6 D 2 , and L 6 D 2 , A = Ala, V = Val, L = Leu) can also form nanotubes with an average diameter of 30-50 nm. [24]Also, a 1D n-type nanotube fabricated by the bolaamphiphile constructed by imitation of 1,4,5,8-naphthalenetetracarboxylic acid dianhydride (NDI) with two equivalents of Boc-l-lysine.The diameter of nanotube is ≈12 nm with the thickness of the wall is ≈2.5 nm. [65]Oligopeptide-based PNTs have found practical applications in various fields, such as material science and tissue engineering.For example, nanotubes self-assembled from dipeptide consisting of flexible -amino acid derivatives, such as Phe-Phe and Phe-,-dehydrophenylalanine, have been used as templates for the synthesis of gold nanoparticles and scaffolds for the growth of cells in tissue engineering applications. [66]Moreover, L-glutamic acid-based bolaamphiphile can also form nanotubes, and the self-assembly pathway is directed by a combination of hydrogen bonding and hydrophobic interactions. [67]yclic Peptides: Cyclic peptides are another class of peptides that have been explored for their ability to form nanotubes.Ghadiri et al. were among the first to demonstrate the development of cyclic peptide nanotubes, which showed that peptides from -sheet-like structures can stack on each other and construct hollow nanocylinders.[26] Also, Ghadiri and colleagues designed peptide nanotubes that not only enable the passage of ions but also facilitate their insertion into lipid bilayers.[68] Pouget et al. described the self-assembly pathway of lanreotide octapeptide into -sheet nanotubes and the role of two stable intermediates in this process.The structural features of these intermediates were related to the final nanotube organization as they set the nanotube wall thickness and curvature radius.[69] The development of a 12-residue cyclic peptide that was able to stack and form nanotubes with controllable pore sizes was also demonstrated, with the ring size of the peptide subunit being a determinant of pore size.[70] Cyclic peptides form nanotubes by stacking peptide rings through backbone-backbone hydrogen bonds between neighboring amide groups.Additionally, cyclic peptide nanotubes self-assembled from (Trp-D-Leu) 4 -Gln-D-Leu have shown potential for use in the delivery of antitumor drug 5fluorouracil.[71] Cyclic peptides have also been explored for their potential use in drug discovery and development due to their unique structural and physiochemical properties, including increased stability and bioavailability, as well as their ability to target specific protein-protein interactions and biological pathways.

Other Proteins
In addition to the peptides mentioned earlier, some other proteins have also been found to form nanotubes.The HIV-1 Rev protein, which plays a key role in the transfection of viral mRNAs from the nucleus, can assemble in vitro into regular, unbranched tubular structures with an outer diameter of 15 nm and an axial channel of 10 nm. [72]This suggests that PNTs could be a potential target for therapeutic intervention against HIV.Brodin and co-workers developed tetrameric protein building blocks called Zn 8 R 4 , which assemble into helical nanotubes via zinc coordination interactions.Three forms of nanotubes were developed, all of which had highly flexible structures despite their crystalline order due to the interactions mediated by metal coordination. [35]This approach opens new possibilities for the development of highly flexible PNTs with defined sizes and shapes for various applications.
Many plant proteins have also been explored for their potential as encapsulation systems.Among them, zein, a byproduct of corn with positive charges, has been reported to form nanotubes upon absorption of the protein on hydrophilic surfaces. [73]Zein is a promising biopolymer for drugs, foods, and nutrient applications due to its biodegradability and biocompatibility. [74]73a] Overall, PNTs hold great promise for various biomedical and biotechnological applications, including drug delivery, sensing, and tissue engineering.

Emerging Routes for Fabricating PNTs
PNTs have garnered significant attention in recent years due to their unique properties and potential applications in various fields, including biomaterials, drug delivery systems, and nanoelectronics.The ability to fabricate PNTs using emerging techniques has opened up new avenues for designing and controlling their physicochemical properties.These novel approaches include bottom-up self-assembly, template LbL, and enzymatic synthesis, among others.In this section, we will discuss the most widely used routes for fabricating PNTs, their advantages and limitations, and provide insights into future directions for the development of PNTs.

Self-Assembly
Self-assembly is a fundamental process that plays a critical role in the formation of PNTs.It involves the spontaneous organization of monomeric peptides or proteins into higher-order structures through non-covalent interactions.8b] In this section, we will review and discuss the main self-assembly mechanisms that contribute to the formation of PNTs.
-Lac has been reported to form PNTs through self-assembly.8a] The resulting PNTs have a diameter of about 21 nm and a cavity size of 8.7 nm. [75]The PNTs grow linearly with time until they reach the gel phase at a rate of 10 nm min −1 or one monomer unit per second.The PNTs are proposed to be composed of dimeric building blocks that selfassemble via template-assisted -sheet stacking perpendicular to the longitudinal axis. [76]Graveland-Bikker et al. demonstrated through atomic force microscopy (AFM) that partially hydrolyzed -lac can organize in a 10-start helix, forming tubes with diameters of only 21 nm. [75]The mechanical strength of these PNTs was also probed, revealing very stable and strong structures with potential practical applications. [75]Furthermore, the formation of tubular structures and transparent gels from partially hydrolyzed -lac was found to be dependent on the concentration of calcium.A minimum calcium concentration ratio (mole calcium per mole -lac) of 1.5 was necessary for the formation of tubular structures, while higher calcium concentrations (up to a ratio of 3) favored the tubular shape.9d] Calcium did not affect the hydrolysis kinetics but significantly influenced the kinetics of self-assembly, facilitating nucleation and accelerating gel formation.
Although new methods for preparing -lac PNTs have been developed, such as acid hydrolysis and V8 protease enzymatic hydrolysis method, [77] the mechanism of -lac PNT formation still requires further exploration and refinement.Recent studies have provided insights into the structural conformations of -lac during hydrolysis and nanotube growth using Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. [78]The conformational changes in the secondary structure of the protein, such as the dominance of -sheets (especially antiparallel), -turns, and random structures, have been observed during nanotube formation.In the helix domain, helix structures also appeared to be important besides -helix.Enzymatic hydrolysis exposes COO − groups at specific Asp and Glu sites, [6a,16,19a,79] where Ca 2+ ions are predicted to bind, leading to nanotube elongation.Further studies on the conformational changes of building blocks (BBs), the kinetics of helical ribbons, and the role of divalent ions will contribute to a better understanding of the formation mechanism of -lac nanotubes.Reproduced with permission. [14]Copyright 2022, Elsevier Ltd.C) Model schematic assembly of amyloid -derived peptides into nanotubes.10a] Copyright 2011, WILEY-VCH.
To study the formation mechanism of nanotubes, Zhang et al. developed a novel procedure that directed the self-assembly of BBs through enzymatic hydrolysis of -lac (Figure 5A). [14]They investigated the formation of nanotubes using peptides with similar secondary structures and revealed the involvement of BB dimers formed by hydrophobic interactions at 50 °C.The presence of Ca 2+ induces conformational change in the BBs, promoting -sheet stacking and electrostatic attraction between them.The electrostatic repulsion of carboxyl groups in BBs resulted in the formation of helical ribbons.10a] These PNTs exhibited a distinct hollow and right-handed helical structure.The elongation and aggregation of filaments in the longitudinal and lateral directions were dominated by caboxyl-Ca 2+ unidentate coordination with a binding stoichiometry of 1:1.The kinetics of nanotube formation, which can be determined using dynamic light scattering (DLS), also play a crucial role in elucidating the nanotube formation mechanism.Further studies focusing on the conformational changes of BBs, the kinetics of helical ribbons, and the influence of divalent ions will provide a deeper understanding of the formation mechanism of PNTs.

Template-Based LbL Deposition
In addition to self-assembly, another commonly used technique for the formation of PNTs is LbL deposition, [15,80] which involves the use of a porous template, such as aluminum oxide (AO) or PC membrane, to facilitate the formation of single or multilayer nanotubes. [81]11b,15] Lu et al. utilized adhesive and electrostatic forces to prepare HSA and L--dimyristoylphosphatidic acid (DMPA)/HSA nanotubes with desirable properties, such as monodisperse size distribution, uniform orientation, and high flexibility.80a] Collagen, a fibrillar protein, can also generate nanotubes through a template-based method combined with LbL deposition.Collagen multilayers with poly (styrene sulfonate) were Copyright 2010, American Chemical Society.B) A LbL deposition strategy for preparing PNTs within the pores of a nanopore alumina template membrane.80b] Copyright 2005, American Chemical Society.C) PNTs of BSA/ALG 3 fabricated by alternate LbL depositions of BSA and ALG into PC templates with pore sizes of 200, 400, 600, and 800 nm.8d] Copyright 2017, Elsevier Ltd.
synthesized onto the pores of a template with a pore size of 200-500 nm.This innovative strategy allows for control over the inside and outside diameters and lengths of nanotubes, providing an alternative to electrospinning. [82]SA, which is a positively charged biopolymer, can be used to fabricate edible complex nanotubes with negatively charged sodium ALG.The nanotubes are formed with the assistance of PC, and the most stable nanotubes are those with 4 bilayers [(BSA/ALG) 4 ] with a rate of addition of 1.0 mL min −1 and biopolymer concentrations of 0.8 and 0.6 mg mL −1 for BSA and ALG, respectively.8d] Cytochrome c (cyto-c), an important redox protein that acts as a key electron transfer agent during the cell respiration process, [83] can also be used to form nanotubes.Tian et al. employed LbL de-position to coat cyto-c and glutaraldehyde onto a polymer film, resulting in the formation of nanotubes that have potential applications as carriers of biocatalysts. [84]inally, glucose oxidase or hemoglobin nanotubes can be fabricated through LbL deposition using an AO template with 200 nmdiameter pores.Glutaraldehyde is employed to cross-link the protein multilayers, enabling the assembly of various proteins.Moreover, the thickness of the nanotubes can be controlled by adjusting the number of deposition cycles, resulting in enhanced enzymatic activity with increased nanotube thickness.However, it is important to consider the potential risk of protein denaturation during the fabrication process, which may impact the functional properties and stability of LbL-deposited PNTs.Nevertheless, LbL-deposited PNTs hold great potential in diverse applications, including biosensors, drug delivery systems, and bioelectronics.
Based on the content discussed above, the methods for preparing PNTs can be classified into two categories: self-assembly and LbL deposition.Self-assembly relies on the intrinsic properties of the proteins or peptides used and is susceptible to environmental factors.Achieving precise control over nanotube dimensions and properties during self-assembly can be challenging.On the other hand, LbL deposition requires the availability of suitable templates and often involves multiple deposition steps, making it a more intricate and time-consuming process.The removal of templates after nanotube formation can also be challenging, affecting the final nanotube structure and integrity.In summary, self-assembly offers simplicity, versatility, and the capability to precisely manipulate nanotube properties, while LbL deposition provides scalability, reproducibility, and the ability to incorporate diverse functional components.The choice of method depends on the specific requirements of the intended application, striking a balance between factors such as control, scalability, and complexity.Future research efforts should concentrate on deepening our understanding of the formation mechanisms, optimizing the methods employed, and exploring novel techniques to enhance the efficiency and versatility of PNT fabrication.

Characterizations and Physicochemical Properties of PNTs
PNTs have attracted considerable attention as a promising nanomaterial owing to their distinctive properties, including high aspect ratio, mono-dispersity and biocompatibility.The characterization and understanding of the physicochemical properties of PNTs are crucial for their potential applications in various fields.In this section, we will discuss the characterization of PNTs and their physicochemical properties.

Morphology and Structural Traits of PNTs
A variety of imaging techniques, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), AFM, and cryogenic electron microscopy (Cryo-EM), have been employed to investigate the physicochemical properties of PNTs.These methods offer valuable insights into the size, shape, surface structure, and internal organization of PNTs.
Additionally, fluorescent labeling coupled with confocal laser scanning microscopy (CLSM) has emerged as a novel approach to explore the construction of nanotubes (Figure 7C), [15,86] even the formation kinetics of PNTs. [15]AFM, known as one of the most versatile single-molecule techniques, is widely used to study the properties of nanostructures. [87]It not only provides morphological information about the nanotubes, such as shape, height, and periodicity, but also offers detailed insights into the formation kinetics of these structures. [88]For instance, Zhang et al. utilized AFM to investigate the self-assembly kinetics of -lac nanotubes, revealing a right-handed helical ribbon and providing detailed information on periodicity, helical angle, and ribbon width. [14]6b,89]

Physicochemical Properties of PNTs
Spectroscopic techniques, such as infrared spectroscopy (IR), Raman spectroscopy, and fluorescence spectroscopy, can provide insights into the chemical composition and properties of nanostructures, [90] including secondary structure and interactions with other molecules.Circular dichroism (CD) is an absorption spectroscopy method that is effective in studying the secondary structure of proteins and protein-based materials (Figure 7F). [14,91]Geng et al. found an increase in -sheet and a decrease in -helix structure of -lac nanotube during aggregation, as determined by CD, indicating the combined effect of hydrolysis of -lac and self-assembly in gelation. [92]FTIR is used to detect changes in different functional groups (Figure 7G). [14,93]he caboxyl-Ca 2+ ion coordination mode during the formation of -lac nanotube was investigated using FTIR, and the results indicated that the coordination was unidentate rather than bridging or bidentate. [94]Thioflavin T is a fluorescent probe that can monitor the increase in -sheet contents during PNT growth formation. [14,95]It was first described in 1959 for the detection of amyloid because of its specificity for -sheet structures, intense fluorescence signal, and micromolar or submicromolar affinity for amyloid fibers. [96]cattering techniques, as a series of experimental methods, among which X-ray diffraction (XRD), small angle X-ray scattering (SAXS), small angle neutron scattering (SANS), DLS, have been used to study the structure, properties, and behavior of PNTs.Mehta et al. found the powder XRD patterns of the A (16-22) peptide nanotubes can diffract typically H-bonded -strand and the -sheet stacking repeats based on the reflections at 4.7 and 9.9 Å. [97] SAXS is another scattering technique commonly used to determine the 3D structure of PNTs. [98]The data from SAXS scattering profiles can be used in conjunction with numerical models to extract structural information (Figure 7H).Structural information is commonly evaluated by least-squares  2 minimization.Graveland-Bikker et al. built a model of -lac nanotube based on SAXS with coexisting polydisperse sphere and polydisperse (10% polydisperse outer radius) hollow cylinders. [99]eng et al. applied the monodisperse hollow cylinder form factor to simulate the structure of -lac nanotube, which was fitted to closely match the experimental data. [100]10e] Additionally, DLS can also be used to characterize the stability of PNTs, and the fitting line based on DLS data can reflect the self-assembly growth kinetics (Figure 7I). [14]ecent advances in solid-state nuclear magnetic resonance (NMR) have permitted the de novo determination of the 3D structures of amyloid materials, including PNTs. [101]The changes in relative positions of the peptide -strand within PNTs were elucidated using solid-state NMR with 13 C- 15 N labeled peptides, confirming PNTs can form identical antiparallel, out-of-register sheets. [97]98e] Figure 7. Main characterization methods for PNTs.A) TEM image of -lac nanotube.Reproduced with permission. [14]Copyright 2022, Elsevier Ltd.B) SEM image of (PLA/HSA) 3 nanotube.C) CLSM images of (PLA/FITC-HSA) 3 nanotubes.Reproduced with permission. [15]Copyright 2008, WILEY-VCH.D,E) AFM height image and Derjaguin-Mueller-Toporov moduli of nanotubes.D,E) Reproduced with permission. [89]Copyright 2012, Royal Society of Chemistry.F) CD spectrum of the formation of -lac nanotube.G) FTIR of -lac nanotube.H) SAXS curve of -lac nanotube.I) DLS curve of -lac nanotube.F-I) Reproduced with permission. [14]Copyright 2022, Elsevier Ltd.

Other Properties of PNTs
The mechanical properties of nanostructures are also an important factor for their applications. [102]Studies have shown that PNTs have high rigidity and stiffness, with a persistence length on the order of micrometers.The mechanical properties of nanostructures can be further characterized by techniques such as AFM or rheology. [103]6b] Interfacial extensional rheology can also determine the stiffness of PNTs as a surfactant at the water-oil interface.9b] The stability and biocompatibility of biomaterials are important considerations for their use in most applications. [104]PNTs can be stabilized through covalent crosslinking, surface modifications, or encapsulation.Biocompatibility can be improved by using non-toxic protein building blocks and through rigorous testing for cytotoxicity and immunogenicity.9b] Chang et al. evaluated the aqueous solubility and stability of PNTs encapsulated with lycopene. [105]6a,16] Moreover, the hemolysis assay can also show the cytotoxicity and biocompatibility of PNTs. [16]n conclusion, PNTs are a versatile and promising class of advanced materials that have attracted considerable attention in various fields, including biomaterials, drug delivery systems, nanoelectronics, and food additives.The successful design and development of PNTs depends on a thorough understanding of their physicochemical properties, which can be characterized using various methods, including electron microscopy, XRD, and spectroscopy analysis.These characterizations can provide insights into the size, shape, surface charge, stability, and other properties of PNTs, which are critical for their performance and applications.Moreover, the physicochemical properties of PNTs can be tuned by changing their building materials, preparation methods, and self-assembly mechanisms, which further increase their versatility and potential applications.More advanced  [6a] Copyright 2020, American Chemical Society.D) In vivo delivery efficiency (Eff) predicted by in vitro experiments and coarse-grained molecular dynamics simulation.6b] Copyright 2021, The Authors, Published by American Association for the Advancement of Science.
instruments, such as cryo-electron microscopy (cryo-TEM) and ultra-high resolution confocal microscopy, [106] are also expected to be used for nanotube characterization.Overall, the characterization and understanding of the physicochemical properties of PNTs will undoubtedly contribute to the continued development of these promising nanomaterials.

Current Applications of PNTs
In this section, we will discuss the current applications of PNTs, which have laid the groundwork for their potential practical applications.Currently, most applications of PNTs are focused on their possible use as delivery systems in the food and pharmaceutical sectors, as their large specific surface area and cavity structure make them capable of transporting charged or hydrophobic bioactive substances through electrostatic or hydrophobic interactions.

Encapsulation of Bioactive Molecules
PNTs show great potential in drug delivery, as their unique size and shape make them easily taken up by target cells.Addi-tionally, the interior of PNTs can be modified to carry different types of drugs or imaging agents, enabling targeted drug delivery to specific locations in the body, potentially improving treatment outcomes and reducing side effects.Bioactive ingredients, such as carotenoids, have attracted a lot of attention due to their health benefits such as antioxidant, anti-obesity, and antiinflammation properties. [107]However, the low solubility, stability, and bioavailability of these compounds have limited their applications. [108]6a] Subsequently, these nanotubes were largely endocytosed by intestinal epithelial cells.The study findings underscore the potential of short, flexible PNTs as a promising platform for enhancing the oral delivery of bioactive compounds.These PNTs exhibited a remarkable capability to efficiently permeate the intestinal mucus barrier, thereby improving the bioavailability and therapeutic efficacy of the administered compounds.6b] They created models based on both experimental data and simulations to investigate how physical properties affect each step of the delivery process and overall efficiency.6b] Furthermore, a PNTbased composite microsphere delivery system exhibited excellent mucoadhesive and mucus-penetrating properties, improving the bioavailability of hydrophobic capsaicin and maintaining the homeostasis of gut microbiota. [109]Overall, PNTs hold great promise for drug delivery in the medical industry due to their unique properties and versatility.Further research and development in this area are necessary to fully realize their potential for clinical use.

Molecular Capture and Virus Trapping
The group of Komatsu employed an approach for a combination of negatively charged proteins (like HSA and ferritin) and positively charged polycations (including PLA, PLG, PLL, and PAH) through LBL depositions. [110]This method created customizable capture and release systems within PNTs, which find promising applications as biological separation devices in the biomedical field.
For example, when alternating the LBL assembly of PLA and HSA within a PC membrane and depositing concanavalin A (ConA) as the final layer, it can capture fluorescein isothiocyanate (FITC)-labeled dextran. [111]In addition, the (PLA/HSA) 3 PNTs serve as efficient captors for HSA ligands, such as zinc(II)protoporphyrin IX (ZnPP) and fatty acids, exhibiting potential applications in blood analysis in biological systems. [112]110b,113] These various applications highlight the great potential of PNTs in molecular capture and virus trapping.

Applications of PNT in Food Fields
In this section, we will discuss potential applications of PNT in the food fields and current limitations.The use of PNTs in functional food development is an area of active research, and their potential applications are still being explored.One potential application of PNTs is as nanocarriers for food bioactive ingredients, such as vitamins, minerals, and antioxidants.By encapsulating these compounds in nanotubes, they could be protected from degradation during processing and storage, and their bioavailability could be improved.6a] This led to a significant improvement in the oral bioavailability of curcumin.6a] Liu et al. fabricated Pickering emulsions utilizing PNTs as the stabilizing agents and incorporated the bioactive compound curcumin as a model molecule within the oil phase of the emulsion (Figure 9A).7a,105] Due to their high aspect ratio, which could increase viscosity effectively thus PNTs could help improving the stability and texture of these foods, as well as provide a smooth mouthfeel and enhanced flavor release.Through hydrophobic interactions, lycopene, a light-sensitive compound, is encapsulated into PNTs, leading to a significant improvement in its photostability and resistance to oxidation (Figure 9B,C).When this delivery system is incorporated into milk beverages, it significantly enhances their viscosity and long-term storage stability (Figure 9D,E). [105]7a] However, as with any new food ingredient, it is important to address safety concerns before commercial use.The safety of PNTs needs to be thoroughly assessed through preclinical and clinical studies to ensure that they do not pose any harm to human health.Regulatory agencies such as the US Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) would also need to evaluate their safety and approve their use in food applications.Currently, there are no specific regulatory policies for the use of PNTs in food, which poses a challenge to their commercialization.Therefore, more research is needed to better understand the safety, toxicity, and potential risks associated with PNTs in food applications.

PNT as Templates for Nanowires in Nano-Electronics
PNTs have also gained attention as promising templates for the synthesis of nanowires in nano-electronics.PNTs are formed from natural or synthetic peptides or proteins, and their unique structural properties, including their nanometer-scale diameter, high aspect ratio, and tunable surface chemistry, make them ideal templates for creating nanowires with desired characteristics.By taking advantage of PNTs as templates, researchers can precisely control the growth and orientation of nanowires, as well as their composition and morphology.For example, researchers observed the self-assembly of a short peptide, the Alzheimer's A diphenylalanine structural motif, into firm and distinct nanotubes. [22]Besides the already discussed technique to cast silver nanowires from peptide nanotubes, [22] -lac nanotubes were also used as templates.Researchers degraded the -lac nanotubes with protease K, resulting in uniform silver nanotubes with a diameter of 20 nm. [43]Additionally, PNTs derived from dipeptides are employed in the on-site generation of gold nanoparticles capped with dipeptides. [25]This approach has potential applications in a wide range of fields, including electronics, sensors, and biomedical devices.

Other Applications
In addition, PNTs can also serve as microreactors and electrode designs.For example, PNTs were applied as microreactors  B-E) Reproduced with permission. [105]Copyright 2022, Elsevier Ltd.
Intriguingly, PNTs can also be modified with gold nanoparticles to act as catalysts for various chemical reactions. [116]These multifunctional PNTs have found applications in microreactors for an array of catalytic processes.Beyond catalysis, the application of PNTs extends to the field of energy storage.Specifically, PNTs self-assembled from diphenylalanine peptides have been ingeniously designed for developing electrodes. [23]6a] and B) human umbilical vein endothelial cells.6a] Copyright 2020, American Chemical Society.C) Effects of different PNTs on lactate dehydrogenase (LDH) release of red blood cells.B,C) Reproduced with permission. [16]Copyright 2022, Wiley-VCH.D) The hemocompatibility of PNTs tested on red blood cells in PBS.Reproduced with permission. [118]Copyright 2023, Elsevier Ltd.E) H&E staining of the organs of healthy mice treated with various PNT formulations.Reproduced with permission. [16]Copyright 2022, Wiley-VCH.

Biocompatibility and Biological Fate of PNTs
As PNTs gain increasing attention in the food and pharmaceutical industries, there is a growing demand for robust scientific research to support their safe and effective integration into these products.The evaluation of the biocompatibility and cytotoxicity of PNTs provides insights into their potential impact on the human body.Furthermore, understanding the mechanisms of cellular internalization is crucial in determining their bioavailability and overall efficacy in delivering bioactive compounds.In this section, we will discuss the biocompatibility and cytotoxicity of PNTs, shedding light on their interactions with biological systems and potential internalization mechanisms.

Biocompatibility and Cytotoxicity of PNTs
Evaluating the biocompatibility and cytotoxicity of PNTs is of utmost importance to ensure their safe integration into biological systems.104a,117] Therefore, investigating the biocompatibility and cytotoxicity of PNTs is essential and critical in assessing their suitability as a platform for applications such as drug delivery and tissue engineering in the medical fields.In recent years, several studies have been conducted to investigate the biocompatibility and cytotoxicity of PNTs, the results of these studies can guide the development of safe and effective  [118] Copyright 2023, Elsevier Ltd.
PNT-based materials for both food and pharmaceutical applications.6a,16,105] Moreover, PNTs have exhibited high biocompatibility, as they showed no hemolytic effect when tested on red blood cells (Figure 10C,D). [16,118]In vivo assays further confirmed the biocompatibility of PNT formulations, as no pathological damage was observed in healthy mice (Figure 10E). [16]Nevertheless, it is essential to conduct clinical trials to thoroughly assess potential toxic side effects and establish regulations for the use of PNTs in the food and medical fields.These trials are particularly important in addressing consumer concerns and ensuring the safe utilization of PNTs in various applications.

Biological Fate of PNTs
Ensuring the safety and efficacy of nanocarriers in drug delivery systems is crucial for maximizing the bioavailability and biological effects of loaded bioactive compounds.The interactions between nanocarriers and biological systems play a vital role in achieving this goal.6a,b,119] Overcoming the intestinal mucus layer, for example, is a significant challenge in oral drug delivery systems, as it acts as a primary obstacle to enterocyte absorption. [120]6a] Their findings suggest that soft PNTs with shorter lengths exhibit superior penetration through the mucus layer and enhanced cellular absorption, which is attributed to the rotational dynamics and movement of short PNTs within the mucus network.6b,16] These findings collectively indicated that PNTs hold great potential in nanocarrier design for effectively overcoming physiological barriers.By harnessing the unique properties of PNTs, such as their nanoscale size, tunable surface chemistry, and structural flexibility, researchers can develop innovative drug delivery systems that effectively penetrate and target specific tissues, leading to improved therapeutic outcomes.Cellular uptake is another crucial factor influencing the efficacy of nanostructure-based drug delivery systems and their biomedical applications. [121]Upon internalization by cells, a majority of nanoparticles are trapped in endocytic vesicles and transported to endosomes, where they undergo lysosomal fusion for degradation, resulting in insufficient drug release. [122]PNTs possess the ability to interact with various cell types, including intestinal epithelial cells, macrophages, and cancer cells, and can be internalized by these cells through endocytosis or other cellular uptake pathways.Understanding the mechanisms of cellular uptake of PNTs is essential for the design of effective drug delivery systems and other biomedical applications utilizing PNTs.Factors such as the size, shape, surface charge, and surface chemistry of PNTs influence their cellular internalization.For instance, Li et al. developed various types of protein nanocarriers loaded with mangiferin (Mgf), differing in size, shape, and rigidity, including nanosphere (NS), long nanotube (LNT), short nanotube (SNT), and cross-linked short nanotube (CSNT) (Figure 11).PNTs exhibited distinct cellular uptake mechanisms, primarily through macropinocytosis, enabling transport to the endoplasmic reticulum and Golgi apparatus escaping from the degradation at lysosomes therefore facilitating a higher cellular Mgf concentration.Furthermore, PNTs can overcome multi-drug resistance by inhibiting P-glycoprotein efflux and facilitating transport across in-tercellular tight junctions, promoting entry into the blood circulation. [118]

Summary and Outlook
In this review, we have discussed PNTs, starting from their building blocks and fabrication mechanisms to their current applications at the research level and beyond, including future prospects.Despite the potential of PNTs in various fields, the lack of efficient and cost-effective methods for their scale-up production remains a significant obstacle.Thus, further studies are needed to achieve a sustainable technology capable of producing PNTs on a larger scale.To translate PNTs into practical applications such as ingestion, injection, and topical applications, a rigorous assessment of their cytotoxicity is still required.A comprehensive evaluation of their cytotoxicity is necessary to assess their biocompatibility with the gastrointestinal digestive behavior and minimize cellular inflammation to achieve an overall beneficial effect.
The potential applications of PNTs in various fields have gained increasing attention in recent years, as PNTs show great potential in developing advanced materials for various purposes.Figure 12 provides an overview of the diverse applications of PNTs as biomaterials in medical and food industries, as well as their use as scaffolds or templates for nano-casting processes.In the field of biomaterials, PNTs have been studied for their biocompatibility and cytotoxicity, as well as their ability to promote cellular adsorption.These properties make PNTs promising materials for tissue engineering, drug delivery, and other biomedical applications.
In drug delivery systems, PNTs have been explored as nanocarriers for a variety of therapeutic agents, including small molecules, proteins, and nucleic acids.The ability of PNTs to protect the cargo from degradation and improve their bioavailability has been studied in vitro and in vivo.Moreover, the physicochemical properties of PNTs can be tailored to achieve the controlled release of drugs, which may lead to more effective and safer drug therapies.However, further studies are needed to investigate the pharmacokinetics and potential toxicity of PNT-based drug delivery systems.
Additionally, PNTs are currently being explored as food additives due to their multiple functionalities, and ability to improve the stability and bioavailability of active compounds in food.The use of PNTs in food products may lead to the development of functional foods with improved health benefits.However, more research is needed to ensure their safety and regulatory compliance.Moreover, PNTs can be used as templates for the synthesis of conductive nanowires and nanotubes, with potential applications in electronic devices and sensors.Future research should focus on optimizing the synthesis and functionalization of PNTbased nanostructures for specific electronic applications.
Despite the promising applications of PNTs, several challenges still need to be addressed in the field, including the development of scalable and cost-effective methods for PNT synthesis, the optimization of PNT properties for specific applications, and the comprehensive evaluation of the safety and toxicity of PNTs.Nonetheless, once and if these potential pitfalls are successfully solved, the future outlook for PNTs as advanced material templates appears to be shining: continued research in this field may accelerate the path to their innovative and appealing applications in a multitude of fields.
Bin Liu is currently a postdoctoral fellow at China Agricultural University.He received his Ph.D. degree in food science at China Agricultural University.He worked as a visiting postdoc at ETH Zurich for 1.5 years.His research is focused on the design and application of micro/nanocarriers derived from proteins and polysaccharides for precise delivery of bioactive compounds.

Figure 3 .
Figure 3. Preparation of various PNTs comprising of positively charged polycation and negatively charged proteins.A) PNTs with different diameters fabricated by BSA and ALG.Reproduced with permission.[8d]Copyright 2017, Elsevier Ltd.B) PNTs comprised of PLA or PEI with different proteins containing HSA, ferritin, and myoglobin.Reproduced with permission.[15]Copyright 2008, WILEY-VCH.

Figure 4 .
Figure 4. Nanotubes formed by protein-derived peptides.A) Molecular structure of the heptapeptide CapFF.Reproduced with permission. [10a]Copyright 2011, WILEY-VCH.B) Schematic illustration of mechanism for fiber ribbon twisted into nanotubes upon screening the surface charge on AAKLVFF -sheets.Reproduced with permission.[55c]Copyright 2010, American Chemical Society.C) Cryo-TEM images of 1 wt% (14.5 mm) solutions of CapFF in 50 mm NaCl.Reproduced with permission.[21]Copyright 2011, American Chemical Society.D) TEM image of nanotubes stained by uranyl acetate.Reproduced with permission.[55a]Copyright 2008, American Chemical Society.

Figure 5 .
Figure 5.The self-assembly of PNTs.A) Schematic illustration of self-assembly of -lac PNTs.B) AFM images of PNTs at different incubation times.Reproduced with permission.[14]Copyright 2022, Elsevier Ltd.C) Model schematic assembly of amyloid -derived peptides into nanotubes.Reproduced with permission.[10a]Copyright 2011, WILEY-VCH.

Figure 6 .
Figure 6.LbL deposition of several PNTs.A) Schematic illustration of PNTs prepared by LbL templating synthesis.Reproduced with permission. [11b]Copyright 2010, American Chemical Society.B) A LbL deposition strategy for preparing PNTs within the pores of a nanopore alumina template membrane.Reproduced with permission.[80b]Copyright 2005, American Chemical Society.C) PNTs of BSA/ALG 3 fabricated by alternate LbL depositions of BSA and ALG into PC templates with pore sizes of 200, 400, 600, and 800 nm.Reproduced with permission.[8d]Copyright 2017, Elsevier Ltd.

Figure 8 .
Figure 8. Kinematic diffusion properties of PNTs in biological mucus systems.A) The experimental mean-squared displacement (MSD) of five protein nanocarriers.B) The calculated MSDs for three PNTs.C) The calculated 3D trajectories of three PNTs.The scale is 10.Reproduced with permission.[6a]Copyright 2020, American Chemical Society.D) In vivo delivery efficiency (Eff) predicted by in vitro experiments and coarse-grained molecular dynamics simulation.Reproduced under the terms of the CC BY-NC 4.0 license.[6b]Copyright 2021, The Authors, Published by American Association for the Advancement of Science.

Figure 9 .
Figure 9.The encapsulation of hydrophobic ingredients in PNTs and potential applications in food systems.A) The construction of curcumin-loaded Pickering emulsion stabilized by PNTs and illustration of their postulated digestion behavior.Reproduced with permission. [9b] Copyright 2021, American Chemical Society.B) Aqueous solubility and stability of lycopene-loaded PNTs (NTs/LYC) or free LYC.C) The photostability LYC after loaded in PNTs.D) Viscosity and E) stability of milk with the addition of NTs/LYC at various concentrations.B-E) Reproduced with permission.[105]Copyright 2022, Elsevier Ltd.

Figure 11 .
Figure11.Illustration of various PNT formulations transported across epithelial cells through multiple cellular barriers.Reproduced with permission.[118]Copyright 2023, Elsevier Ltd.

Figure 12 .
Figure 12.A comprehensive overview of the current advances in PNTs for various potential applications.
Li received his B.S. degree from Jilin University in 2014 and his Ph.D. degree from Harbin Institute of Technology in 2021.He then joined the college of food science and nutritional engineering at China Agricultural University as postdoctoral fellow.His current research interests are protein self-assembly and their food and biomedical applications.Yuan Li is the professor of College of Food Science and Nutritional Engineering at China Agricultural University.She received her Ph.D. degree from the Lab of Physical Chemistry and Soft Matter at Wageningen University.Her research focuses on food colloid science and precise nutrition delivery systems.Her work explores the self-assembly of -lactalbumin nanotubes, revealing their remarkable mucus permeability and enhanced bioavailability for compounds like curcumin and polyphenols.She has also developed intestinal-targeted microgel platforms for functional proteins, oils, and probiotics, improving their stability and viability in challenging processing and physiological conditions.Raffaele Mezzenga's research focuses on the fundamental understanding of self-assembly principles in proteins, polymers, liquid crystals, food, and biological colloidal systems.He has co-authored more than 400 publications and 20 patents.His work has been recognized by several prestigious international distinctions, such as the 2017 Fellowship and the 2013 John H. Dillon Medal of the American Physical Society, the 2013 Biomacromolecules/Macromolecules Young Investigator Award of the American Chemical Society, the 2011 American Oil Chemists' Society Young Scientist Research Award, and the 2004 Swiss Science National Foundation Professorship Award.