Recombinant Reflectin Nanoparticles

Abstract

Provided are a reflectin polypeptide comprising an amino acid sequence sharing at least 70% sequence identity or at least 80% sequence homology with the amino acid sequence set forth in SEQ ID NO:1, a nucleic acid molecule encoding the reflectin polypeptide, a host cell comprising the nucleic acid molecule; recombinant reflectin nanoparticles comprising the reflectin polypeptide, a method for synthesizing recombinant reflectin nanoparticles, a substrate surface-functionalized with recombinant reflectin nanoparticles, and a skin care product comprising the recombinant reflectin nanoparticles.

Description

[Technical field]

[0001] One aspect of the present disclosure relates to a reflectin polypeptide. Another aspect of the present disclosure relates to recombinant reflectin nanoparticles. Another aspect of the present disclosure relates to surface-immobilized recombinant reflectin nanoparticles and methods of producing and immobilizing recombinant reflectin nanoparticles on surfaces. Another aspect of the present disclosure relates to skin care products comprising recombinant reflectin nanoparticles. [Background technology]

[0002] Cephalopods (octopuses, squids and cuttlefish) are the masters of camouflage in the animal kingdom. They use color change to dynamically control the morphology of dermal cells (chromatophores and iridophores) to modulate their body color and pattern. In fact, paralarvae of Sepioteuthis lessioniana (Bigfin reef squid) can produce highly complex yet dazzling body patterns from the moment they hatch. Signaling using reflective tissues is common in nature and typically serves important survival functions such as deterring predators, capturing prey and signaling. These iridescent light reflecting-refracting structures often rely on Bragg reflectors that exploit the periodic spacing and constructive interference of thin films of photonic crystals. Such tissues can typically be found in butterfly wings, peacock feathers or the specialized tapetum lucidum reflective tissue found in the eyes of some vertebrates.

[0003] The squid of the Loliginidae family (including Sepioteuthis lessionia) has the unique ability to dynamically regulate the iridescent properties of their skin by tuning and controlling the internal assembly and periodicity of breg-like reflectors made entirely of proteins called reflectins within iridophores. This is in contrast to the reflectors of other animals, which are composed of purine crystals. Previous studies have demonstrated that such dynamic photonic properties are regulated by phosphorylation / dephosphorylation of condensed reflectin nanoparticles in the reflectors. Phosphorylation of multiple tyrosine (Tyr), serine (Ser), and threonine (Thr) residues, controlled by the neurotransmitter acetylcholine (ACh), rapidly imparts negative charges to the positively charged reflectin, leading to charge neutralization and a reduction in nanoparticle size, ultimately resulting in a blue shift in the emission wavelength. This results in dynamic iridescence, i.e., angle- and wavelength-dependent reflection, resulting in a variety of vibrant colors.

[0004] The remarkable dynamic camouflage capabilities of cephalopods result from precisely orchestrated structural changes within the chromatophores and iridophore photocytes. This dazzling display of colour, still unmatched in synthetic coatings, is regulated by the expansion / de-expansion of reflectin nanoparticles, which alter the dimensions of the iridophore reflectors and control the light pattern according to Bregg's law.

[0005] Early studies on reflectin A1 suggested that the formation and self-assembly of reflectin nanoparticles occurs due to the presence of repeated motifs, however, this study did not use the complete sequence of the reflectin protein, instead only working with mutated and truncated sequences.

[0006] Reflectins are likely to have a wide variety of applications, and therefore there is a need for the provision of said reflectins. Summary of the Invention [Means for solving the problem]

[0007] In a first aspect, there is provided a reflectin polypeptide comprising an amino acid sequence sharing at least 70% sequence identity or at least 80% sequence homology with the amino acid sequence set forth in SEQ ID NO:1, wherein the reflectin polypeptide substantially retains the activity of reflectin B1 (SEQ ID NO:1).

[0008] In a second aspect, there is provided a nucleic acid molecule encoding the reflectin polypeptide described above. In a third aspect, there is provided a host cell comprising the above-described nucleic acid molecule, the host cell being a bacterial cell.

[0009] In a fourth aspect, there is provided a recombinant reflectin nanoparticle comprising a reflectin polypeptide as described above. In a fifth aspect, there is provided a method of synthesizing a recombinant reflectin nanoparticle as described herein.

[0010] In a sixth aspect, there is provided a substrate surface-functionalized with recombinant reflectin nanoparticles as described herein. In a seventh aspect, there is provided a method for immobilizing the above-described recombinant reflectin nanoparticles on a substrate.

[0011] In an eighth aspect, there is provided a skin care product comprising the recombinant reflectin nanoparticles described above. The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which: [Brief description of the drawings]

[0012] [Figure 1A] One-step purification of unconjugated S. lessoniana B1 reflectin (SlRF-B1) using strong cation exchange chromatography. Figure 1A shows a chromatogram of SlRF-B1 crude extract purification at pH 6.0, with the protein peak occurring between 21 and 27 min. [Figure 1B]One-step purification of unconjugated S. lessoniana B1 reflectin (SlRF-B1) using strong cation exchange chromatography (Figure 1B) shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the pooled protein peak, indicating high purity of the monomer and its dimer. [Figure 1C] Figure 1C shows the one-step purification of unconjugated S. lessoniana B1 reflectin (SlRF-B1) using strong cation exchange chromatography.C shows matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectra confirming the molecular weight and purity of the collected fractions. [Figure 1D] One-step purification of unconjugated S. lessoniana B1 reflectin (SlRF-B1) using strong cation exchange chromatography. Figure 1D shows the average particle size, measured by dynamic light scattering (DLS), of SlRF-B1 nanoparticles assembled for different concentrations of acetonitrile (ACN) before and after dibenzocyclooctyne (DBCO) conjugation. Error bars represent the final nanoparticle size averaged across different batches of produced samples (n = 50), not the size dispersity of individual samples; a color scheme of visible wavelengths from 380 to 780 nm was used as a reference for comparing comparable nanoparticle sizes. [Figure 2A] Figure 2: DLS monitoring and schematic diagram of different stages in the growth of DBCO-SlRF-B1 nanoparticles. Figure 2A shows DLS scattering plots and curve fittings of 360 nm and 660 nm DBCO-SlRF-B1 nanoparticles as a function of time, showing that the growth is rapid, saturating within a few minutes, with the particle size being fully developed by 30 minutes. [Figure 2B] Figure 2B shows DLS monitoring and schematic diagram of different stages in the growth of DBCO-SlRF-B1 nanoparticles. Figure 2B shows the growth of 660 nm nanoparticles and their respective polydispersity index (PDI), divided into four steps. [Figure 2C] Figure 2C shows DLS monitoring and schematic diagram of different stages in the growth of DBCO-SlRF-B1 nanoparticles. Figure 2C is a schematic diagram of nanoparticle growth shown in steps 1-4. Step 1: Growth is initiated by the addition of DBCO-sulfo-NHS ester. Step 1: Nanoparticles initially coalesce to form large anisotropic nanoparticles with increased PDI. Step 2: Equilibrium between inward Laplace forces and outward elastic energy results in significantly lower PDI and maximum achievable nanoparticle size. Step 3: Large unstable nanoparticles undergo digestive ripening or coalescence destruction resulting in smaller particles and therefore increased PDI. Step 4: Size-concentrated region of stabilized quasi-monodisperse DBCO-SlRF-B1 nanoparticles controlled by ACN concentration. [Figure 3A] Atomic force microscopy (AFM) and transmission electron microscopy (TEM) images showing the difference in surface profile of DBCO-SlRF-B1 nanoparticles in the fully coalesced state (FIG. 3A) and the binary arrested coalescence state (FIG. 3B). [Figure 3B] Atomic force microscopy (AFM) and transmission electron microscopy (TEM) images showing the difference in surface profile of DBCO-SlRF-B1 nanoparticles in the fully coalesced state (FIG. 3A) and the binary arrested coalescence state (FIG. 3B). [Figure 3C] Graph of the surface profile of a fully coalesced particle. The interface between two independent particles is sharp, as depicted by the black arrow, whereas the stopped coalescence shows a smooth transition between the two joined particles. [Figure 3D] A series of AFM and TEM images depicting various states of stopped coalescence, from dyads to tetrads. [Figure 3E] AFM phase contrast image of multiple self-assembled globules. [Figure 3F] A close-up of a single 200 nm nanoparticle, showing that each globule is approximately 20 nm in diameter. [Figure 3G]TEM image of 150 nm nanoparticles showing the contrast regions of the different globules. [Figure 3H] A digitally enhanced image of Figure 3G, with surface blurring, noise removal, and HDR toning to improve contrast and clarity of the interior globular elements. [Figure 4] Structural color and reflectance of DBCO-SlRF-B1 nanoparticle monolayer films using Langmuir-Schaefer deposition. The structural color of nanoparticles with average sizes of A) 170 nm, B) 240 nm, C) 270 nm, and D) 310 nm were observed under an optical microscope along with AFM images (center) and reflectance measurements (bottom). [Diagram 5] Structural color and reflectance of DBCO-SlRF-B1 nanoparticle monolayer films using drop-cast deposition method. Average nanoparticle sizes of A) 400 nm, B) 460 nm, C) 520 nm, and D) 660 nm drop-cast onto azide-functionalized wafers are shown along with the corresponding AFM topology image (second from top) and reflectance measurements (third from top) of the monolayer. The white spots and lines are scratch marks on the soft nanoparticle coating caused by repeated handling. E)-F) show the spectral shift due to hydration of the DBCO-SlRFB1 nanoparticle monolayer film at 460 nm. In the dehydrated state E), the monolayer film has a blue reflection color, whereas in state F), the reflection color is orange-red. Figure 5G shows reflectance measurements showing a spectral red shift due to hydration, with a peak reflectance at 700 nm. This shift is reversible and can be obtained multiple times without any change in the reflectance spectrum. [Figure 6]Figure 6 shows the effect of coumarin 343X azide and 5-FAM azide with absorbance at 430-500 nm on a 660 nm nanoparticle coating. Figure 6A shows a 660 nm nanoparticle coating with a blue structural color at 429 nm and an attenuated red peak at wavelengths of about 700 nm. Figure 6B shows a 660 nm nanoparticle coating conjugated with coumarin 343X azide for 1 hour, which exhibits a purple color due to a mix of red and blue wavelengths. Figure 6C shows that extending the conjugation time to 24 hours significantly suppresses the secondary resonant peak at 429 nm, with the red wavelengths dominating. [Figure 7] Liquid-liquid phase separation of SlRF-B1 protein in the presence of chaotropic agents. The concentration is below the critical concentration required to completely solubilize SlRF-B1. Coacervate microdroplets of SlRF-B1 are visible with a diameter of 1-3 μm under transmitted light microscopy. The inset shows a dense viscous protein phase (orange) at the bottom of the centrifuge tube after 2 days; the protein can only be resolubilized by heating above 80 °C. [Figure 8] FIG. 13 shows DLS intensity size distribution of DBCO-SlRF-B1 nanoparticles self-assembled from 10 mM (3-(N-morpholino)propanesulfonic acid) (MOPS) buffered to pH 7.0 with 20-35% v / v acetonitrile and 5 mM DBCO-sulfo-NHS ester. [Figure 9] Figure 14. Growth of 360 nm nanoparticles and their respective PDI. The pattern is not stochastic and shows similarities to the growth of 660 nm particles, where the largest average size corresponds to a decrease in PDI. Data was extrapolated to 800 seconds later. [Figure 10] TEM image of long chain coalescence necking between multiple particles observed when the coalescence process was stopped early after 10 minutes. The final average particle size for this particular sample was 280 nm. [Figure 11]Schematic of Langmuir-Blodgett and Langmuir-Schaefer deposition methods using sodium polytungstate as a carrier medium. Langmuir-Blodgett deposition was unable to form a monolayer due to a thin layer of sodium polytungstate between the sample and the wafer surface. A custom-designed Langmuir-Blodgett / Schaefer mini device was used to prepare Langmuir films on the wafer. The Langmuir-Schaefer deposition method was found to be more suitable for the fabrication of DBCOSIRF-B1 monolayer PAS. [Figure 12] Surface functionalization of wafers verified by contact angle measurements and ellipsometry. The thickness of each molecular layer was obtained by subtracting the thickness of the previous layer from the total thickness. A) Piranha etched wafer was used as a negative control. B) Using N-[3-(trimethoxysilyl)propyl]ethylenediamine (AEAPTMS). C) Using azide-dPEG®4-TFP. D) Fluorescent 6-FAM DBCO was used as a positive control to verify the presence of azide on the surface. E) Non-functionalized wafers did not fluoresce at an excitation wavelength of 488 nm. F) Green fluorescence was observed for wafers functionalized with 6-FAM-DBCO. [Figure 13] AFM of DBCO-SlRF-B1 nanoparticles with average particle sizes of A) 215 nm, B) 270 nm, and C) 320 nm, and D) 375 nm, along with their respective fast Fourier transform (FFT) images (bottom row), showing that all monolayers are arranged as photonic amorphous structures. [Figure 14]Thermally assisted colloidal self-assembly of 215 nm DBCO-SlRF-B1 nanoparticles on azide-functionalized 100 mm2 wafers at different temperatures. A) At room temperature, the nanoparticles are concentrated near the center of the wafer and a blue halo ring surrounds the sample. B) The coffee ring effect of the nanoparticles at 40 °C is seen as concentric rings that form during evaporation of the buffer. Structural color is visible. C) Self-assembly of the nanoparticles at 50 °C shows attenuated structural color with a pale blue hue in the lower half of the sample. D) No structural color was observed in the sample heated to 60 °C. The coating had surface irregularities with structural defects. [Figure 15] AFM images of DBCO-conjugated SlRF-B1 nanoparticles of various diameters drop-cast onto azide-functionalized 225 mm2 wafers at 40 °C and 23 °C. At higher temperatures, the large empty patches are likely due to rapid evaporation of acetonitrile forming convection pockets that prevent uniform distribution of the nanoparticles. Samples prepared at room temperature show improved dispersion and smaller interparticle spacing. No structural color was observed under direct illumination in any of the samples due to the high concentration and irregular arrangement of the particles. [Figure 16] Optical microscope images of A) 400 nm and B) 460 nm DBCO-SlRF-B1 monolayer PAS using the drop-cast deposition method to produce structural color on silicon wafers. The respective AFM images of the region where structural color is observed (α) show the topology of the monolayer film with small interparticle spacing of less than 1 μm, while the region without structural color (β) shows large interparticle spacing. [Figure 17] Figure 1 shows UV-Vis spectra of DBCO-conjugated and unconjugated SlRF-B1 nanoparticles at 400 nm and 600 nm in solution. The peak at 310 nm corresponds to the presence of DBCO. The peaks at 200 nm and 280 nm for unconjugated SlRF-B1 nanoparticles correspond to peptide bonds and aromatic amino acid signatures. DBCO-conjugated nanoparticles show a decrease in absorbance observed at visible wavelengths. [Figure 18] Figure 1 shows a custom-made setup to utilize track-etched membranes for large-scale desalting. The pore size of 100-800 nm allowed us to improve the polydispersity index by filtering out small nanoparticles. [Figure 19] FIG. 1 illustrates a custom-made wafer support platform for simultaneously functionalizing multiple 100 mm2 or 225 mm2 wafers. [Figure 20] Reaction scheme for functionalization of silicon dioxide surface and conjugation of SlRFB1-DBCO nanoparticles by copper-free click chemistry. A) Piranha-etched silicon dioxide surface has generated hydroxyl groups and is functionalized with [3-(2-aminoethylamino)propyl]trimethoxysilane at 130° C. for 16 hours. B) Azido-dPEG®4-TFP ester has specificity for free amine groups and is allowed to react for at least 4 hours. C) Free amine groups are further reacted with Azido-dPEG®4-TFP ester to generate click chemistry-compatible azide groups. D) SlRF-B1-DBCO nanoparticles are introduced to the surface and the copper-free click chemistry reaction is allowed to proceed for 24 hours. E) The “clicked” DBCO-SlRF-B1 nanoparticles form covalently bound 1,4-disubstituted triazoles with azide-functionalized silicon dioxide surface. [Figure 21] Comparison of non-functionalized coverslips (left) and functionalized coverslips (right) with APTES, 4-ethynylbenzoic acid or propiolic acid, and 1-azidomethylpyrene as a fluorescent probe excited with UV light to confirm successful functionalization. [Figure 22] FIG. 1 shows the reaction scheme of triethoxy(ethynyl)silane on a hydroxylated glass surface after piranha etching and fluorescent probe reaction with 1-azidomethylpyrene. [Diagram 23]FIG. 13 is an exemplary reaction scheme for protein nanoparticles with surfaces occupied by either carboxylic acids or amines that can be conjugated with azidoproylamine or azidoacetic acid by using EDC / NHS or DIC / HOBt chemistry. [Figure 24] Simple self-contained Langmuir-Blodgett and Langmuir-Schaefer setups using small glass petri dishes or 6-well polystyrene plates. The density of sodium polytungstate is adjusted accordingly based on the density of the sample and the type of O-ring used. The image on the left is a schematic of the setup with the substrate masked along the edges. The image on the right is the actual setup using a 6-well polystyrene plate. SlRF-B1 nanoparticles are contained within the FKM O-ring and exhibit a faint purple hue (in this example only) after the particles have been adequately compressed into a monolayer. [Diagram 25] FIG. 1 shows the wavelengths of UV-A, -B and -C. [Figure 26] FIG. 2 shows the absorbance of bulk titanium dioxide and zinc oxide at room temperature. [Figure 27] FIG. 1 shows the particle size dependence of UV-A and UV-B properties of TiO2. [Figure 28] FIG. 2 shows UV-Vis absorption spectra for TiO2 control and commercial sunscreens. [Figure 29] Figure 1 shows the UV absorbance of DBCO-conjugated reflectin nanoparticles (400 nm) in solution. The concentration of 0.3 mg mL-1 was diluted 250-fold (1.2 μg mL-1). The UV profiles of DBCO-sulfo-NHS ester and native SlRF-B1 were used as controls. [Diagram 30] 1-day cytocompatibility study of DBCO-SlRF-B1 (400 nm) and ZnO (250 nm) nanoparticles at concentrations of 2, 20 and 200 μL. DBCO-SlRF-B1 shows better keratinocyte tolerance at higher concentrations. [Diagram 31]Alamar blue staining of cell control and incubation with different concentrations of reflectin and ZnO nanoparticles. [Diagram 32] FIG. 13 shows keratinocyte cell uptake of 400 nm DBCO-SlRF-B1 nanoparticles confirmed by 5-FAM azide staining. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The following detailed description refers to the accompanying drawings, which show, by way of example, specific details and embodiments in which the present disclosure may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the present disclosure. Other embodiments may be utilized, and structural and logical changes may be made without departing from the scope of the present disclosure. Various embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments.

[0014] In one aspect, the present disclosure provides recombinant reflectin nanoparticles. Advantageously, by controlling the average size of the reflectin nanoparticles, the color of the film / coating made from the recombinant reflectin nanoparticles can be tuned. Thus, providing recombinant reflectin nanoparticles provides a biomimetic approach to color modulation. Furthermore, the recombinant reflectin nanoparticles can be substantially monodisperse and / or have a controllable size to mimic the photon response of iridophores. For example, it is possible to provide recombinant reflectin nanoparticles with tunable sizes ranging from about 100 to about 1000 nm. By immobilizing recombinant reflectin nanoparticles on a surface, it is possible to provide monolayer photonic structures with tunable structural colors, thereby enabling the fabrication of environmentally friendly, bioinspired color-changing coatings that mimic the dynamic camouflage of cephalopods.

[0015] The recombinant reflectin nanoparticles may include polypeptides similar to naturally occurring polypeptides from cephalopods. The naturally occurring polypeptides may be fully sequenced and then recombinantly expressed by bacteria, such as E. coli, prior to self-assembly. The naturally occurring polypeptides from cephalopods may include polypeptides called "reflectins", which are typically composed of conserved amino acid sequences. Each sequence may include a combination of standard and sulfur-containing amino acids. The light-interacting properties of reflectin polypeptides may be due to their ordered hierarchical structure and hydrogen bonds.

[0016] In one embodiment, the reflectin polypeptide is fully sequenced and recombinantly expressed. The fully sequenced reflectin polypeptide can be obtained from any member of the cephalopod family. In one embodiment, the reflectin polypeptide is obtained from Sepioteuthis lessioniana, and the reflectin polypeptide may be referred to as reflectin B1. The sequence of Sepioteuthis lessoniana reflectin B1 is identified in Table 1 as SEQ ID NO: 1.

[0017] A reflectin polypeptide of Reflectin B1 (SEQ ID NO:1) for use in the present disclosure can be any reflectin family member or homologue thereof that substantially retains the activity of Reflectin B1 (SEQ ID NO:1).

[0018] Thus, according to another aspect, (a) the amino acid sequence set forth in SEQ ID NO:1; (b) an amino acid sequence sharing at least 65%, preferably at least 70%, or 75%, more preferably at least 85%, and most preferably at least 95% sequence identity, or at least 80%, preferably at least 90%, and more preferably at least 95% sequence homology, with the amino acid sequence set forth in SEQ ID NO:1; (c) a functional fragment of (a) or (b); or (d) a reflectin polypeptide comprising or consisting of an amino acid sequence containing any one of (a), (b) or (c) as an essential component, Reflectin polypeptides are provided that substantially retain the activity of reflectin B1 (SEQ ID NO: 1). In some embodiments, amino acid sequences related to the repeat motifs of reflectin A1 are specifically excluded.

[0019] According to various embodiments, a reflectin polypeptide may be said to "substantially retain" the activity of reflectin B1 (SEQ ID NO: 1) if the monolayer structure of the recombinant reflectin nanoparticles on the surface exhibits 80% of the structural color activity shown herein at temperatures below 40°C.

[0020] According to various embodiments, the reflectin polypeptide comprises or consists of an amino acid sequence that is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.25% or 99.5% identical or homologous over its entire length to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the reflectin polypeptide has an amino acid sequence that shares at least 60, at least 65, preferably at least 70, at least 75, more preferably at least 80, and most preferably at least 90% sequence identity over its entire length with the amino acid sequence set forth in SEQ ID NO:1, or has an amino acid sequence that shares at least 80, preferably at least 90, and more preferably at least 95% sequence identity over its entire length with the amino acid sequence set forth in SEQ ID NO:1.

[0021] The identity of nucleic acid or amino acid sequences is generally determined by sequence comparison. Sequence comparison is based on the BLAST algorithm, which is established and commonly used in existing technology, and is performed in principle by correlating consecutive sequences (successions) of similar nucleotides or amino acids in nucleic acid sequences and amino acid sequences, respectively. Tabular association of related sites is called "alignment". Sequence comparison (alignment), especially multiple sequence comparison, is generally prepared using computer programs available and known to those skilled in the art.

[0022] This type of comparison also allows for a statement of the similarity of the compared sequences to each other. This is usually indicated as a percentage of identity, which is calculated relative to the reference sequence and its entire length. The term "sequence identity" refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a window of comparison. Thus, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of sites where identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matched sites, dividing the number of matched sites by the total number of sites in the window of comparison (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. In the context of amino acid sequences, the term "homology" more broadly interpreted also incorporates conservative amino acid substitutions (i.e., amino acids with similar chemical activity). This is because these amino acids usually have similar chemical activity in proteins. Thus, the similarity of the compared sequences can also be indicated as "percent homology" or "percent similarity". Indications of identity and / or homology may occur over an entire polypeptide or gene, or over only individual regions. Thus, homologous and identical regions of various nucleic acid or amino acid sequences are defined by sequence matches. Such regions often exhibit the same function. Such regions may be small and may encompass only a few nucleotides or amino acids. Small regions of this kind often perform functions essential to the overall activity of the protein. Thus, it may be useful to refer to sequence matches only to individual, and optionally small, regions. However, unless otherwise indicated, indications of identity and homology herein refer to the full length of the indicated nucleic acid or amino acid sequence, respectively.

[0023] All amino acid residues are generally referred to herein by their one-letter code, and in some cases their three-letter code, this nomenclature being well known to those of skill in the art and is used herein as understood in the art.

[0024] Reflectin polypeptides substantially retaining the activity of Reflectin B1 (SEQ ID NO: 1) according to the present application may contain amino acid modifications, in particular amino acid substitutions, insertions or deletions. Such reflectin polypeptides can be further developed, for example by targeted genetic modification, i.e. mutagenesis, and optimized for specific purposes or with respect to special properties, for example with respect to the ability to form nanoparticles and / or with respect to providing tunable structural color. The aim may be, for example, to introduce targeted mutations, such as substitutions, insertions or deletions, into known molecules in order to improve their ability to form nanoparticles and to show structural color in the form of monolayers. For this purpose, in particular the surface charge and / or isoelectric point of the molecule and thus its interaction with the substrate may be modified. The advantageous properties of the individual mutations, for example the individual substitutions, may complement each other.

[0025] In various embodiments, the reflectin polypeptide can be characterized as being derived from such a reflectin as an initial molecule by single or multiple conservative amino acid substitutions. The term "conservative amino acid substitution" refers to the exchange (substitution) of one amino acid residue with another, such that such an exchange does not result in a change in polarity or charge at the site of the exchanged amino acid, e.g., the exchange of a non-polar amino acid residue with another non-polar amino acid residue. Conservative amino acid substitutions in the context of the present disclosure include, for example, G=A=S, I=V=L=M, D=E, N=Q, K=R, Y=F, and S=T.

[0026] The reflectin polypeptide may be a recombinant reflectin polypeptide, i.e. a reflectin produced in a genetically engineered organism that does not naturally produce said reflectin polypeptide. The term "recombinant expression" as used herein refers to the expression of said reflectin polypeptide by recombinant DNA technology using a nucleic acid molecule. The nucleic acid molecules encoding the reflectin polypeptides described herein, and vectors containing such nucleic acids, in particular copy vectors or expression vectors, also form part of the present disclosure.

[0027] Thus, also provided is a nucleic acid molecule encoding a reflectin polypeptide as described herein. In some embodiments, the nucleic acid molecule may be included in a vector. The vector may further include a regulatory element for controlling expression of the nucleic acid molecule.

[0028] A "vector" is understood for the purposes of this specification as an element composed of nucleic acid, containing a nucleic acid contemplated herein as a characterizing nucleic acid region. A vector allows the establishment of said nucleic acid as a stable genetic element in a species or cell line over multiple generations or cell divisions. In particular when used in bacteria, a vector is a special plasmid, i.e. a circular genetic element. In the context of this specification, the nucleic acid contemplated herein is cloned into a vector. Vectors include, for example, those that are bacterial plasmids in origin, or mainly synthetic vectors, or plasmids with elements of widely different origins. With the aid of additional genetic elements present in each case, the vector can be established as a stable unit over multiple generations in the relevant host cell. A vector can be present extrachromosomally as an independent unit or can be integrated into a chromosome or chromosomal DNA.

[0029] An expression vector may include a nucleic acid sequence capable of replicating in a host cell, preferably a bacterium, that contains the expression vector and capable of expressing the contained nucleic acid in said host cell. Thus, in various embodiments, the vectors described herein also contain regulatory elements that control the expression of the nucleic acid encoding the reflectin polypeptide described herein. An example of such a vector may be a pET vector. Expression is particularly influenced by one or more promoters that regulate transcription. Expression can in principle be caused by the native promoter that is originally present in front of the nucleic acid to be expressed, but also by a host cell promoter placed in the expression vector, or by a modified promoter of another organism or another host cell, or by a completely different promoter. The expression vector can further be regulated, for example, by changing the culture conditions, when the host cell containing the vector reaches a certain cell density, or by the addition of certain substances, in particular activators of gene expression. An example of such a substance is the galactose derivative isopropyl-β-D-1-thiogalactopyranoside (IPTG), e.g., the T7 promoter.

[0030] In some embodiments, the isoelectric point of the reflectin polypeptide can be greater than 7, or greater than 8, or about 8-10. Advantageously, such high isoelectric points provide an opportunity to modulate the zeta potential, and therefore the colloidal properties, by screening buffer types and additives during the purification (e.g., dialysis) process. For example, during the dialysis step, the following criteria can be simultaneously achieved: (i) mitigation of aggregation, (ii) control of nanoparticle size, (iii) narrow size distribution, and (iv) particle stability.

[0031] The expressed reflectin polypeptide may be allowed to self-assemble into nanoparticles, thereby forming a recombinant reflectin nanoparticle. "Nanoparticle" refers to a particle having a characteristic length, e.g., diameter, in the range of less than 1000 nm. The recombinant reflectin nanoparticle may be of a regular or irregular shape. For example, the recombinant reflectin nanoparticle may be a sphere, a rod, a cube, or an irregular shape. The size of the recombinant reflectin nanoparticle may be characterized by its average diameter. As used herein, the term "diameter" refers to the maximum length of a line segment passing through the center of the shape and terminating at the perimeter. The term "average diameter" refers to the average diameter of the nanoparticles and can be calculated by dividing the sum of the diameters of each nanoparticle by the total number of nanoparticles. The term "diameter" is usually used to refer to the maximum length of a line segment passing through the center of a nanosphere and connecting two points on the perimeter, but is also used herein to refer to the maximum length of a line segment passing through the center of a nanoparticle having other shapes, such as a nanocube, nanotetrahedron, or irregular shapes, and connecting two points on the perimeter.

[0032] Self-assembly into nanoparticles can be performed on either unconjugated reflectin polypeptides or on reflectin polypeptides that have been conjugated (e.g., ligated) to a ligand. For example, reflectin polypeptides ligated according to the present application can be modified by conjugation to a ligand before or after the reflectin polypeptide self-assembles into nanoparticles. The unconjugated ligand (i.e., the chemical structure of the ligand before conjugation) can include a functional group for reacting with a reflectin polypeptide. More specifically, in various embodiments, the functional group of the unconjugated ligand can include a leaving group that is commonly used to make peptide bonds. The functional group can be a succinimide ester or a fluorinated phenyl ester. The succinimide of the succinimide ester or the fluorinated phenyl of the fluorinated phenyl ester can function as a leaving group in the reaction with a free amine of the reflectin polypeptide. Thus, in some embodiments, conjugation of a reflectin polypeptide to a ligand can result in a covalent bond between the ligand and the reflectin polypeptide. The covalent bond between the reflectin polypeptide and the ligand may be a peptide bond, ie an amide bond.

[0033] In embodiments where the functional group of the unconjugated ligand for reaction with a reflectin polypeptide is a succinimide ester, the succinimide may be modified with an electron-withdrawing group. The electron-withdrawing group may be SO3 - It may contain a group.

[0034] The ligand may further comprise a linking group for immobilizing the recombinant reflectin nanoparticle to a substrate. The linking group may comprise or may be a triple bond. The triple bond may react with an azide that may be attached to a substrate to form a covalent bond between the substrate and the ligand. Alternatively, the linking group may be an azide and the triple bond may be attached to a surface that is functionalized to covalently bind the ligand to the surface. Thus, in some embodiments, immobilizing the recombinant reflectin nanoparticle to an azide via the ligand may result in a covalent crosslink between the recombinant reflectin nanoparticle and the surface via the ligand.

[0035] In some embodiments, the ligand (after conjugation) has the following formula (I):

[0036] [ka]

[0037] (In the formula, CG is a linking group, and n is an integer selected from 1 to 5. For example, n may be 1, 2, 3, 4, or 5.

[0038] [ka]

[0039] indicates the point of attachment to a nitrogen atom of the reflectin polypeptide to form a peptide bond. The linking group CG has the following formula (II):

[0040] [ka]

[0041] (In the formula,

[0042] [ka]

[0043] is the -(CH2) of the ligand of formula (I) n The linking group CG may be an azide (-N3), which indicates the point of attachment to the moiety. In one example, the unconjugated ligand can be dibenzocyclooctyne-sulfo-NHS ester (DBCO-sulfo-NHS ester), or a sodium salt thereof.

[0044] In various embodiments, the recombinant reflectin nanoparticles are controlled to be essentially the same size. That is, they can be substantially monodisperse. To measure the heterogeneity of a sample based on size, the polydispersity index (PDI) is often used. Polydispersity can be caused by particle size distribution in a sample, or agglomeration or aggregation of the sample during isolation or analysis. PDI can be obtained from an instrument using dynamic light scattering (DLS) or can be determined from electron micrographs. The PDI of the recombinant reflectin nanoparticles can be less than 0.5. Advantageously, in embodiments where the recombinant reflectin nanoparticles are conjugated to a ligand, the PDI can be less than 0.1, or less than 0.09.

[0045] In various embodiments, the recombinant reflectin nanoparticles may have a high negative zeta potential. Zeta potential may be understood as a measurable indicator of the stability of a colloidal dispersion, and the magnitude of the zeta potential may indicate the degree of electrostatic repulsion between adjacent similarly charged particles in the dispersion. In other words, the higher the zeta potential, the more stable the solution or dispersion may be and the less likely it is to aggregate. In contrast, with a low zeta potential, the attractive forces may outweigh the repulsive forces and the dispersion may break down or aggregate. Thus, colloids with a high zeta potential (negative or positive) are electrically stabilized, while colloids with a low zeta potential tend to coagulate or flocculate. According to various embodiments, the zeta potential of the recombinant reflectin nanoparticles may be in the range of about -30 to -100 mV, or in the range of -35 to -45 mV, which indicates a stable colloidal suspension.

[0046] In various embodiments, the recombinant reflectin nanoparticles may be crystalline. The photonic crystal structure may provide long-range order that results in iridescence. In alternative embodiments, the recombinant reflectin nanoparticles may be amorphous. The amorphous photonic structure may provide short-range order that results in structural color. In one example, the recombinant reflectin nanoparticles may have an amorphous photonic structure.

[0047] In a further aspect, the present disclosure also relates to a host cell, preferably a non-human host cell, containing the nucleic acid molecule contemplated herein or the vector contemplated herein. The nucleic acid contemplated herein or the vector containing said nucleic acid is preferably transformed into a microorganism, which then represents a host cell according to one embodiment. Methods for transforming cells are established in the existing technology and are well known to the skilled person. As a host cell, in principle all cells are suitable, i.e. prokaryotic or eukaryotic cells. The host cell can be genetically manipulated in an advantageous manner.

[0048] Preferred host cells are prokaryotic or bacterial cells, such as E. coli cells. Bacteria are notable for their short generation time and low demands on culture conditions. As a result, economical culture or production methods can be established. In addition, those skilled in the art have ample experience with bacteria in fermentation technology. Gram-negative or Gram-positive bacteria may be suitable for a particular production example for a variety of reasons, such as nutrient sources, product formation rate, time required, etc., which are confirmed experimentally in each individual case. In various embodiments, the host cell may be an E. coli cell.

[0049] The host cells contemplated herein may be modified with respect to their requirements for culture conditions, may contain other or additional selectable markers, or may express other or additional proteins.

[0050] The host cells contemplated herein are cultured and fermented in the usual manner, for example in discontinuous or continuous systems. In the former case, a suitable nutrient medium is inoculated with the host cells and the product is harvested from the medium after an experimentally determined period. Continuous fermentation is notable in that over a relatively long period of time, a flux equilibrium is achieved during which the cells are partially killed but partially replaced and the proteins formed can be simultaneously removed from the medium. The host cells contemplated herein are preferably used to produce the reflectins described herein.

[0051] Thus, a further aspect of the present disclosure is a method for synthesizing a reflectin polypeptide as described herein, comprising culturing a host cell as contemplated herein; and isolating the reflectin polypeptide from the culture medium or the host cell. Culture conditions and media can be selected by the skilled artisan using general knowledge and techniques known in the art based on the host organism used.

[0052] In a further aspect, a method for synthesizing recombinant reflectin nanoparticles may be provided. The method may include a first step of recombinantly expressing a reflectin polypeptide as described herein. The expression may be performed from E. coli. In other words, the method may include the steps of providing an E. coli host cell culture, altering the growth rate of the E. coli host cells, and inducing expression of the recombinant reflectin polypeptide as inclusion bodies. The recombinantly expressed inclusion bodies may then be extracted from the E. coli host cell culture. In a next step, these inclusion bodies may be solubilized. The solubilization may be performed under strong denaturing conditions, for example using a urea or dimethylurea solution with a molar concentration of about 5 to 10. Having a molar concentration in this range may be advantageous for the transparency of the recombinant reflectin solution. Concomitantly or alternatively, the solution may be heated to a temperature of about 80° C. or higher, or from 80° C. to about 100° C.

[0053] After the recombinant reflectin polypeptide is solubilized, the next step may be purification by chromatography. Advantageously, the purification by chromatography may be carried out in a pH range higher than pH 5.0, which may advantageously be beneficial for the stability of the recombinant reflectin polypeptide. In various embodiments, the chromatography may be carried out using ion exchange chromatography, for example, cation exchange chromatography. After purification, a purity of the recombinant reflectin polypeptide higher than 95% or higher than 98% may be obtained.

[0054] In some embodiments, the recombinant reflectin polypeptide may be conjugated to a ligand as described hereinbefore, which may be performed prior to self-assembly. The conjugation of the ligand may include adding an unconjugated ligand to the solution. The unconjugated ligand may have a molar concentration of about 5 mM to 10 mM in the solution. Below this molar concentration range, the solution may not be stabilized, whereas a molar concentration above this range may not further affect the size or stability of the subsequent recombinant reflectin nanoparticles.

[0055] Chromatography of the recombinant reflectin polypeptide and optional conjugation to a ligand may be followed by a dialysis step to remove urea or dimethylurea. The dialysis step may advantageously include a self-assembly step.

[0056] Thus, in the next step, the purified recombinant reflectin polypeptide can be induced to self-assemble into nanoparticles. This step can be performed in pure water. Alternatively, in some embodiments, a buffer can be added. The buffer can have a molar concentration of about 5 to about 20 mM. The type of buffer can be a Good's buffer or can be selected from the group consisting of MOPS, MES, HEPES, and combinations thereof. The pH of the buffer can be adjusted to 4 to 10, or 6 to 8, in some embodiments, about 7.0 to 7.4.

[0057] The buffer may comprise an organic solvent. The organic solvent may be a polar solvent. More particularly, the organic solvent may be selected from the group consisting of a polar aprotic solvent or an alcohol. The polar protic solvent may be selected from the group consisting of acetone, acetonitrile, dimethylformamide, dimethylpropyleneurea, dimethylsulfoxide, hexamethylphosphoric triamide, pyridine, sulfolane, tetrahydrofuran, and combinations thereof. The alcohol may be selected from the group consisting of methanol, ethanol, iso-propanol, tert-butanol, and combinations thereof.

[0058] The concentration of the organic solvent in the buffer may be about 5-50%, or about 20-35%, or about 5-15%. Advantageously, the size of the recombinant reflectin nanoparticles can be controlled by the concentration of the organic solvent. For example, when acetonitrile is used as the organic solvent, it may be possible to obtain a substantially linear relationship of the size of the nanoparticles with an increase in acetonitrile concentration of 10-20%, 20-30%.

[0059] If no organic solvent is added for self-assembly, the pH of the aqueous buffer can be about 8-10, for example, by using Good's buffer or by using 5-50 mM sodium borate or 50-200 mM imidazole. Optionally, sodium chloride may be added.

[0060] In some embodiments, a surfactant may be added to the solution in which the self-assembly is carried out. The surfactant may be either zwitterionic or neutral.

[0061] In some embodiments, an antioxidant may be added to the solution in which the self-assembly is carried out. The antioxidant may be ascorbic acid and / or sodium ascorbate.

[0062] In some embodiments, methyl-β-cyclodextrin may be added to the solution in which the self-assembly is carried out. Advantageously, the addition of methyl-β-cyclodextrin can control the size of the resulting recombinant reflectin nanoparticles to less than 200 nm.

[0063] In another embodiment, a substrate is provided that is surface-functionalized with recombinant reflectin nanoparticles. Advantageously, by using recombinant reflectin nanoparticles with specific nanoparticle sizes, it is possible to induce reflectance that shows tunable response from violet (400 nm) to near infrared (800 nm). Also, the recombinant reflectin nanoparticles immobilized on the substrate allow dynamic color change caused by the swelling of the recombinant reflectin nanoparticles due to hydration.

[0064] In some embodiments, the recombinant reflectin nanoparticles may be assembled on the surface as a substantially monolayer. In some embodiments, the recombinant reflectin nanoparticles may be covalently immobilized on the surface, optionally using a drop-cast deposition method. In some embodiments, the distance between one recombinant reflectin nanoparticle and another recombinant reflectin nanoparticle is less than 1 micrometer.

[0065] In another aspect, a method is provided for immobilizing recombinant reflectin nanoparticles on a substrate, the method comprising: providing a substrate comprising hydroxy groups; reacting the hydroxy groups with surface-bound spacer chains; providing recombinant reflectin nanoparticles; and reacting the recombinant reflectin nanoparticles with the surface-bound spacer chains.

[0066] The substrate may comprise any material, for example glass, so long as it has hydroxy groups on the surface for surface treatment. These hydroxy groups may be surface treated with organosilanes, which may be an example of surface-bound spacer chains. Advantageously, surface treatment of the substrate with organosilanes may promote hydrogen bonding and / or covalent bonding between the substrate and the recombinant reflectin nanoparticles. Surface treatment with organosilanes may be used to covalently bond the surface-treated substrate to the recombinant reflectin nanoparticles. When the organosilanes are present and bound to the recombinant reflectin nanoparticles, a "covalent bridge" may be formed that extends from the substrate through the surface-bound spacer chains to the recombinant reflectin nanoparticles.

[0067] The organosilane may include an active functional group selected from the group consisting of octyl, amine, vinyl, ethynyl, hydroxyl, thiol, and combinations thereof. The organosilane may be an aminoalkylsilane, such as APTES, or triethoxy(ethynyl)silane. In some embodiments, the organosilane may be further functionalized with an additional linker. For example, a click chemistry functional group (i.e., azide or triple bond) may be present at a terminal site, allowing for covalent bond formation via click chemistry with the recombinant reflectin nanoparticle, for example, via the linking group of the ligand. Thus, both the surface-functionalized substrate and the recombinant reflectin nanoparticle may have complementary click chemistry functional groups before reacting with each other. In other words, one of the components may have a triple bond functional group and the other an azide functional group.

[0068] In another aspect, a skin care product is provided that includes the recombinant reflectin nanoparticles. Advantageously, the absorption ability of the recombinant reflectin nanoparticles can be utilized in skin care products such as sunscreens. Even more advantageously, the recombinant reflectin nanoparticles have been found to be less toxic than ingredients traditionally used in sunscreens, such as titanium dioxide. Thus, in another aspect, a skin care product for use in treatment is further provided. In particular, a skin care product for use in preventing skin cancer and / or inflammatory responses to ultraviolet (UV) radiation damage to the outermost layer of the skin (e.g., sunburn) is provided. Also provided is the use of the skin care product in the manufacture of a medicament for skin care and / or preventing inflammatory responses to ultraviolet (UV) radiation damage to the outermost layer of the skin.

[0069] Nanoparticles that may be beneficial for use as skin care products may have a size of about 350 nm to about 450 nm, or about 400 nm. The term "comprising" should be understood to have the same broad meaning as the term "including" and to mean the inclusion of a stated integer or operation, or group of integers or operations, but not the exclusion of any other integers or operations, or group of integers or operations. This definition also applies to variations of the word "comprising", such as "comprise" and "comprises".

[0070] "About" with respect to a given numerical value, such as concentration and composition, is meant to include the numerical value within 10% of the specified value. Features described in the context of one embodiment may be applicable to the same or similar features in other embodiments. Features described in the context of one embodiment may be applicable to the other embodiments even if they are not explicitly described in the other embodiments. Furthermore, additions and / or combinations and / or substitutions as described for features in the context of one embodiment may be applicable to the same or similar features in the other embodiments.

[0071] In the context of the various embodiments, the articles "a," "an," and "the" used in reference to features or elements include a reference to one or more of the feature or element. As used herein, the term "and / or" includes any and all combinations of one or more of the listed items associated with that term. EXAMPLES

[0072] Reflectin from the squid Sepioteuthis lessioniana was sequenced, and the sequenced reflectin B1 was used to prepare coatings with tunable structural color. Reflectin B1 was conjugated to click chemistry ligands and self-assembled into quasi-monodispersed nanoparticles with tunable sizes in the range of 100-1000 nanometers (nm). Using Langmuir-Schaefer and drop-cast deposition methods, the ligand-conjugated reflectin B1 nanoparticles were immobilized on azide-functionalized substrates by click chemistry to create monolayer amorphous photonic structures with tunable structural color, paving the way for the fabrication of environmentally friendly, bioinspired color-changing coatings that mimic the dynamic camouflage of cephalopods. In one embodiment of the present disclosure, a rechargeable energy storage system is based on the safe, environmentally friendly, and sustainable use and functionalization of rainwater as an electrolyte.

[0073] Since the size of reflectin nanoparticles governs the iridescence properties, the color of the film / coating made from recombinant reflectin nanoparticles can be tuned by controlling the average size of the nanoparticles and subsequently immobilizing the nanoparticles into photonic structures that mimic the 1D Bragg lamellar photonic lattice in vivo. To this end, we first sequenced the reflectin from S. lessoniana by next-generation RNA sequencing (RNA-seq) of dermal tissue (for clarity, we refer to the unconjugated S. lessoniana B1 reflectin as SlRF-B1). Full-length SlRF-B1 was then recombinantly expressed in E. coli and purified by one-step strong cation exchange chromatography. A systematic approach to self-assemble SlRF-B1 into distinct nanoparticle sizes followed. The nanoparticles showed a polydispersity index (PDI) of less than 0.1 when conjugated with the click chemistry ligand dibenzocyclooctyne-sulfo-NHS ester (DBCO-sulfo-NHS ester), which was achieved by varying the solvent conditions during the self-assembly process.

[0074] These quasi-monodisperse DBCO-SlRF-B1 nanoparticles were immobilized on azide-functionalized wafer substrates by combining click chemistry with Langmuir-Schaefer and drop-cast deposition techniques. The result was a monolayer assembly that behaved as a photonic amorphous structure (randomly packed particles), with reflectance controlled by nanoparticle size and showing a tunable response from the violet (400 nm) to the near-infrared (800 nm). A dynamic color change of the monolayer film caused by hydration-induced swelling of the immobilized DBCO-SlRF-B1 nanoparticles was also demonstrated. The formation and self-assembly of reflectin B1 nanoparticles with low polydispersity index and highly controlled tunable diameter was carried out without any equipment, only by dialysis buffer at room temperature (23 °C, humidity 60%) and atmospheric pressure. All chemicals used are low toxic and biocompatible.

[0075] Advantages and improvements over existing methods, devices or materials Self-assembly of protein nanoparticles can be achieved by supercritical fluid techniques, emulsification, desolvation, complex coacervation, electrospray and sol-gel, but is difficult because of their susceptibility to chemical and physical degradation during processing involving stresses (heat, pressure, organic solvents) that may be detrimental to protein structure and function. Chemical methods for producing protein nanoparticles involve lyophilization with methyl-β-cyclodextrin and resuspension in ethyl acetate. There is limited control of nanoparticle size and significant aggregation of proteins in organic solvents. The present invention involves only gentle, uncomplicated procedures for self-assembly of protein nanoparticles that do not compromise protein integrity.

[0076] Technical Description of the Invention Example 1: Expression, purification, self-assembly and conjugation The recombinantly expressed SlRF-B1 (Table 1) can be characterized by the following sequence:

[0077] [Table 1]

[0078] Recombinantly expressed SlRF-B1 inclusion bodies extracted from E. coli could only be fully solubilized under strong denaturing conditions (8 M urea or 6 M dimethylurea, T = 85 °C). Below this chaotropic concentration, the initially clear protein solution became turbid and then phase separated by a liquid-liquid phase separation (LLPS) process, resulting in a condensed colloidal phase over a few days. When the turbid solution was imaged by transmitted light microscopy, spherical coacervated SlRF-B1 microdroplets with diameters in the 1–3 micrometer (μm) range were observed (Figure 7). The precipitated protein phase was highly viscous and sticky (Figure 7, inset) and could only be solubilized by heating the solution above 80 °C. This process was reversible upon subsequent cooling / heating cycles, indicating that SlRF-B1 exhibits upper limit solution temperature (UPST) phase behavior. Solubilized inclusion bodies were purified by one-step strong cation exchange chromatography, with an estimated purity of >98% based on SDS-PAGE and MALDI-ToF analysis (Figure 1A-C). The use of reversed-phase high-pressure liquid chromatography (RP-HPLC) for purification was precluded because the reflectin protein was found to degrade at pH ≤ 5.0.

[0079] To allow the self-assembly of SlRF-B1 into nanoparticles, a dialysis protocol was used to slowly remove urea from the ion-exchange purified sample. One of the unique properties of SlRF-B1 is that it is a highly charged protein (isoelectric point 8.8), which provides an opportunity to modulate the zeta potential and therefore the colloidal properties by screening buffer types and additives during the dialysis process. Thus, four criteria were simultaneously achieved during dialysis: (i) mitigation of aggregation, (ii) control of nanoparticle size, (iii) narrow size distribution, and (iv) particle stability. Click chemistry was subsequently used to immobilize SlRF-B1 nanoparticles on the selected surfaces. The free amines on the surface of SlRF-B1 nanoparticles were first functionalized with different NHS ester-containing click chemistry molecules (see Table 2). The copper-free click chemistry DBCO-sulfo-NHS ester gave the best results in terms of size distribution.

[0080] [Table 2]

[0081] To control particle size, the acetonitrile (ACN) concentration was varied during dialysis as a way to modulate the hydrophobic interactions between SlRF-B1 and the solvent (Figure 8). The polydispersity index (PDI), measured by dynamic light scattering (DLS), improved from 0.40 ± 0.20 (unconjugated) to less than 0.08 ± 0.02 (conjugated) for most samples.

[0082] As shown in Figure 1D, the final average size of the conjugated DBCO-SlRF-B1 nanoparticles could be precisely tuned between 200 nm and 1000 nm by increasing the ACN concentration from 20-35% v / v, with a nearly linear correlation between particle size and ACN content. The ζ potential of all samples was consistently ζ = -39.4 ± 1.95 mV, indicating stable colloidal suspensions. The conjugation reaction was complete in only 30 min for particles smaller than 300 nm and 2 h for larger nanoparticles.

[0083] Example 2: Growth mechanism of DBCO-SlRF-B1 nanoparticles To determine which growth mechanism (LaMer burst nucleation, Ostwald / digestive ripening, or coalescence) best explains the formation of DBCO-SlRF-B1 nanoparticles, DLS was used to monitor the growth of the 360 ​​nm and 660 nm nanoparticles. Scatter plots of particle size as a function of time are shown in Figure 2A. The growth of the 360 ​​nm nanoparticles was completed within a few minutes, and the final size obtained after 1000 s did not change further over time. No additional peaks of smaller particle sizes consistent with an Ostwald ripening mechanism were detected. For the 660 nm nanoparticles, an initial rapid growth was observed, resulting in a maximum average particle size of approximately 900 nm after 500 s. The particle size then gradually decreased, reaching a saturation plateau at a final average particle size of 660 nm. The data suggest that while the initiation of growth with DBCO is rapid, the formation of monodisperse particles takes longer at larger particle sizes. The growth curves of the DBCO-SlRF-B1 660 nm nanoparticles were further supplemented with the PDI at each time point (Figure 2B). Interestingly, the PDI was observed to first increase and then decrease, a pattern that was not stochastic and was also observed for the 360 ​​nm nanoparticles (Figure 9).

[0084] The growth of DBCO-SlRF-B1 nanoparticles occurs mainly by a simplified four-step coalescence mechanism, as outlined in Figure 2C, with a possible minor contribution of digestive ripening at later stages. To clarify the schematic model, only binary coalescence (fusion of two particles) is illustrated. The self-assembled SlRF-B1 suspension was initially transparent, and growth began upon addition of DBCO-sulfo-NHS ester. In step 1, the growth was rapid and exponential, forming a turbid but milky yellow-orange solution. The large fluctuating PDI can be explained by the anisotropic particle shape during coalescence. In step 2, the maximum particle size was reached when the inward Laplace force and the outward elastic energy reached equilibrium. Thus, particles in this region have a low PDI. In step 3, the large unstable particles underwent digestive ripening, coalescence breakage, and collisional breakage, which removed excess protein material from the large particles. The small particles grew again by coalescence, and the PDI decreased. Finally, step 4 was the final plateau region, where DBCO-SlRF-B1 stabilized to the final nanoparticle size with a narrow size distribution regulated by the ACN concentration.

[0085] Further evidence of coalescence was observed by the presence of arrested coalescence, which resulted in incomplete fusion. Different states of arrested coalescence were identified using AFM. Coalesced particles had a surface profile showing a clear boundary between each other (Figure 3A). In contrast, in nearly complete coalescence, no clear boundary was observed, but consisted of a smooth transition between adjacent particles (Figure 3B). Both profiles are compared in Figure 3C. Arrows indicate the boundaries between particles. The complexity of the growth system may result in arrested coalescence in triplet, quartet, or higher configurations, which are more frequently seen in the self-assembly of nanoparticles larger than 600 nm. AFM and TEM images of arrested coalescence are shown in Figure 3D. When the coalescence process was prematurely stopped within 10 min by adding 10 mM TRIS buffer (pH 7.0), long-chain coalescence necking between multiple particles was also observed by TEM (Figure 10).

[0086] The internal structure of the 200 nm nanoparticles was further investigated using AFM and TEM observations and was found to consist of small spherical units of approximately 20 nm, as shown in the AFM phase images (Figures 3E-F). TEM imaging revealed similar features (Figure 3G), and post-imaging processing further improved the clarity of the structural details (Figure 3H).

[0087] Example 3: Fabrication of photonic structures Structural colors with low angular dependence are common in nature, and the structural colors depend on the dielectric refractive index, colloid diameter, thickness of the structural layers, and lattice distance. The controllable DBCO-SlRF-B1 nanoparticle size has provided an opportunity to fabricate photonic structures using bottom-up self-assembly techniques such as physical confinement and gravitational sedimentation. The self-assembled photonic structures are either in the form of photonic crystal structures (PCS) that exhibit long-range order or photonic amorphous structures (PAS) that have only short-range order, resulting in iridescence and structural colors, respectively.

[0088] Example 4: Langmuir-Schaefer surface monolayer immobilization method The use of the Langmuir-Blodgett / Schaefer deposition technique was investigated to produce either PCS or PAS using DBCO-SlRF-B1 nanoparticles. A concentrated, turbid DBCO-SlRF-B1 nanoparticle suspension was carefully added to the air-water interface required for deposition, but due to its high mass density it settled over time. To ensure that the nanoparticles remained at the air-carrier interface, an inert, low-toxicity, solids-to-water ratio-dependent density (1.0–3.1 g / cm) was used instead of water. 3Sodium polytungstate, a heavy liquid with tunable solubility, was used as the carrier medium. A customized Langmuir-Blodgett / Schaefer minidevice was fabricated by precision CNC machining (Figure 10), allowing either the Langmuir-Blodgett or Langmuir-Schaefer method to be used for DBCO-SlRF-B1 monolayer deposition. The specificity of click chemistry was exploited to covalently immobilize DBCO-SlRF-B1 nanoparticles on silicon wafer surfaces. The wafer was functionalized with aminoalkylsilane [3-(2-aminoethylamino)propyl]trimethoxysilane (AEAPTMS), followed by conjugation of the free amines with azido-dPEG®4-TFP ester. Functionalization was confirmed by ellipsometry, contact angle and fluorescence labeling (Figure 12).

[0089] The Langmuir-Blodgett technique formed a very thin layer of highly hygroscopic sodium polytungstate between the wafer surface and the nanoparticles, forming a partial monolayer. The Langmuir-Schaefer deposition technique proved to be superior for forming a monolayer of DBCO-SlRF-B1 nanoparticles on the wafer surface. Acceptable monolayer Langmuir coatings were also produced from nanoparticle sizes of 170 nm, 240 nm, 270 nm, and 310 nm, and theoretical reflectances in the blue (λ = 442 nm), orange (λ = 624 nm), red (λ = 702 nm), and near-infrared (λ = 806 nm) were calculated using a random close-packed volume fraction f = 0.64. All coatings exhibited structural colors as shown in Figure 4A-4D (top panels), along with corresponding AFM images of the coatings and measured reflectance values. The reflectance peak wavelength of the amorphous structure can be approximated by the following equation:

[0090]

number

[0091] Here, λ maxis the maximum reflection wavelength of the particle array, d is the diameter of the DBCO-SlRF-B1 nanoparticles and n eff is the average refractive index of the DBCO-SlRF-B1 nanoparticles, which also takes into account the dielectric-air composite. The average refractive index n eff can be derived using the following formula:

[0092]

number

[0093] Here, the reflectance of reflectin nSlRF-B1 = 1.44, n air = 1, and f is the volume fraction occupied by the nanoparticles. The distribution, volume fraction, and fast Fourier transform (FFT) of the nanoparticles were analyzed using ImageJ (Figure 13), confirming that the particles are quasi-monodisperse and that there is no long-range order in the monolayer assembly. With light incident normal to the surface, structural color was observed in all coatings at visualization angles between 0 and 35°.

[0094] Example 5: Drop-cast immobilization method Temperature is an important factor in the thermally assisted colloidal self-assembly of long-range PCS with iridescence, as it affects nanoparticle diffusion in suspension. We investigated whether thermally assisted colloidal self-assembly (TACSA) can be applied to DBCO-SlRF-B1 nanoparticles by fabricating multilayer iridescent PCS at high temperatures using a simple drop-casting method. Because proteins are prone to denaturation at high temperatures, the temperature was limited to a maximum of 60 °C to investigate the effect of TACSA on the average nanoparticle size of 215 nm. Samples prepared at 23 °C and 40 °C produced structural colors, and defects in coverage were evident at higher temperatures (Figure 14). No iridescence was observed at any temperature, indicating the absence of long-range PCS. The investigation was extended to other different sizes of DBCO-SlRF-B1 nanoparticles obtained from Figure 1D. AFM imaging (Figure 15) revealed that samples prepared at 40 °C and above had uncontrolled thicknesses of 2.5 μm. 2The results showed that the nanoparticles self-assembled at temperatures below 40 °C exhibited randomly stacked particles with patches of voids. Samples prepared at temperatures below 40 °C showed better nanoparticle dispersion across the surface. The larger nanoparticle size self-assembled from 30-35% v / v ACN buffer was not significantly affected by temperature.

[0095] The drop cast volume is 225 mm 2 The volume was optimized by reducing the volume to 300 μL per nanoparticle and using a maximum temperature of 35 °C for 400 nm DBCO-SlRF-B1 nanoparticles. Instead of the near-infrared reflectance signature expected at λ = 850 nm based on the Bragg-Snell equation, a purple hue was observed under normal incidence light. Using the optimized conditions, the experiment was repeated with particle sizes of 400 nm, 460 nm, 520 nm and 660 nm, which showed purple, blue, green and red coloration, respectively (Figures 5A-5D), but no coloration was observed at the periphery of the drop-cast film. This is due to the interparticle spacing in this region being 2.5 μm. 2 The random close-packed coatings had a stronger reflection intensity than the random sparsely packed samples prepared from drop-cast volumes of less than 300 μL.

[0096] Finally, attention was focused on inducing a dynamic shift in reflectance, which was hypothesized to be achieved by inducing swelling of the nanoparticles. maxUsing a spectrophotometer (T = 453 nm), condensed water vapor was directed onto the dehydrated monolayer (Figure 5E). When monitored by a spectrometer (Figure 5G), the color spontaneously red-shifted to 700 nm (Figure 5F). As the surface water gradually evaporated, the spectrum blue-shifted back to the original spectrum. This process could be repeated multiple times in quick succession without any change in the sample surface or spectral peaks. With light incident perpendicular to the surface, structural color was observed in all coatings at visualization angles between 0 and 45°.

[0097] The structural colors observable when nanoparticles have diameters comparable to visible wavelengths are due to incoherent scattering effects via isotropic photonic pseudoband gaps. Interestingly, this effect is similar to cephalopod pigment granules found in chromatophores whose size (~500 nm) is comparable to visible wavelengths. The lack of wavelength absorption in reflectin nanoparticles, coupled with their random close-packed monolayer arrangement, results in single-particle (cavity) scattering properties that depend on the individual nanoparticle size, shape, refractive index, and volume fraction of scatterers.

[0098] The formation of a red-colored coating due to the absorbance of the second resonance peak No red structural color morphology was observed, even though the measured wavelength in the hydrated monolayer was 700 nm. A model proposed in the literature describes the challenge of obtaining saturated red structural color in amorphous photonic structures, which is due to the shift and intensification of higher-order resonance modes (second-order resonance peaks) to blue wavelengths as the particle size increases. The leaky resonances found are consistent with the observations showing the absence of saturated red coloration in hydrated films (Figure 5F, Figure 5G) because blue wavelengths have stronger optical confinement in the resonator, but blue and green were unaffected because the second-order resonance peaks are in the UV region. Chromatophores in cephalopod skin can exhibit a deep red color because they contain ommochrome pigments, including xanthommatin and decarboxylated xanthommatin. The absorbance of xanthommatin pigments peaks at 430 nm, effectively removing the second-order higher-order resonance peaks from the blue spectrum, making the red color dominant. The goal was to remove this secondary resonant blue peak from the nanoparticle coating at 660 nm and mimic the sample scattering at red wavelengths (600-700 nm). As shown in Figure 5D, a secondary resonant peak at 429 nm was present. Hydrogel or metal core-shell coatings have been designed to weaken or eliminate this secondary higher order resonant peak by matching the refractive index of the nanoparticle boundary to the refractive index of the surroundings, but because protein nanoparticles are soft biomaterials and processing conditions for core-shell fabrication are limited, the goal was to mimic the use of absorbing molecules by cephalopods.

[0099] For this purpose, the fluorescent molecule coumarin 343X azide, which absorbs effectively at blue wavelengths around 430 nm, was studied. Using the same click chemistry principle to conjugate reflectin nanoparticles to wafer substrates, coumarin 343X azide (1 mM in dimethyl sulfoxide, 50 μL drop cast) was conjugated to 660 nm nanoparticle coatings (shown in Figure 4D) for at least 4 hours to investigate whether red structural color could be obtained. With coumarin 343X azide, the effect of increasing particle size should be negligible, since the nanoparticle surface would only be thickened by one molecule. Structural color and reflectance measurements before (Figure 6A) and after (Figure 6B and Figure 6C) conjugation confirmed that the secondary resonance peak at 430 nm was significantly suppressed after 24 hours, resulting in a dominance of red color. For conjugation times less than 1 hour, the samples appeared purple, which was due to the mixing of blue and red wavelengths by partial conjugation. This was verified by reflectance measurements (Figure 6B). Because coumarin 343X azide was used, the fluorescence emission wavelengths may result in unwanted peaks in the reflectance spectrum. However, no characteristic emission peaks were detected between 477 and 520 nm.

[0100] [Table 3]

[0101] The reflectance FWHM values ​​of the 400-660 nm reflectin nanoparticle coatings were relatively consistent from approximately 180-200 nm, as summarized in Table 3. Furthermore, these values ​​were independent of nanoparticle size, unlike the increase in FWHM reflectance values ​​of coatings made with 170-310 nm nanoparticles. The broadening of the FWHM with increasing nanoparticle size is attributed to the leakage of bound photons from the resonator. Photons in higher order resonant modes are more tightly bound to the resonant nanoparticles, and even more so for blue resonances, resulting in a sharper spectral response (smaller FWHM values). From the data in Figures 4 and 5, it can be inferred that the reflectance spectrum of the 400-660 nm nanoparticle coating is a second order resonance, red-shifted from the 170-310 nm nanoparticles with increasing intensity, explaining the narrower FWHM. This is further supported by the fact that the near-infrared reflectance at λ = 1000 nm for a nanoparticle size of 400 nm was significantly attenuated, and the purple color at λ = 404 nm dominated the spectrum. Assuming that the reflectance of the nanoparticle coating from 170 to 310 nm is the first resonance, the reflectance of the nanoparticle coating from 400 to 660 nm is the second resonance. The resonance peak in Figure 5D is a third-order mode with a FWHM of 130 nm. Finally, it has also been shown that structural colors can still be produced from polydisperse nanoparticles, even at the expense of multiple scattering peaks. Thus, structural colors are based on a weighted average of the particle size distribution of the particles. Taking this into account, a quasi-monodisperse 400-660 nm nanoparticle coating with a standard deviation within 15% was able to produce structural colors with good FWHM, despite not having a completely monodisperse size.

[0102] The use of naturally occurring xanthomanthin by cephalopods occurs through careful selection of organic molecules that specifically absorb the second-order higher-order resonance mode at approximately 430 nm. Reflectin nanoparticles were initially identified in iridophores and were responsible for iridescence, but have recently been found in chromatophores along with other structural proteins, including S-crystallin and r-opsin. It has been suggested that the S-crystallin protein, which has a high refractive index and inhibits aggregation, has a high affinity for xanthomanthin and functions as a light scatterer. This data indicates that self-assembled reflectin nanoparticles can also function as light scatterers.

[0103] Consideration For the first time, it is possible to modulate the growth of quasi-monodisperse reflectin-based nanoparticles simply by varying the dialysis conditions after purification. The self-assembly and conjugation method is based on simple colloid chemistry, with a process carried out at room temperature (23 °C, 60% RH), pressure, and physiological conditions (pH 7.0). Furthermore, these DBCO-SlRF-B1 nanoparticles are click chemistry-enabled, allowing surface modification strategies to be implemented and properties to be altered using a wide variety of azide-functionalized molecules. The data demonstrate that DBCO-conjugated SlRF-B1 can self-assemble into nanoparticles of various sizes with controlled size distribution, a process that does not depend on the presence of the repeating motifs found in reflectin A1. The formation of larger particles also reveals the coalescence behavior of the protein, elucidating the self-assembly mechanism of reflectin. This method was applied to recombinantly expressed and purified Doryteuthis pealeii reflectin A1 (GenBank: FJ824804, 6x His-tagged) and self-assembly of nanoparticles was achieved, although a complete systematic study was not performed.

[0104] Colloidal self-assembly experimental conditions did not yield long-range periodic PCS. Instead, quasi-monodisperse nanoparticles self-assembled into photonic amorphous structures. While Langmuir Schaefer is useful for producing monolayers of small nanoparticle sizes, TACSA drop casting is a simpler method for producing either long-range periodic lattices or random close-packed monolayers for nanoparticles larger than 400 nm. With either method, DBCO-SlRF-B1 coatings yielded vibrant structural colors on silicon wafers when the nanoparticles were arranged with interparticle distances of less than 1 μm. Interestingly, the structural colors observed at particle sizes larger than 400 nm suggest that these nanoparticles resemble the behavior of chromatophores. The granules of about 500 nm act as bandpass filters through light absorption and scattering, and this effect is enhanced by the presence of high refractive index proteins and xanthommatins. Thus, DBCO-SlRF-B1 nanoparticles with an average size on the same scale as the visible wavelength may partially mimic the structure and function of chromatophore granules.

[0105] The coatings prepared by click chemistry immobilization were stable for more than a year at room temperature without special storage. It is favorable that the single molecule ligand DBCO-sulfo-NHS ester could initiate controllable nanoparticle growth and reveal the time-resolved self-assembly of reflectin nanoparticles. Overall, this work provides a broader platform for protein-based photonic structures and can be expanded to other fields, such as nanocarriers for controlled drug delivery applications.

[0106] Commercial Applications of the Disclosure Defense - Mitigating Thermal Detection for Camouflage Commercial - Building windows that reflect NIR wavelengths (reducing heat build-up and saving energy costs) Industrial - Paints and Coatings Structural Color of Nanoparticle Coatings for Photonics-Optoelectronic Displays Cosmetics - colored nail polish, skin lotions that reflect NIR (sunscreens) or absorb UV (DBCO ligands) Medical - Drug-encapsulating nanocarriers for drug delivery and other bioconjugates of therapeutic molecules Experimental Method Example 6: Sample Collection A live Sepioteuthis lessioniana squid was caught off the coast of Keppel Bay, Singapore, and sedated for at least 30 minutes in a 20-liter bucket filled with seawater supplemented with 0.15 M magnesium chloride. The body was washed twice with Milli-Q® water and dissected immediately on the spot. Three skin specimens, each 3 × 3 cm, were excised from different sites of the mantle with a sterile scalpel, washed twice with Milli-Q® water to remove excess pigment, and immediately stored in RNAlater solution. They were then stored in a -80°C freezer.

[0107] Example 7: RNA sequencing of Sepioteuthis lessoniana iridophores S. lessoniana skin (100 mg wet weight) was rapidly cut into small pieces and transferred to a sterile 2 mL tube. For every 100 mg of skin tissue, 1 mL of Trizol solution was added. The mixture was thoroughly vortexed for 5 min and incubated at room temperature for an additional 5 min. Two methods can be used to break down the skin tissue: A) The tissue was sonicated on ice for 3-5 cycles at 20% power and 1 s pulses at 50% duty cycle. Insoluble material was centrifuged at 15,000 rpm for 10 min and the supernatant was transferred to a new sterile 2 mL tube. B) The skin tissue incubated in Trizol was transferred to a sterile bead beater tube and filled with 0.5 mm zirconia beads to half the tube volume or until the solution nearly reached the rim of the tube. The vial was placed in the bead beater and run at maximum speed for 30 s, after which the vial was cooled on ice for 1 min. This step was repeated 2-3 times.

[0108] Lysates from either method A or B were transferred to a QiaShredder and spun at 15,000 rpm for 2 minutes, and the flow-through was transferred to a new sterile 2 mL tube. 200 μL of chloroform was added for every 1 mL of Trizol and vortexed for 30 seconds. The mixture was incubated at room temperature for 5 minutes and centrifuged at 15,000 rpm for 15 minutes. The upper aqueous fraction was transferred to a new 2 mL, 1 volume of freshly prepared 70% ethanol was added, and mixed gently with a pipette.

[0109] The solution was transferred to an RNeasy® Mini Kit column and centrifuged at 15,000 rpm for 1 minute, and the flow-through was discarded. 700 μL of solution RW1 was added to the column, centrifuged for 1 minute, and the flow-through was discarded. 500 μL of solution RPE was added to the column, centrifuged for 2 minutes, and the flow-through was discarded. This RPE step was repeated once. The column was transferred to a new sterile collection tube and spun dry for an additional minute. 40 μL of DEPC-treated water or RNase-free water was carefully added to the column, incubated for 1 minute, and centrifuged at 15,000 rpm. The extracted RNA was stored at -80°C.

[0110] PolyA selection of mRNA was performed with DynaBeads® Oligo dT and subsequently sequenced using an Illumina-compatible NEXTflex™ Rapid Directional RNA-Seq kit according to the manufacturer's protocol.

[0111] Transcriptome assembly Pooled libraries were sequenced on a HiSeq 2000 with a read length of 2 × 151. Raw fastq reads were checked with FastQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) and quality trimmed with Trimomatic. Paired-end reads were then pooled and denovo transcript assembly was performed in Trinity. Each library was then analyzed individually against this reference using RSEM to estimate the expression level of each transcript.

[0112] Protein expression A plasmid encoding the gene for Sepioteuthis lessoniana reflectin B1 (SlRF-B1) was purchased from Genscript (New Jersey, USA). The pET-28a(+) plasmid conferred kanamycin resistance but did not encode a 6x His-tag. The restriction sites selected were NcoI and XhoI. The plasmid was transformed into BL21(DE3) E. coli and protein expression was induced by the T7 promoter (isopropyl β-D-1-thiogalactopyranoside, IPTG). Tartoff Hobbs Terrific broth was used for sustained growth of E. coli with minor modifications: glycerol was increased to 10 mL per L and potassium phosphate buffer was adjusted to pH 7.4. 25 mL of preculture was supplemented with 50 μg / mL kanamycin and incubated at 37°C overnight for 16 h.

[0113] 25 mL of preculture was centrifuged at 5000xg for 5 min and the supernatant was discarded. The bacterial pellet was resuspended in 10 mL of fresh Terrific Broth and added to 1 L of Terrific Broth medium supplemented with 50 μg / mL kanamycin and 100 μL of Antifoam 204. Bacteria were grown for an additional 6 h at 37°C. Recombinant protein expression was induced with 1 mM IPTG for the next 16 h. 1 L of bacterial culture was harvested by centrifugation at 20,700xg for 5 min at 4°C (Hitachi Koki Himac CR22N, Tokyo, Japan). The cell pellet was resuspended in 50 mL of ice-cold 50 mM HEPES pH 7.4 lysis buffer and 1 mM PMSF and 10 mM DTT were added immediately before cell lysis. Cell lysis was performed using a microfluidizer (M110P, Microfluidics International Corporation, Massachusetts, USA) with three passes at 20,000 psi (137.46 MPa).

[0114] Lysed cells were centrifuged at 20,700xg for 5 min at 4°C and inclusion bodies were resuspended in 20 mL ice-cold lysis buffer supplemented with 1 mM DTT. This washing step was repeated three times, followed by one wash with 1% w / v CHAPS zwitterionic detergent and 10 mM DTT. The suspension was vortexed briefly and incubated on ice for 10 min. To remove excess detergent, the washing step was repeated three more times with lysis buffer alone and the inclusion bodies were solubilized with 15 mL of 6 M N,N'-dimethylurea alone in an ultrasonic bath at 50°C for 1 h with occasional brief vortexing. The solution was centrifuged at 20,700xg for 30 min at 4°C and the clarified supernatant was transferred to a 10 kDa regenerated cellulose dialysis membrane. The extra length of the dialysis membrane was required for at least a two-fold increase in volume. The supernatant was dialyzed for 24 h with Milli-Q® water as the only external solution, changing the water every 3 h. Soluble SlRF-B1 protein was lyophilized for at least 48 h, purged with argon gas, and stored at -20°C.

[0115] optical microscopy Optical microscopy was performed on a Zeiss A1 upright microscope. Coacervates were imaged in light transmission mode. The coacervate suspension was pipetted onto a clean glass slide with a piece of cover slip, and the magnification was set at 50x using an EC "Epiplan-Neofluar™" 50x / 0.8 HD objective. The structural color of DBCO-SlRF-B1 nanoparticles immobilized on silicon wafers was imaged in reflected light mode using an Epi Brightfield, and the magnification was set at 2.5x using an EC "Epiplan-Neofluar™" 2.5x / 0.06 HD objective. All images were taken at full resolution (2584 x 1936 pixels) using a Zeiss AxioCam MRc5 5 MP color microscope camera attached to a 60N-C 2 / 3 inch 0.63x C-mount camera adapter.

[0116] Dynamic Light Scattering (DLS) size Nanoparticle size was determined on a Malvern Panalytical Zetasizer™ Nano ZS and data was analyzed using Zetasizer software v8.01. Acquisition settings were set at a backscattering angle of 173° and three repeated measurements were performed, with each measurement having 10 acquisitions lasting 5 seconds at 25°C. Each set of acquired data was repeated three times from each batch and from three different dialysis batches. This was to ensure batch-to-batch reproducibility and stability of the nanoparticles in the buffer. Samples were pipetted into disposable UV microcuvettes (cat. no. 759200).

[0117] Size growth of SlRF-B1 with added DBCO The growth of SlRF-B1 nanoparticles doped with DBCO-sulfo-NHS ester was monitored for a total of 800 seconds. A total of 80 measurements were recorded, each acquisition lasting 2 seconds, with a 5 second delay between each measurement. The particle size at time t = 0 seconds was measured with SlRF-B1 nanoparticles after centrifugation, and 500 μL was pipetted into a cuvette. A minimum amount of dialysis buffer was used to dissolve 5 mM (1.33 mg) of DBCO-sulfo-NHS ester, which was then added to the cuvette. Homogeneous mixing was performed by rapidly pipetting the solution twice, followed by analysis. Particle number averages were used for data plotting, and three additional data points were added to extrapolate the data: 1800 seconds (30 minutes), 3600 seconds (1 hour), and 7200 seconds (2 hours). Curve fitting was performed for the scatter plots in OriginPro 2016.

[0118] Zeta potential Zeta potential measurements of nanoparticles are an important and measurable indicator of colloidal stability in dispersions. A high positive or negative (≥ ±30 mV) zeta potential confers stability to particles due to charge repulsion that resists aggregation. Samples were measured for electrophoretic mobility using a DTS1070 cuvette and converted to zeta potential using the Smoluchowski equation. The number of measurements was fixed at 50, with no delay between the three sets of measurements.

[0119] Strong cation exchange HPLC (SCX-HPLC) Strong cation exchange buffers are listed in Table 4. Ultrapure urea is dissolved in Milli-Q® water and equilibrated to room temperature. 8M urea solutions were deionized using AG 501-X8 or Bio-Rex® MSZ 501(D) mixed bed resin (Bio-Rad Laboratories, California, USA) in nylon tea bags for at least 1 hour according to the manufacturer's protocol. The resin bags were removed and buffered to pH 6.0 with MES using piperidine. The solutions in Table 4 were vacuum degassed through a Corning® 0.22 μm PES bottle top vacuum filter (φ45 mm neck, part number 431118) and placed in a vacuum safe amber glass bottle. The solution is stable at room temperature for one week. Lyophilized SlRF-B1 protein was weighed and dissolved in SCX buffer A to a stock concentration of 30 mg / mL, 10 μL of β-mercaptoethanol (1% v / v) was added, and heated at 85 °C until the protein was completely solubilized. The solution was centrifuged at 21,500 x g for 10 min at room temperature (Hitachi Koki Himac CT15E, Tokyo, Japan) and syringe filtered through a 4 mm 0.45 μm regenerated cellulose membrane. Purification was performed on a 5 μm, 1000 Å polysulfoethyl ATM semi-preparative column (PolyLC Inc., Maryland, USA). The flow rate was set to 2 mL / min, and the UV detector was set to acquire chromatograms at 254 nm and 280 nm wavelengths. The buffer gradient was set from 0% A to 20% B in 30 min. The maximum sample injection was 15 mg per 500 μL. The collected fractions were pooled together and stored at 4 °C.

[0120] [Table 4]

[0121] * Deionized Urea ** pH adjusted with piperidine Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption / ionization time-of-flight Electrophoresis was performed using a neutral PAGE system according to the manufacturer's protocol. MALDI-ToF was performed using the sinapinic acid sandwich method as previously described.

[0122] Self-organization of Sepioteuthis lessoniana reflectin B1 protein Buffers containing 10 mM MOPS at pH 7.0 and various amounts of acetonitrile (HPLC grade) were prepared accordingly based on the desired nanoparticle size. 10 mM MOPS free acid powder was added to Milli-Q® water first, followed by the required amount of acetonitrile. The buffers were mixed homogeneously on a magnetic stir plate at 200 rpm for at least 1 hour to equilibrate to room temperature. Sodium hydroxide was added to the buffer until pH 7.0, and finally brought to a final volume of 1800 mL with Milli-Q® water. Three milliliters of HPLC-purified SlRF-B1 were pipetted into a 3.5 kDa regenerated cellulose dialysis membrane and dialyzed against one of the seven buffers for 16 hours at room temperature with continuous stirring at 200 rpm. The dialyzed protein was transferred to a sterile microcentrifuge tube and centrifuged at 2,500 x g for 5 minutes at room temperature. The clarified supernatant was carefully transferred to a new sterile microcentrifuge tube. All buffer volumes were measured with graduated cylinders.

[0123] Conjugates of self-assembled SlRF-B1 with the click chemistry ligand dibenzocyclooctyne-sulfo-NHS ester Dibenzocyclooctyne-sulfo-NHS ester (DBCO-sulfo-NHS ester) was used as a ligation molecule for the self-assembly of SlRF-B1 at a concentration of 5 mM. Excess ligand (10-20 mM) did not further affect the size and stability of the nanoparticles, although lower concentrations did not stabilize the particles (the particles aggregated after a few hours). The reaction was allowed to react for 2 hours, forming a cloudy but milky yellow-orange solution indicating the presence of nanoparticles. The excess of unreacted DBCO-sulfo-NHS ester was desalted by dialysis with the same dialysis buffer used to self-assemble the specific SlRF-B1 size as the external solution. For sample volumes of 5 mL-30 mL, a home-made setup was fabricated using a 25 mm diameter, GVS or Oxyphen (Unique-Mem / Rotrack) track-etched polyester membrane (pore size 200-1000 nm) (Figure 18). Dialysis and protein concentration were performed by gravity filtration by inverting the vial and were completed within 1 hour (Table 5). The use of a large pore membrane allowed for rapid dialysis while filtering out smaller sized nanoparticles and improving overall monodispersity.

[0124] For desalting of small sample volumes of 1-3 mL, Merck Millipore Ultrafree®-MC Durapore® PVDF centrifugal filters can be used instead of dialysis. Desalting was performed three times by adding the same volume of fresh buffer (the same dialysis buffer used for self-assembly of the desired SlRF-B1 size) filtered through a Whatman® Anotop® 0.02 μm syringe filter. Alternatively, samples may be desalted for 16 h using 10 kDa regenerated cellulose dialysis bags. In case of dust particles or weak aggregates, samples were cleaned up (centrifuge at 10 × g for 30 s; resuspend and repeat) using a 5.0 μm filter.

[0125] [Table 5]

[0126] Surface functionalization of SiO2 substrates with [3-(2-aminoethylamino)propyl]trimethoxysilane and azido-dPEG®4-TFP ester A silicon P-type wafer with a native oxide film of about 3 nm was 2 Or 225mm 2 The wafers were cleaved into 1000 μm pieces and first cleaned by sonication for 5 min in 5% v / v Decon90™ detergent, followed by two washes each in anhydrous toluene, 2-propanol, and Milli-Q® water. The cleaned wafers were then immersed in 40 mL of piranha solution (3:1 ratio of sulfuric acid to 30% hydrogen peroxide) in a PFA container for 1 h. The wafers were rinsed thoroughly with Milli-Q® water and dried on a hot plate at 80° C. for 1 min. Wafers were prepared fresh for each experiment and used immediately.

[0127] The cleaned silicon wafer was placed face down on a 5 cm high homemade platform with 8 mm or 12 mm square holes to support the wafer (Figure 19). A 20 mm diameter PFA screw cap containing 500 μL of [3-(2-aminoethylamino)propyl]trimethoxysilane (AEAPTMS) was placed on the bottom of a 180 mL PFA container. The hand-tightened container was placed in a preheated oven at 130°C for 16 hours. The AEAPTMS coated substrate was sonicated twice each in anhydrous toluene, 2-propanol, and Milli-Q® water. For immediate use, the wafer was dried on a hot plate at 80°C for 1 minute.

[0128] The AEAPTMS-functionalized wafer was reacted with 10 mM azido-dPEG®4-TFP ester in an incubation buffer consisting of 10 mM HEPES and 150 mM NaCl (pH 8.0, DMSO to buffer ratio 9:1). The wafer was reacted for at least 4 hours at room temperature in a closed PFA container. The substrate was sonicated twice for 5 minutes each in anhydrous DMSO and Milli-Q® water, dried on a hot plate at 80° C. for 1 minute, and used immediately.

[0129] Azide-functionalized wafers were confirmed using 6-FAM-DBCO as a labeling fluorescent probe. 500 μM of the fluorescent probe in 10 mL was first dissolved in 1 mL of anhydrous DMSO and then added to 9 mL of 10 mM HEPES, 150 mM NaCl pH 8.0 buffer. The substrate was incubated in this solution at room temperature in the dark overnight for 16 hours. The substrate was sonicated three times for 1 min each in 50% DMSO-water and air-dried. A schematic reaction scheme is shown in Figure 20.

[0130] Fluorescence microscopy was performed using a Nikon microscope and imaged through a Plan Fluor™ 4x / 0.13 WD 16.5 objective with a Nikon DS-Ri2 CMOS camera. The imaging software used was NIS Elements D v4.5 (Build 1117). A FITC 488 nm laser wavelength was selected for excitation of the 6-FAM-DBCO fluorescent probe. Exposure was set for 5 seconds with an analog gain of 64x.

[0131] Transmission electron microscopy 4 μL of the nanoparticle dispersion was pipetted onto a TEM copper grid (Ted Pella product no. 01824) with Ultrathin C Film on Lacey Carbon support and allowed to sit for 5 min before excess sample solution was removed by absorbing with a piece of filter paper. Imaging was performed using a JEOL 2010 TEM equipped with a Gatan 794 MSC CCD and an ultra-high resolution (UHR) pole piece operated at an accelerating voltage of 200 kV.

[0132] Langmuir-Schaefer deposition of DBCO-SlRF-B1 nanoparticles A custom computer numerically controlled (CNC) machined Langmuir-Schaefer deposition trough was fitted to the 225 mm 2The trough reservoir was fabricated using aluminum grade AA 5083 for small wafers up to 100 mm in diameter. The inner edge of the trough reservoir was lined with PTFE tape. Sodium polytungstate was used as the carrier medium and the density of the solution was 2.8 gcm. -3 The solution was fixed at 100° C. A weight of 22.97 g of sodium polytungstate was added to 5.04 g of water (total volume 10 mL) and mixed uniformly with a magnetic stirrer. The pH was then adjusted to pH 7.0 with 6 M NaOH. In each experiment, 1.2 mL of sodium polytungstate was added to the trough reservoir, followed by 60 μL of nanoparticle suspension (10x concentrated). The ACN was allowed to evaporate for at least 30 min before compressing the trough. The interfacial tension (surface pressure) of the Langmuir film was monitored using a DyneProbe (perimeter 1.59 mm) using a Kibron Force Sensor KBN320 (Kibron Inc., Helsinki, Finland). These values ​​were used to plot the compression area isotherm. The azide-functionalized wafer was masked with polyester seal or aluminum tape along the edges of the square cutout. A vacuum support wafer (Pisco φ 8 mm vacuum pad (VPB8PFS-4B) connected to a 12V KnF micro gas diaphragm pump (NMP830KPDC-B-HP)) was lowered to contact the nanoparticle surface using an Edmund Optics XYZ manual stage (stock number 36-034). The click reaction was carried out for 16 hours, after which the wafer was immersed in Milli-Q® water for at least 1 hour, rinsed with Milli-Q® water, and air-dried.

[0133] Drop-casting method for DBCO-SlRF-B1 nanoparticles The as-prepared DBCO-SlRF-B1 nanoparticles were added to a 225 mm 2The wafer was drop-cast onto an azide-functionalized wafer. The wafer was warmed to 35°C and the sample solution was mixed homogeneously by very gentle pipetting every hour. This step was performed in case the settled nanoparticles formed crosses on the wafer surface, preventing proper monolayer self-assembly. The whole process took about 6 hours and mixing should be stopped when the crosses could no longer be observed. This is also the time when the sample meniscus angle is near zero. The sample solution was allowed to dry for 24 hours, after which it was immersed in Milli-Q® water for at least 1 hour, rinsed with Milli-Q® water and air-dried.

[0134] Atomic force microscopy The nanoparticle coatings were imaged using a Parks NX10 AFM (Parks System, Suwon, Republic of Korea) equipped with a NanoWorld Pointprobe NCSTR probe. Imaging was performed in non-contact mode with a focus range of 1–30 μm. 2 The desired scan area was 100 nm, image size 512 × 512 pixels, scan speed 0.25 Hz, and under ambient conditions. Image analysis and processing were performed in XEI 4.3.4.Build22. For accurate measurements on the X and Y axes, tip deconvolution estimation was performed in the software. Briefly, an AFM calibration standard with a highly defined pitch of 300 nm was used. Based on the manufacturer's recommendations, the Z height is not accurate because the probe tip may not reach the bottom of the calibration standard. The calibration image was processed in the AFM software and the data was used to perform tip estimation. This data was saved and applied to images scanned with the same probe.

[0135] Ellipsometry The thickness of successively deposited molecular layers on the wafer was measured using a JAWoollam VASE® ellipsometer controlled by WVASE32 v3.77 software. The refractive index of the materials was obtained from published literature or estimated using known properties of the molecules. The refractive indexes of AEAPTMS, azido-dPEG® 4-TFP and 6-FAM-DBCO were estimated to be n=1.444, 1.454 and 1.816, respectively. The unknown layer thickness of the nanoparticle coating on the substrate was loaded with Cauchy.mat layers. The thickness and refractive index of the Cauchy layers were fitted and the lowest mean square error (MSE) value between 1 and 20 was used.

[0136] Reflectance measurement Reflectance data were measured using an Avantes Avaspec ULS2048 spectrometer equipped with a 200 nm to 1100 nm grating mounted on a Zeiss A1 upright microscope. The fiber optic end was attached to a 60N-C 2 / 3 inch 0.63x C-mount camera adapter. A Zeiss HAL 100 microscope lamp was used as the light source. For measurements at near infrared wavelengths up to 900 nm, the infrared filter on the lamp housing was removed. The spectrometer software version used was Avasoft® 8.12.0.0. Calibration was performed using calibration tile WS-2.

[0137] Variations of the method The following summary describes other changes in experimental parameters that led to nanoparticle formation. These parameter changes were performed with the click chemistry ligand dibenzocyclooctyne-sulfo-NHS-ester during the experimental method screen unless otherwise stated. Other method parameters for surface functionalization and Langmuir-Schaefer setup are included here. The parameters described here are not optimized and there is no complete systematic study that renders the data as reliable and usable. The parameters described here provide alternatives that may be used in the future after optimization.

[0138] Nanoparticle formation 1. Changes in organic solvents The organic solvent can be replaced by alcohol (HPLC grade); ethanol and 2-propanol have been tested at concentrations of 5-15% in 5-20 mM (usually 10 mM) MOPS, MES or HEPES buffers at pH 7.0-pH 7.4, and the organic solvent concentration can be increased up to 15-50%. Nanoparticle formation was induced, but investigations into the effect of varying the alcohol concentration were not performed as thoroughly as seen with acetonitrile.

[0139] 2. Change in buffer pH with various organic solvent concentrations The SlRF-B1 self-assembly buffer was varied between pH 4.0 and pH 10.0 with 10 mM Good's buffer, and the acetonitrile concentration was varied between 5 and 50%. Nanoparticle size could be varied, but size control is unpredictable and nanoparticle stability is not guaranteed.

[0140] 3. Fully Aqueous Buffers Self-assembly of SlRF-B1 can be achieved in pure water or in any Good's buffer at 10 mM, or in aqueous buffers varying between pH 8.0 and pH 10.0 using 5-50 mM sodium borate (usually 10 mM) or imidazole (50-200 mM), with the addition of sodium chloride (0-150 mM) for ionic charge screening, and one of the two surfactants listed in Section 3.1. Buffers containing surfactants.

[0141] 3.1 Detergent-containing buffers Self-assembly of SlRF-B1 was carried out using either of two detergents, the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or the neutral detergent n-octyl-β-D-glucopyranoside, at concentrations between 0.5 and 10 mM. Detergents may not work well in buffers containing organic solvents.

[0142] 3.2 Buffers containing antioxidants Aqueous buffers were spiked with 1-10 mM ascorbic acid as an antioxidant and buffer at pH 6.0, titrated to pH 6.0 with sodium hydroxide (as sodium ascorbate) or piperidine, and either one of the two surfactants listed in Section 3.1. Buffers with Surfactants. Dibenzocyclooctyne-sulfo-NHS-esters were not used in this method, but their use could be encompassed since pH 6.0 is the lower limit for NHS ester reactivity.

[0143] 3.3 Buffers containing other organic and inorganic acids Aqueous buffers at pH 4.0 with 1 mM acetate or 0.61 mM phosphate were tested, as well as one of the two detergents listed in Section 3.1, "Buffers with Detergents." This method does not use dibenzocyclooctyne-sulfo-NHS-ester, which may not work well with NHS-ester reactions.

[0144] 4. Nanoparticle formation in organic solvents SlRF-B1 nanoparticles formation with size less than 200 nm were synthesized using methyl-β-cyclodextrin (MβCD) without dibenzocyclooctyne-sulfo-NHS-ester. 1 mg mL -1Purified SlRF-B1 protein at a concentration of 0.01% was pipetted into a 3.5 kDa regenerated cellulose dialysis membrane and dialyzed against water alone. Protein to MβCD ratios of 1:1, 1:2, and 1:4 (w / w) were investigated. No nanoparticles were detected at ratios of 1:6, 1:8, and 1:10. Samples were inverted several times until the MβCD was dissolved and then lyophilized for 24 h.

[0145] One mL of anhydrous ethyl acetate was added to the lyophilized samples and sonicated for 1 min to resuspend the protein nanoparticles. The aggregates were centrifuged at 5000 x g for 20 min at 4 °C. The supernatant was carefully transferred to a new Eppendorf tube and the centrifugation was repeated once more with the same parameters.

[0146] [Table 6]

[0147] 5. Conjugation of SlRF-B1 nanoparticles with other click chemistry ligands Nanoparticle self-assembly was tested using the click chemistry ligands listed in Table 6 below taken from Table 2, other than dibenzocyclooctyne sulfo-NHS ester. The ligands were tested in place of dibenzocyclooctyne sulfo-NHS in all experimental conditions described in Sections 1, 2 and 3. The term "dPEG" in Azido-dPEG4-TFP (Product No. 10567) is an acronym for Quanta BioDesign's "discrete polyethylene glycol" or "discrete PEG" and refers to their single molecular weight PEG technology.

[0148] [Table 7]

[0149] surface functionalization Surface functionalization has been successfully achieved using azide-dPEG4-TFP on solid substrates and dibenzocyclooctyne sulfo-NHS ester on nanoparticles, with covalent immobilization between the two occurring via copper-free strain-promoted azide-alkyne cycloaddition. Prior to this, copper-assisted click chemistry (CuAAC) and other functionalization schemes detailed below were first explored. The following functionalization method can be applied to any material surface that can be hydroxylated. Although surface functionalization with amines using APTES is described herein, the same procedure can also be applied to functionalized carboxylic acid surfaces. The choice of carboxylated or aminated ligands and their derivatives is simply reversed.

[0150] Stabilization of Reflectin B1 Nanoparticles Purified reflectin B1 (SlRF-B1) self-assembles in acetonitrile buffer at pH 7.0 and then into controlled quasi-monodisperse nanoparticles upon addition of the click chemistry ligand DBCO-sulfo-NHS ester.

[0151] The nanoparticles settle after a period of time. Nanoparticles larger than 500 nm settle within 1 day, and nanoparticles smaller than 500 nm settle within 2-5 days. The settled and compacted reflectin nanoparticles cannot be resuspended homogeneously in the same buffer, even with sonication. This is due in part to hydrophobic aggregation of the DBCO ligands on the nanoparticles, despite their zeta potential of -38 mV (borderline stability).

[0152] Nanoparticles also aggregate in solution after dialysis against fully aqueous buffers such as the DMEM buffer used in keratinocyte cell uptake studies, where acetonitrile must be removed. To mitigate aggregation, DBCO conjugated to reflectin B1 can be end-capped with an azide hydrophilic ligand.

[0153] Three ligands were selected for conjugation to DBCO-SlRF-B1 nanoparticles: Azide-PEG3-OH Azido-PEG3-phosphonic acid Azido-PEG2-sulfonic acid It should be noted that end-capping of DBCO on SlRF-B1 nanoparticles with the above-mentioned ligands effectively prevents further conjugation or modification to the surface chemistry.

[0154] Two nanoparticle sizes were tested: 360 nm and 500 nm, and each ligand (1 μL or 1 mg) was added to 500 μL of nanoparticle suspension. The click chemistry reaction was allowed to proceed for 4 hours at 40° C. Once the nanoparticle suspension had cooled, it was transferred to a 3.5 kDa regenerated cellulose membrane and dialyzed for 16-24 hours against DMEM buffer pH 7.4 as the external solution.

[0155] The nanoparticle suspension (1) that was not conjugated with any of the above ligands and dialyzed against DMEM as the external solution precipitated. Most of the precipitated nanoparticles could not be resuspended by gentle agitation or sonication.

[0156] The liganded nanoparticles (2) show slight aggregation, and most of the settled nanoparticles could be resuspended by gentle agitation. (1) The aggregates were centrifuged at 2500xg for 10 min, and the supernatant was dialyzed against water for 16-24 h and lyophilized. The non-aggregated protein content was only 30% or less.

[0157] (2) The aggregates were centrifuged at 2500 x g for 10 min, and the supernatant was dialyzed against water for 16–24 h and lyophilized. Non-aggregated protein was greater than 90% (minimal loss). The screening results in Table 7 below are preliminary and should be interpreted as a reference only. The results may even vary beyond the values ​​in the above table due to the presence of residual aggregates that still need to be optimized for removal. It was analyzed that negatively charged phosphonic and sulfonic acids induce charge repulsion, impart hydrophilicity to the PEG arms, reduce aggregation in fully aqueous buffers, and reduce the overall nanoparticle size. After dialysis and acetonitrile removal, the nanoparticles swell with increased water uptake (acetonitrile dehydrates the nanoparticles).

[0158] The ligand is not limited to the above molecules. PEG4 to PEG 36 Longer PEG arm linkers of -OH may be tested further. Phosphonic and sulfonic acids do not have longer PEG arms, but tests with carboxyl end groups with various PEG arm lengths may also be screened.

[0159] [Table 8]

[0160] 1. Surface functionalization with (3-aminopropyl)triethoxysilane (APTES) Clear glass slides and cover slips were first treated with piranha solution (sulfuric acid-30% hydrogen peroxide in a 3:1 ratio) and functionalized with aminoalkylsilane (3-aminopropyl)triethoxysilane (APTES).

[0161] 1.1 APTES functionalization by solution method The concentration of APTES in anhydrous acetone was 3 mM and cleaned coverslips were immersed in this solution for 3 hours. The coverslips were washed twice each with acetone and methanol and annealed in an oven at 150°C for 16 hours. The concentration of APTES in anhydrous ethanol was 10% v / v and cleaned coverslips were immersed in this solution for 15 minutes. The coverslips were washed five times in ethanol and annealed in an oven at 150°C for 16 hours. The concentration of APTES in anhydrous toluene was 2% v / v and cleaned coverslips were immersed in this solution for 3 hours. The coverslips were washed twice each with toluene and methanol and annealed in an oven at 150°C for 16 hours.

[0162] 1.2 APTES functionalization by vapor deposition method 1.2.1. Diluted deposition method The APTES concentration in anhydrous toluene was 1% v / v, and a cleaned coverslip was suspended in a 180 mL PFA bottle using Kapton tape. The diluted APTES solution was added to a small metal cap and placed in the PFA bottle. The bottle was purged with argon gas and placed in a 150°C oven for 16 hours.

[0163] 1.2.2. Concentrated vapor deposition method A clean coverslip was suspended in a 180 mL PFA bottle using Kapton tape. 50 μL of APTES was added to a small metal cap and placed in the PFA bottle. The bottle was purged with argon gas and placed in a 150° C. oven for 16 hours.

[0164] Contact angles for vapor-deposited APTES-coated glass slides were observed to be around 80-90°, whereas solution-based APTES coatings varied between 40-60°. Since the amines themselves are hydrophilic, a smaller contact angle suggests that more amines are present on the surface. While most of the literature has consistent results with vapor deposition, a larger contact angle could mean that the APTES molecules are oriented sideways, exposing more of the alkyl chains, or that the amines are buried below the surface due to their higher tendency to bond with hydrophilic silanol groups.

[0165] 2. Amide formation by carboxyl-amine coupling of aminoalkylsilanes with ethynyl or propargyl functional groups Carboxyl-bearing ethynyl or propargyl functional groups can be coupled with amines using standard DIC / HOBt or EDC / NHS chemistry. The following are the two molecules that were tested: 4-ethynylbenzoic acid (hydrophobic molecule); propiolic acid (hydrophilic molecule). 4-ethynylbenzoic acid at a concentration of 2 mM was dissolved in 10 mL of anhydrous N,N-dimethylformamide, followed by the addition of 2.5 mM N,N'-diisopropylcarbodiimide (DIC) and 3 mM hydroxybenzotriazole (HOBt). The reaction was carried out for 30 minutes under argon. APTES-coated glass was immersed in the solution and allowed to react at room temperature under argon for at least 6 hours.

[0166] Propiolic acid at a concentration of 20 mM was dissolved in 1 mL of 5 mM MES, 0.5 M NaCl, pH 6.0 buffer, followed by the addition of 30 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 40 mM N-hydroxysuccinimide (NHS). The reaction was carried out for 15 minutes under argon. This coupling solution was added to 29 mL of 5 mM HEPES buffer, 0.15 M NaCl (pH 7.2) containing APTES-coated glass, and the reaction was carried out at room temperature under argon for at least 6 hours.

[0167] 3. Copper(I)-catalyzed alkyne-azide cycloaddition of ethynyl or propargyl to the azide functionality Here, 1-azidomethylpyrene is used in the method as an example, and can be replaced by any azide-conjugated sample or molecule. Cycloaddition utilized copper ligand trishydroxypropyltriazolylmethylamine (THPTA) as a catalyst. Biological samples containing carboxylic acids and / or amines can be conjugated with azidopropylamine (for carboxylic acids) or azidoacetic acid (for amines), or their equivalents, using EDC / NHS (aqueous) or DIC / HOBt (non-aqueous) chemistry. However, the method must be optimized to prevent the activated carboxylic acid on one nanoparticle from reacting with the amine on another nanoparticle, which can cause aggregation.

[0168] 10 mL of 1-azidomethylpyrene at a concentration of 0.4 mM was first dissolved in 2 parts anhydrous N,N-dimethylformamide and 1 part Milli-Q® water (1 mL). 0.5 mM anhydrous copper sulfate, 0.5 mM copper ligand trishydroxypropyltriazolylmethylamine (THPTA), and 5 mM sodium ascorbate were weighed and added to the PFA vessel. 2 parts anhydrous N,N-dimethylformamide and 1 part Milli-Q® water (9 milliliters) were added to the vessel and mixed thoroughly by repeated pipetting to dissolve the solids. An ethynyl or propargyl functionalized APTES glass was immersed in the solution, followed by the addition of the 1-azidomethylpyrene solution. The vessel was purged with argon and sealed. The reaction was carried out at room temperature with stirring at 200 rpm for at least 2 hours. Anhydrous N,N-dimethylformamide may be replaced with anhydrous DMSO.

[0169] 3. Simplified coupling with triethoxy(ethynyl)silane The aminoalkylsilane APTES may be replaced by triethoxy(ethynyl)silane, eliminating the process outlined above using 4-ethynylbenzoic acid and propionic acid as intermediate molecules. The reaction scheme is shown below, using 1-azidomethylpyrene as an example. The functionalization of hydroxylated surfaces with triethoxy(ethynyl)silane is similar to that of APTES in solution and deposition processes.

[0170] Monolayer coverage of SlRF-B1 nanoparticles on functionalized wafers Another simple method for monolayer coating using only minimal materials was previously devised with ideas and references from the following publication: Ryan van Dommelen, Paola Fanzio, Luigi Sasso, Surface self-assembly of colloidal crystals for micro- and nano-patterning, Advances in Colloid and Interface Science, Volume 251, 2018, Pages 97-114, https: / / doi.org / 10.1016 / j.cis.2017.10.007. The density of sodium polytungstate solution is ρ = 1.0-3.1 g / cm. 3 The density of the substrate needs to be higher than that of the prepared sodium polytungstate solution to allow the Langmuir-Blodgett method. For the Langmuir-Schaefer method, the density of the substrate needs to be lower than that of the prepared sodium polytungstate solution. This can be applied to any substrate. The density of any protein, regardless of its molecular weight, is between 1.22 and 1.43 g / cm. 3 It has previously been determined that the density of the sodium polytungstate solution is therefore at least 1.5 g / cm 3 FKM O-ring (1.85g / cm 3 ) or general nitrile O-ring (1.00g / cm 3) were floated in the sodium polytungstate solution. FKM O-rings are preferred due to the acetonitrile content of the nanoparticle solution. The nanoparticle solution was slowly added to the O-ring and placed inside it. The acetonitrile was allowed to evaporate for at least 30 minutes. The amount of nanoparticles added must be determined experimentally using this method. The azide-functionalized wafer was masked with a polyester sticker frame along the edges and placed very gently inside the O-ring. The masking limits the nanoparticles from moving due to vibration during the click chemistry reaction and confines the loaded particles within the substrate area. The click reaction was carried out for 24-72 hours, after which the wafer was immersed in Milli-Q® water for at least 1 hour, removed, and air-dried.

[0171] Toxic effects and UV absorption properties of reflectin nanoparticles in keratinocytes Introduction: Ultraviolet (UV) rays are composed of UV-A, -B and -C (Figure 25) and are abundant in natural sunlight. UV-A and UV-B can bypass the atmosphere and accelerate skin aging. The former can penetrate deep into the dermis layer, whereas the latter causes sunburn. UV-C is mostly blocked by an ozone atmosphere, but its germicidal properties, used in germicidal lamps, pose a potential health risk. These UV wavelengths cause eye injuries (e.g., corneal irritation and inflammation) as well as skin damage (e.g., erythema). Chronic exposure to UV rays can also accelerate the skin aging process and increase the risk of skin cancer.

[0172] To mitigate the harmful effects of UV from natural and artificial sources, personal care products such as sunscreens with high SPF numbers are effective in absorbing UV rays (Figure 26). The active compounds in these sunscreens are either nano-sized zinc oxide, titanium dioxide, or a combination of both. However, in the past decade, the widespread use of these metal oxide nanoparticles has been found to have some toxicity even when applied externally to the epidermis, but these compounds have been disproved as safe when used at concentrations found in commercial products, typically 1-3%. It has been suggested that uptake of these metal oxide nanoparticles in keratinocyte cells occurs when the healthy stratum corneum is compromised. Nevertheless, the production of these nanoparticles in factories inadvertently exposes workers to nanoparticle powders, which are highly damaging to the lungs and raise concerns about bioaccumulation in target organs and long-term circulation in the body. Although there have been conflicting studies on the toxicity of zinc oxide and titanium dioxide when used in sunscreens, it is still worthwhile to identify direct alternatives to these compounds. Alternatives would likely help further reduce exposure to fine powders during manufacturing, provide a renewable and sustainable source of UV-absorbing material, and reduce reliance on titanium metal.

[0173] Commercially available formulations of zinc oxide and titanium oxide have nanoparticle sizes between 0.1-10 μm, especially less than 100 nm, because the smaller the size, the more efficient the absorption of UV wavelengths (Figure 27).

[0174] In this report, we currently only test the toxic effects of DBCO-SlRF-B1 on keratinocytes in vitro using ZnO as a control. ZnO nanopowder is commercially available below 130 nm, therefore this preliminary study is limited to a narrow range of ZnO nanoparticle sizes.

[0175] In the following sections, UV absorbance and DLS were performed with a TiO2 nanoparticle control. Additionally, toxicity studies of DBCO-SlRF-B1 in keratinocyte cells were compared to a ZnO control.

[0176] result The size of the TiO2 nanoparticle controls was first analyzed using dynamic light scattering (DLS) to determine the hydrodynamic radius. Titanium dioxide nanoparticles (10 μL) were extracted from a commercial sunscreen (Biore, Japan, SPF 50+) and resuspended in 1 mL of Milli-Q® water. The results are shown in Table 8.

[0177] [Table 9]

[0178] The UV absorbance of the TiO2 control and the sunscreen was then analyzed using a UV-Vis spectrometer (Nanodrop™ 2000c), as shown in FIG. It was observed that as the nanoparticle size increases, the absorbance red-shifts to visible wavelengths. TiO2 nanoparticle sizes above 200 nm no longer have UV absorbing properties. Therefore, for effective UV absorption, the nanoparticle size needs to be at least 200 nm or less. It was also noted that as the nanoparticle size increases, the absorbance intensity decreases (due to the reduced surface area) when compared at the same concentration.

[0179] It has been previously reported that reflectin nanoparticles conjugated with dibenzocyclooctyne (DBCO-SlRF-B1) form quasi-monodisperse nanoparticles with controllable sizes between 170 and 1000 nm. We synthesized 400 nm DBCO-SlRF-B1 and recorded its UV absorbance (Figure 29). The spectrum shows a similar UV absorbance profile to TiO2 at 100-200 nm.

[0180] A comparison of the unconjugated reflectin nanoparticle DBCO-SlRF-B1 with the larger DBCO-SlRF-B1 nanoparticle sizes of 400 nm and 660 nm is shown in Figure 17. The UV absorbance is prominent in the UV-B and UV-C regions.

[0181] Consequences of toxic effects on keratinocytes In the following preliminary studies, zinc oxide alone was used as a control compared to 400 nm DBCO-SlRF-B1. Cell viability was tabulated after 1 day of cytocompatibility testing and plotted in Figure 30. 400 nm DBCO-SlRF-B1 shows better tolerance of keratinocytes compared to the same concentration of ZnO nanoparticles (hydrodynamic radius 250-350 nm). A serum-free DMEM solution was used to allow the cells to properly uptake the nanoparticles. As a result, the experiment could not proceed beyond 2 days due to cell death.

[0182] Live / dead Alamar Blue staining and 5-FAM azide staining were performed on the cells and imaged as shown in FIG. 31 and FIG. conclusion DBCO-conjugated reflectin nanoparticles show promising results that may be further explored for use as an alternative to TiO2 and ZnO nanoparticle formulations in cosmetics and personal care products. First, DBCO-SlRF-B1 results in a similar UV absorbance profile compared to the combination of TiO2 and ZnO. Second, 400 nm DBCO-SlRF-B1 nanoparticles show better tolerance and lower cytotoxicity in keratinocytes compared to 250 nm ZnO.

[0183] The current data suggests that the UV absorption properties of DBCO-SlRF-B1 originate from DBCO itself and are independent of reflectin nanoparticle size. Therefore, further studies will be carried out to determine the toxic effects of reflectin nanoparticles in keratinocyte cells, based on the size and concentration of DBCO-SlRF-B1, compared with different TiO2 controls.

[0184] Materials and Methods Reflectin Nanoparticles Concentration 0.2μg mL -1Zinc oxide nanoparticles with a hydrodynamic radius of 250 nm were used as a control. Reflectin nanoparticles were synthesized using 17.5% v / v acetonitrile (285 ± 18 nm) and dialyzed against an aqueous DMEM solution (385 ± 26 nm) as the external solution. The final concentration was approximately 0.2 μg mL -1 .

[0185] UV absorbance of titanium dioxide nanoparticles The UV absorbance of titanium dioxide controls was analyzed using a Nanodrop (Nanodrop™ 2000c) at wavelengths of 200-800 nm. The sizes of titanium dioxide nanoparticles tested were 21 nm (anatase, Sigma Aldrich), 200 nm (rutile, Nanografi) and 490 nm (rutile, Nanografi). A weight of 1 mg of powder was weighed into a glass vial and resuspended in 1 mL of Milli-Q® water in an ultrasonic bath for 10 min.

[0186] UV absorbance of DBCO-SlRF-B1 nanoparticles DBCO-SlRF-B1 nanoparticles of 400 nm and 660 nm (0.3 mg mL -1 The UV absorbance of the nanoparticles was analyzed at wavelengths of 200-800 nm using a Nanodrop (Nanodrop™ 2000c). To improve the UV absorbance profile (signal desaturation), the 400 nm nanoparticle suspension was diluted 250 times with water.

[0187] Keratinocyte seeding Keratinocytes (HaCaT passage 20) were cultured at 75,000 cells cm using 10% FBS DMEM medium. -2 Cells were seeded onto 48-well plates at a cell density of 1000 x g / ml. Cells were incubated overnight at 37°C with 5% CO2 for 16 h. Seed medium was aspirated and washed with serum-free medium (DMEM). After washing and removal of DMEM, control and nanoparticle solutions were added. Zinc oxide nanoparticles were weighed, freshly prepared using DI water, UV sterilized for 10 min, diluted to the desired concentration, sonicated for 10 min, exchanged into DMEM buffer, and sonicated again.

[0188] Reflectin in DMEM: 2, 20, 200 μg mL -1 ZnO in DMEM: 2, 20, 200 μg mL -1 Control DMEM solution Acetonitrile 17.5% v / v was used in this experiment. Acetonitrile is cytotoxic at this concentration. 3 mL of 17.5% ACN, 82.5% HO, 10 mM MOPS, pH 7.0 was dialyzed for 24 hours at 200 rpm using a 10 kDa regenerated cellulose membrane with 500 mL of DMEM solution as the external solution. The final acetonitrile concentration in the DMEM solution after dialysis was 0.35 μL mL -1 It becomes.

[0189] Sample allocation to well plates: Negative and positive controls were allocated to 48-well plates as shown in Table 9, along with reflectin and zinc oxide nanoparticles.

[0190] [Table 10]

[0191] Legend: A1–C1, blank medium A2–C2: Cells with added control DMEM solution A3-C3, 2 μg mL -1 Reflectin A4-C4, 20μg mL -1 Reflectin A5-C5, 200μg mL -1 Reflectin D3-F3, 2 μg mL -1 ZnO D4-F4, 20 μg mL -1 ZnO D5-F5, 200 μg mL -1 ZnO Control solutions and nanoparticles in DMEM were incubated with cells for 24 hours at 37°C with 5% CO2. Cells were imaged using a bright field microscope at 4x magnification. The nanoparticle solution was removed and 200μL of 1x Alamar Blue in DMEM was added to check cell viability according to the manufacturer's protocol (Thermofisher product DAL1025). This was incubated for 1 hour and 100μL of the assay solution was analyzed using a plate reader at excitation 560nm and emission 590nm. The average results were calculated by the following formula:

[0192] Mean {(X μg / mL value-value without blank cells) / [mean (0 μg / mL value-value without blank cells)]} x 100%. The assay solution was discarded and the nanoparticle solution was added to the cells and incubated for 24 h. Afterwards, the nanoparticle solution was removed and one well was stained with 200 μL of live dead and another well was stained for DBCO-SlRF-B1 nanoparticles with the 5-FAM azide fluorescent probe. Live dead staining was performed according to the manufacturer's protocol (https: / / ibidi.com / img / cms / support / AN / AN33_Live_Dead_staining_with_FDA_and_PI.pdf). Hoescht was performed simultaneously with live dead according to the manufacturer's protocol (https: / / www.thermofisher.com / sg / en / home / references / protocols / cell-and-tissueanalysis / protocols / hoechst-33342-imaging-protocol.html).

[0193] Cell fixation and fluorescein staining Cells were fixed with 4% paraformaldehyde for 1 hour and washed three times with PBS buffer. 0.1% Triton® X-100 was added for cell permeabilization and washed three times with PBS buffer. 5-Fluorescein azide isomer (5-FAM azide) 0.1 mM was dissolved in 10 mM HEPES (pH 8.0). 5-FAM azide was incubated with cells overnight at 4°C for 16 hours, washed three times with PBS, and imaged.

[0194] Although the present disclosure has been shown and described in detail with reference to specific embodiments, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is therefore indicated by the appended claims, and all changes that come within the meaning and range of equivalents of the claims are therefore intended to be embraced.

Claims

1. A reflectin polypeptide comprising an amino acid sequence that shares at least 70% sequence identity or at least 80% sequence homology with the amino acid sequence set forth in SEQ ID NO: 1, wherein the reflectin polypeptide substantially retains the activity of reflectin B1 (SEQ ID NO: 1).

2. The reflectin polypeptide of claim 1, wherein the amino acid sequence shares at least 75%, at least 80, at least 85, at least 90, at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:

1.

3. The reflectin polypeptide according to claim 1 or 2, wherein the amino acid sequence is identical to SEQ ID NO:

1.

4. A nucleic acid molecule encoding the reflectin polypeptide of claim 1.

5. The nucleic acid molecule of claim 4 , wherein the nucleic acid molecule is contained in a vector.

6. The nucleic acid molecule of claim 5 , wherein the vector further comprises a regulatory element for controlling expression of the nucleic acid molecule.

7. A host cell comprising a nucleic acid molecule according to claim 4, which is preferably a bacterial cell.

8. A recombinant reflectin nanoparticle comprising the reflectin polypeptide of claim 1.

9. The recombinant reflectin nanoparticle of claim 8 , wherein the reflectin polypeptide is conjugated to a ligand.

10. The recombinant reflectin nanoparticle of claim 9 , wherein the ligand is covalently bound to the reflectin polypeptide via a peptide bond.

11. The ligand has the following formula (I): 【Chemical 1】 (In the formula, CG is a linking group; n is an integer selected from 1 to 5; 【Chemistry 2】 The recombinant reflectin nanoparticle of claim 10, wherein: indicates a point of attachment to a nitrogen atom of the reflectin polypeptide to form a peptide bond.

12. CG is represented by the following formula (II): 【Chemistry 3】 (In the formula, 【Chemistry 4】 is the —(CH ２ ) ｎ The recombinant reflectin nanoparticle of claim 11, wherein the ribonucleotide is a nucleotide sequence of the ribonucleotide sequence (SEQ ID NO: 1), which is a nucleotide sequence of the ribonucleotide sequence of the ribonucleotide sequence (SEQ ID NO: 2), which is a nucleotide sequence of the ribonucleotide sequence of the ribonucleotide sequence (SEQ ID NO: 3), which is a nucleotide sequence of the ribonucleotide sequence of the ribonucleotide sequence (SEQ ID NO: 4),

13. The recombinant reflectin nanoparticle according to claim 11 or 12, wherein n is 4.

14. CG is -N ３ The recombinant reflectin nanoparticle of claim 11,

15. The recombinant reflectin nanoparticle of claim 8, wherein the reflectin polypeptide comprises an isoelectric point greater than 7.

16. The recombinant reflectin nanoparticle of claim 8, wherein the nanoparticle has a polydiversity index of less than 0.

5.

17. 9. The recombinant reflectin nanoparticle of claim 8, wherein the recombinant reflectin nanoparticle has a zeta potential in the range of about -30 to about -100 mV.

18. The recombinant reflectin nanoparticle of claim 8, wherein the recombinant reflectin nanoparticle is amorphous.

19. 9. A method for synthesizing recombinant reflectin nanoparticles according to claim 8, said method comprising: recombinantly expressing a reflectin polypeptide; purifying the reflectin polypeptide by chromatography; and allowing the purified reflectin polypeptide to self-assemble into nanoparticles; A method comprising:

20. 20. The method of claim 19, further comprising conjugating the purified reflectin polypeptide to a ligand.

21. 20. The method of claim 19, wherein the chromatography is carried out in a pH range above pH 5.

0.

22. 20. The method of claim 19, wherein the self-assembly is carried out in a buffer containing an organic solvent.

23. The method of claim 22, wherein the concentration of the organic solvent determines the size of the recombinant reflectin nanoparticles.

24. 24. The method of claim 23, wherein the organic solvent is acetonitrile.

25. 25. The method of claim 24, wherein the size of the recombinant reflectin nanoparticles is substantially linearly related to the concentration of the acetonitrile.

26. A substrate surface-functionalized with the recombinant reflectin nanoparticles of claim 8.

27. 27. The substrate of claim 26, wherein the recombinant reflectin nanoparticles are covalently immobilized on the surface of the substrate.

28. 28. The substrate of claim 27, wherein the recombinant reflectin nanoparticles are assembled on the surface as substantially a monolayer.

29. 27. The substrate of claim 26, wherein the distance from one recombinant reflectin nanoparticle to another is less than 1 micrometer.

30. 9. A method for immobilizing recombinant reflectin nanoparticles according to claim 8 on a substrate, the method comprising: providing a substrate containing a hydroxy group; reacting said hydroxy group with a surface-bound spacer chain; providing a recombinant reflectin nanoparticle according to claim 8 and reacting the recombinant reflectin nanoparticle with the surface-bound spacer chain; A method comprising:

31. The method of claim 30 , wherein the surface-bound spacer chain comprises an organosilane.

32. The method of claim 30, wherein the recombinant reflectin nanoparticle comprises a triple bond functional group, the surface-bound spacer chain comprises an azide functional group, and the method further comprises reacting the triple bond functional group with the azide functional group to form a covalent bond.

33. A skin care product comprising the recombinant reflectin nanoparticles of claim 8.

34. 34. The skin care product of claim 33, wherein the recombinant reflectin nanoparticles have a size of about 350 to about 450 nanometers.

Images