Edible non-clonable functional components

Abstract

A method of generating a physically unclonable function component (PUF) for authentication of a pharmaceutical is disclosed, the method comprising generating an edible (PUF), attaching the edible PUF to the pharmaceutical in a randomly distributed manner, and generating an encryption key based on the attached randomly distributed edible PUF. The present application generally relates to anti-counterfeiting measures, and in particular, to arrangements for edible unclonable function component anti-counterfeiting measures.

Description

[0001] Cross-references to related applications

[0002] This patent application relates to and claims priority to U.S. Provisional Patent Application No. 62 / 915,667, entitled "EDIBLE UNCLONABLE FUNCTIONS", filed October 16, 2019, and U.S. Provisional Patent Application No. 62 / 915,666, entitled "IMAGE PROCESSING AND AUTHENTICATION OF EDIBLE UNCLONABLE FUNCTIONS", filed October 16, 2019, the contents of each of which are hereby incorporated herein by reference in their entirety.

[0003] Statement regarding government funding

[0004] This invention was developed with government support under license number FA2386-17-1-4072 granted by the U.S. Air Force Office of Scientific Research. The government holds certain rights to this invention. Technical Field

[0005] This application generally relates to anti-counterfeiting measures, and in particular to the arrangement of anti-counterfeiting measures for edible, non-clonable functional components. Background Technology

[0006] The information presented in this section is intended to help in a better understanding of aspects of this application. Therefore, these statements should be understood in this light and should not be construed as an admission of what is prior art or not prior art.

[0007] Counterfeit medicines have become ubiquitous, causing countless problems. This problem is not new, but is becoming a huge burden on societies in all countries. While “fake” pharmaceutical products can be clearly classified into several categories, including substandard, counterfeit, fake, and transferred products, they are often referred to as a single group as counterfeit medicines. They pose a significant threat to patient safety and public health, and cause substantial economic losses in both developed and developing countries. As a particularly devastating example, counterfeit drugs used to treat malaria and pneumonia cause an estimated 250,000 deaths in children each year. The increasing production of counterfeit medicines for both lifestyle medications (e.g., for erectile dysfunction) and life-saving medications (e.g., for cancer, malaria, diabetes, etc.) in both developed and developing countries is partly due to the increasing use of online pharmacies. Furthermore, as a violation of intellectual property rights, the scientific innovation and economic returns of pharmaceutical companies are undermined by widespread counterfeit medicines. In low- and middle-income countries, the health and economic consequences of counterfeit medicines are far more severe. It is estimated that counterfeit drugs account for 10% of the global pharmaceutical trade and more than 20%-30% of all pharmaceuticals in Africa, Asia and the Middle East.

[0008] There are various methods available for detecting counterfeit drugs and for providing potential solutions to reduce the threat. Traditionally, analytical chemistry and spectroscopic techniques have been used to identify counterfeit drugs by detecting the chemical characteristics of the main ingredients. However, these techniques require complex and expensive machinery and have limited accuracy based on the identification of such main ingredients. Other techniques include marking and printing on the surface of drugs at various resolution levels using lasers and other proprietary techniques. These markings and printings modify the outer surface or coating of tablets or capsules; however, this technique is easily copied by counterfeiters. Recently, digital anti-counterfeiting technologies have played a more significant role in authentication and the supply chain. Packaging-level barcodes and radio frequency identification (RFID) are commonly used for instant remote authentication. Several mobile technologies have been introduced for authentication services, tracking and tracing solutions, and drug identification. Disadvantageously, such authentication and security technologies are symmetrical; that is, if an illegal manufacturer or distributor has access to the same technology, they can create clones. The ideal authentication technology should be asymmetrical, with a dose-dependent (on-dose / in-dose) authentication format that can be swallowed and digested directly. Specifically, dosage certification means verifying the authenticity of each individual pill or dose without the original packaging. This essentially eliminates the possibility of ingesting counterfeit medications, even if the pharmacist or patient does not retain the original packaging. In practice, packaging information is often unavailable; pills are sold in small quantities and dispensed in individual strips by pharmacists. Dosage certification minimizes the opportunity for illicit sellers to use expired, counterfeit, or substandard medications.

[0009] In this regard, several promising technologies with digital authentication potential have recently been introduced, including: digitally encoded polymers, QR-coded microtaggants and advanced wrinkle-based labels, QR code printing of active pharmaceutical ingredients, encoded multifunctional hydrogel microparticles, large-scale microparticle arrays, encoded metal nanomaterials, and silica microtags. However, such materials are generally not ideal from an oral safety perspective. These methods rely on biocompatible and biodegradable but exogenous materials such as polystyrene, cellulose acetate phthalate (CAP), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), poly(ethylene glycol) diacrylate (PEGDA), silver, gold, and silica. It should be noted that exogenous nanoscale food additives may potentially have dangerous and adverse effects (e.g., carcinogenic and cytotoxic), which currently leads to limited applications. Furthermore, due to the limited safety level, such simple labeling technologies may be vulnerable to attack.

[0010] Therefore, there is an unmet need for a novel approach to provide drugs with non-clonable safety against the widespread availability of counterfeits. Summary of the Invention

[0011] A method for generating physically unclonable functional parts (PUFs) for drug authentication is disclosed. The method includes: generating edible physically unclonable functional parts (PUFs); attaching the edible PUFs to a drug in a random distribution; and generating an encryption key based on the attached randomly distributed edible PUFs.

[0012] According to one implementation of the above method, edible PUF includes silk protein.

[0013] According to one embodiment of the above method, the edible PUF includes an edible polymer.

[0014] According to one implementation of the above method, edible PUF is based on randomly distributed edible fluorescent proteins.

[0015] According to one implementation of the above method, the edible PUF is based on a randomly distributed edible fluorescent dye.

[0016] According to one embodiment of the above method, edible PUF is based on randomly distributed fluorescent protein expression filament particles.

[0017] According to one embodiment of the above method, edible PUF is based on randomly distributed edible polymer particles containing edible fluorescent dyes.

[0018] According to one implementation of the above method, fluorescent protein-expressing silk can be produced by genetically engineered domesticated silkworms.

[0019] According to one implementation of the above method, gene splicing is used to express transgenes through germline transformation.

[0020] According to one implementation of the above method, gene splicing is based on piggyBac.

[0021] According to one embodiment of the above method, the fluorescent filament protein is selected from the group including enhanced cyan fluorescent protein (eCFP), enhanced green fluorescent protein (eGFP), enhanced yellow fluorescent protein (eYFP), and mKate2 (far-red) fluorescent protein.

[0022] According to one embodiment of the above method, the edible polymer is selected from the group consisting of starch, cellulose derivatives, chitosan, pectin, alginate, gum, carrageenan, and combinations thereof.

[0023] According to one embodiment of the above method, the edible polymer is selected from the group consisting of gelatin, collagen, albumin, milk protein and combinations thereof.

[0024] According to one embodiment of the above method, the edible polymer is selected from the group consisting of zein, soybean, wheat gluten, lectins and combinations thereof.

[0025] According to one embodiment of the above method, the edible polymer is selected from the group consisting of fatty acids, triglycerides, phospholipids and combinations thereof.

[0026] According to one embodiment of the above method, the edible fluorescent protein is selected from the group consisting of red fluorescent protein (DsRed), orange fluorescent protein (mKO), and combinations thereof.

[0027] According to one embodiment of the above method, the edible fluorescent dye is selected from the group consisting of Brilliant Blue FCF, Indigo, Fast Green FCF, Erythrosine, Allura Red AC, Tartrazine, Sunset Yellow FCF, and combinations thereof.

[0028] A method for manufacturing physically unclonable functional components and applying them to a drug is also disclosed. The method includes constructing a plasmid vector DNA for silkworm transgenesis, thereby generating one or more fluorescent silk proteins. The method further includes:

[0029] The method involves removing sericin from one or more fluorescent silk proteins. Furthermore, the method includes cutting the one or more fluorescent silk proteins from which sericin has been removed into multiple pieces. Additionally, the method includes washing the cut pieces with a solution, filtering the dissolved solution, dialyzing the filtered dissolved solution, and centrifuging the dialyzed solution to provide a regenerated silk protein solution. Furthermore, the method includes freeze-drying the regenerated silk protein solution, mechanically grinding the freeze-dried silk protein into granular silk microparticles, sieving the mechanically ground silk protein to generate fluorescent silk microparticles, and a mixture of the generated fluorescent silk microparticles. Furthermore, the method includes dispersing the mixture of fluorescent silk microparticles onto the surface of a polystyrene petri dish to generate a silk membrane suitable for drug adhesion.

[0030] According to one embodiment of the above method, one or more fluorescent filament proteins include eCFP, eGFP, eYFP, and mKate2.

[0031] According to one embodiment of the above method, the washing of one or more fluorescent silk proteins is carried out at a temperature below 50°C in a mixed solution of Na2CO3 (0.2%).

[0032] According to one embodiment of the above method, the cut sericin-removed one or more fluorescent silk proteins are in the range of 2 mm to 5 mm.

[0033] According to one embodiment of the above method, the cut blocks are dissolved in an aqueous solution of LiBr (9.5M) at 45°C and stirred at 400 rpm for four hours.

[0034] According to one embodiment of the above method, the dissolved solution is filtered through a Mirabu filter.

[0035] According to one embodiment of the above method, the filtered solution is dialyzed in deionized water at room temperature using a cellulose semipermeable tube for about two days to remove LiBr.

[0036] According to one embodiment of the above method, the dialyzed solution is centrifuged at about 4°C at a speed of 9000 rpm for about 20 minutes to form a regenerated silk core protein solution.

[0037] According to one embodiment of the above method, the regenerated silk protein solution is freeze-dried at approximately -18°C for seven days.

[0038] According to one embodiment of the above method, the freeze-dried fluorescent filament is mechanically ground into granular particles using a mortar and pestle, a power mixer, or a combination thereof.

[0039] According to one embodiment of the above method, the sieving is performed to generate fluorescent filament particles with a predetermined size range by shaking the fluorescent filament particles through a stack of two standard test sieves with corresponding openings.

[0040] According to one embodiment of the above method, fluorescent filament particles are mixed in a microcentrifuge tube in a ratio of 1:1:1:1 (eCFP, eGFP, eYFP and mKate2 filaments) and then shaken by hand.

[0041] According to one embodiment of the above method, the dispersion of the mixture of fluorescent filament particles onto the surface of a polystyrene petri dish is carried out by mechanically shaking the particles through a sieve with a predetermined opening size. Attached Figure Description

[0042] Figure 1a This is a schematic diagram illustrating the application of edible, physically non-clonable functional parts (PUFs) used in conjunction with pharmaceuticals.

[0043] Figure 1b A schematic diagram is shown of the photoluminescence properties of a fluorescent silk protein for achieving multiple challenge-response pairs in an edible PUF platform to obtain improved safety, according to the contents of this application.

[0044] Figure 1c This is the output of a scanning electron microscope (SEM) of an edible PUF device in which fluorescent filament particles are embedded in a thin filament membrane.

[0045] Figure 1d These are the emission spectra of granular eCFP, eGFP, eYFP, and mKate2 filaments, which cover a relatively wide wavelength range in the visible light spectrum, with non-overlapping emission peak positions.

[0046] Figure 1e Confocal fluorescence microscopy images of the corresponding fluorescent filament particles of eCFP, eGFP, eYFP and mKate2 filaments under excitation at 405 nm, 458 nm, 514 nm or 561 nm are provided.

[0047] Figure 2 It is a transformation vector p3xP3-DsRed2-FibH-(eCFP, eGFP, eYFP or mKate2) used to produce fluorescent filaments in transgenic silkworms.

[0048] Figure 3 White light and fluorescence images of eCFP, eGFP, eYFP, and mKate2 silk cocoons produced by transgenic silkworms via the piggyBac transposase method are provided.

[0049] Figure 4 and Figure 5The graphs show the normalized absorption and normalized intensity versus wavelength (in nm) for eCFP, eGFP, eYFP, and mKate2, respectively.

[0050] Figure 6 This is a schematic diagram of a silk protein regeneration and edible PUF device manufactured according to the contents of this application, using fluorescent filaments (i.e., eCFP, eGFP, eYFP and mKate2 filaments) and natural white filaments.

[0051] Figure 7 This is a bar graph showing the size distribution of fluorescent filament particles in edible PUFs according to the contents of this application.

[0052] Figure 8 These are photographs of 30 different edible PUF devices used to characterize the overall performance of PUF, according to the contents of this application.

[0053] Figure 9 A series of fluorescence intensities versus time (seconds) are shown, depicting the time-photostability of eCFP, eGFP, eYFP, and mKate2 filament particles under excitation at 440 nm for eCFP, eGFP, and eYFP filaments and at 514 nm for mKate2 filaments.

[0054] Figure 10a and Figure 10b This describes the scalability (Figure 10a) and flexibility (of the proposed edible PUF for mass production and easy integration with pharmaceuticals) of the PUF. Figure 10b Two photos.

[0055] Figure 11 This is a schematic diagram of an exemplary custom imaging setup for obtaining fluorescent images of an edible PUF device, according to the contents of this application.

[0056] Figure 12 A graph of probability versus normalized Hamming distance (HD) is provided to illustrate intra-device (reproducibility) and inter-device (uniqueness) variability at a cutoff threshold of 0.1808.

[0057] Figure 13 This is a schematic diagram illustrating the feasibility of dosage (on-dose or in-dose) certification for the use of edible PUF.

[0058] Figure 14 A series of photographs are provided to depict the water solubility of the edible PUF composed of silk protein and fluorescent protein according to the contents of this application.

[0059] Figure 15This is a schematic diagram of a dosage certification concept, in which each individual drug product in a solid oral dosage form (e.g., tablets and capsules) is combined by the pharmaceutical manufacturer with an edible PUF device. Detailed Implementation

[0060] The patent or application document contains at least one color drawing. A published copy of this patent or patent application with color drawings will be provided by the official upon request and after payment of the necessary fees.

[0061] To facilitate an understanding of the principles of this application, reference will now be made to the embodiments shown in the accompanying drawings, and these embodiments will be described using specific language. However, it will be understood that this is not intended to limit the scope of this application.

[0062] In this application, the term "about" may allow for a certain degree of variability in the value or range, for example, within 10%, 5%, or 1% of the limits of the value or range.

[0063] In this application, the term "substantially" may allow for a certain degree of variability in the value or range, for example, within 90%, 95%, or 99% of the limits of the value or range.

[0064] An excellent method for ensuring high safety in dosage authentication and preventing counterfeit pharmaceuticals is to utilize Physically Unclonable Functional Components (PUFs). A PUF relies on the uniqueness of the physical microstructure that defines it. This uniqueness depends on numerous random physical factors introduced during manufacturing; and assuming these factors are unpredictable and uncontrollable, replication is essentially impossible. Instead of using a single encryption key that could be decoded and used without authorization, a PUF implements challenge-response authentication to authenticate the associated microstructure. When a physical stimulus is applied to this structure, it responds in a reproducible manner, although in an unpredictable way. The applied stimulus is called a challenge, and the PUF's response is called the associated response. Such specific attacks and their corresponding responses are maintained in a secure database, and authentication can therefore be checked against such a database. To further enhance security, the challenge-response and its communication with the secure database can be encrypted.

[0065] Importantly, PUFs can be asymmetric, making them easy to manufacture but extremely challenging for counterfeiters to create clones. Once an output response is read from the database, it cannot be reused. Information regarding dosage, frequency, and warnings can be coded for user compliance by tagging individual medications.

[0066] For digital dosing PUF, this application proposes silk protein and fluorescent protein as edible and digestible photonic biomaterials. From an edibility perspective, important considerations include digestibility and non-allergenicity. Therefore, endogenous natural or biomaterials are selected for dosing applications. Importantly, silk protein (i.e., filamentin) possesses excellent intrinsic functionality, biocompatibility, and low immunogenicity, while exhibiting minimal inflammatory and immune responses. Naturally derived filamentin is dissolved in aqueous solution without any external treatment. Silk protein is also biodegradable, and the degradation rate can be controlled using various silk regeneration and manufacturing methods. More relevantly, silk protein is edible and digestible. Additionally, fluorescent proteins have been introduced into food supplies from genetically modified foods. The potential toxicity and allergenicity after ingestion of green fluorescent protein are minimal. Fluorescent proteins do not possess common allergen epitopes compared to common food allergens and are degraded during gastric digestion. From an engineering perspective, the processing of silk protein allows for easy construction of structures and patterns ranging from nanoscale to microscale. In particular, the polymeric properties of silk proteins allow for the easy fabrication of various types of rigid or flexible structures with tunable mechanical and optical properties. Furthermore, silk proteins containing recombinant fluorescent proteins can be produced through transgenic modification of domesticated silkworms using genetic engineering. Transgenic modifications of multiple fluorescent proteins can be expressed via germline transformation using the piggyBac gene splicing method. This hybridization method can produce multiple generations of transformed silkworms and generate abundant fluorescent filaments.

[0067] To this end, this application provides a fully protein-based PUF that generates cryptographic keys with multiple interactive challenge-response pairs for dosage authentication and anti-counterfeiting of pharmaceuticals. The edible PUF is made of silk (i.e., silkworm (Bombyxmori)) protein microparticles fused with different fluorescent protein genes, including enhanced cyan fluorescent protein (eCFP), enhanced green fluorescent protein (eGFP), enhanced yellow fluorescent protein (eYFP), and mKate2 (far-red) fluorescent protein. Granular fluorescent filaments are embedded in a thin film of natural white silk protein, which can be directly attached to the surface of a solid oral dosage form of pharmaceutical product. The entropy source is a randomly dispersed (or scattered) mixture of fluorescent microparticles of eCFP, eGFP, eYFP, and mKate2 filaments, as the behavior of granular or particulate materials inherently exhibits complex spatiotemporal fluctuations during manufacturing. For the challenge-response requirements of the PUF, a unique set of excitation and emission bands of different fluorescent proteins serves as the input challenge. The edible PUF produces a truly inherent output response pattern of spontaneous emission (i.e., fluorescence). The reported protein-based PUF exhibits strong randomness, ensuring a unique and unpredictable cryptographic key with a relatively large coding capacity. Basic PUF functionality, including homogeneity, uniqueness (for security), and reproducibility (for reliability), is further characterized by calculating inter-device Hamming distance (HD), intra-device HD, false positive rate, and false negative rate. The reported protein-based PUF can provide a highly secure and immediate solution for dosage authentication and anti-counterfeiting of pharmaceuticals.

[0068] Fluorescent filaments were generated by fusing the genes of fluorescent proteins (eCFP, eGFP, eYFP, and mKate2) via germline transformation (i.e., piggyBac transposons) and native white filaments. The following chemicals were used: dialysis tubing (12000 DaMWCO pore size), lithium bromide (LiBr, ≥99%), miracloth (22 μm to 25 μm pore size), sodium carbonate (Na2CO3, ≥99%), and Triton X100 from Sigma-Aldrich Co. (Milwaukee, Wisconsin, USA). To select appropriate particle sizes and disperse the fluorescent filament particles, two standard test sieves with opening sizes of 90 (No. 170) μm and 106 (No. 140) μm from Cole-Parmer (Niles, Illinois, USA) were used. All experiments were conducted under ambient conditions (22 ± 2 °C and 40 ± 10% relative humidity). It should be noted that, for the sake of edibility and safe consumption, any organic solvents and synthetic polymers should be completely avoided.

[0069] Information can be extracted from the PUF using various image processing techniques and securely checked against a database for authentication. Such a novel technique is described in a sister patent application filed on the same day as this patent application, entitled “IMAGE PROCESSING AND AUTHENTICATION OF UNCLONABLE FUNCTIONS”.

[0070] Reference Figure 1a The diagram shows an edible PUF used in conjunction with a drug. Figure 1a A schematic illustration of a dose-dependent PUF is shown, with a photograph of a concealed and transparent PUF attached to the surface of the drug substance. The PUF device is composed of proteins derived from edible and digestible filaments and fluorescent proteins. The different photoluminescent properties of the fluorescent proteins in the filaments provide parametric support for unique challenge-response pairs. In response to an input challenge, the edible PUF generates its corresponding output response. Figure 1b The photoluminescent properties of fluorescent silk proteins are demonstrated for use in implementing multiple challenge-response pairs in an edible PUF platform to achieve improved safety. Figure 1c This is a photograph of an edible PUF device in which fluorescent filament microparticles are embedded in a thin filament membrane. An SEM image of the fluorescent filament microparticles with a zeolite-like shape is shown. Figure 1d These are the emission spectra of granular eCFP, eGFP, eYFP, and mKate2 filaments, which cover a relatively wide wavelength range in the visible light spectrum, with non-overlapping emission peak positions. Figure 1e These are confocal fluorescence microscopy images of the corresponding fluorescent filament particles under excitation at 405 nm, 458 nm, 514 nm, or 561 nm. The scale bar is 100 μm. The size of the fluorescent filament particles is 99.3 ± 7.9 μm (mean ± standard deviation). Importantly, the challenge-response pair distinguishes the protein-based PUF of this application from other common unique objects and tags. As a response to an optical challenge defined by a unique set of excitation and emission bands of different fluorescent proteins, an edible PUF made of silk proteins (i.e., silkcore proteins) and fluorescent proteins generates different output responses. The source of entropy is the randomly distributed fluorescent filament particles seamlessly embedded in a concealed thin transparent silk membrane. First, four different fluorescent proteins (i.e., eCFP, eGFP, eYFP, and mKate2) with specific excitation and emission peaks in the visible wavelength range (provided in Table 1) were used.

[0071] Table 1. Optical properties of fluorescent proteins hybridized with silk genes.

[0072]

[0073] Specifically, via the piggyBac transposase method (see...) Figure 2(As described below) The recombinant protein is produced from the silk of transgenic silkworms expressing fluorescent protein. Silk protein is an excellent biopolymer that genetically hybridizes with the fluorescent protein gene. Secondly, in order to manufacture fluorescent filament microparticles (see discussion below)... Figure 6 The fluorescent filamentin was regenerated into an aqueous solution using a low-temperature process, freeze-dried, and then gently ground into zeolite-like particles with a size of 99.3 ± 7.9 μm (mean ± standard deviation) (see discussion below). Figure 1b and Figure 1c as well as Figure 7 Third, the mixture of fluorescent filament particles was spread on a large, flat surface, and a white silk protein solution was poured onto it. After ambient drying in the dark, the thin, transparent filament film, which was 150 μm thick, was punched to a size of 7 × 7 mm. 2 A square shape is formed, thus yielding an edible PUF device that is entirely protein-based (see discussion below). Figure 6 and Figure 8 The eCFP, eGFP, eYFP, and mKate2 cocoons exhibit light blue, light green, slightly yellow, and slightly reddish hues, respectively, under white light (see discussion below). Figure 3 However, after filament regeneration, each type of filament particle is indistinguishable to the naked eye while retaining its fluorescence properties (see discussion below). Figures 1b to 1e and Figure 9 The manufacturing process can be scaled up for mass production without the use of any complex equipment and is safe for oral administration without the use of any organic solvents or synthetic polymers (such as methanol, ethanol, isopropanol, or polyvinyl alcohol) (see Figure 10 discussed below).

[0074] Reference Figure 1b This study demonstrates regenerated granular eCFP, eGFP, eYFP, and mKate2 silk, produced via piggyBac transposase method through silkworm transgenic synthesis, in which fluorescent proteins and silk (i.e., Bombyx mori) are fused into recombinant proteins. Figure 1b In, as specified at the top of each image, a set of excitations (λ) is used. ex ) and emission (λ) em Fluorescence images were acquired using wavelengths specified. The scale bar is 5 mm. Figure 1c Photographs of an edible PUF device in which fluorescent filament particles are embedded in a thin filament membrane are provided. SEM images of the fluorescent filament particles with a zeolite-like shape are also provided. Figure 1d Emission spectra of granular eCFP, eGFP, eYFP, and mKate2 filaments are provided, covering a relatively wide wavelength range in the visible light spectrum, with non-overlapping emission peak positions. Figure 1eThese are confocal fluorescence microscopy images of the corresponding fluorescent filament particles excited at 405 nm, 458 nm, 514 nm, or 561 nm. The scale bar is 100 μm. See below for further discussion. Figure 7 As shown, the size of the fluorescent filament particles is 99.3 ± 7.9 μm (mean ± standard deviation).

[0075] According to this application, when optically challenged, an encryption key is extracted from the output response. This encryption key includes the original output measurement, bitstream extraction, and a final digitized security key. According to one embodiment, four representative challenge-response pairs (n=4) are used based on the excitation and emission peak wavelengths of individual fluorescent proteins in the filament. The input challenge (C... n The combination of excitation and emission bands is selected for a specific wavelength, for example, λ. ex =415nm and λ em =460nm; λ ex =470nm and λ em =510nm; λ ex =470nm and λ em =560nm; λ ex =530nm and λ em =630nm, which correspond to eCFP, eGFP, eYFP, and mKate2 in the filament, respectively. After photoexcitation, the raw fluorescence image is recorded by a charge-coupled device (CCD) camera equipped with a conventional zoom lens via a tunable color filter that can be used for downstream optical recognition arrangements (see discussion below). Figure 11 ).

[0076] Reference Figure 3 The image shows white light and fluorescence images of silk cocoons containing eCFP, eGFP, eYFP, and mKate2 transgenic silk produced via the piggyBac transposase method from silkworms. The fluorescence images are taken at a set of excitation wavelengths (λ) specified on each photograph. ex ) and emission wavelength (λ) em (Photos taken by a group. Scale bar is 10mm.)

[0077] Reference Figure 4 and Figure 5 The normalized absorption and fluorescence spectra of eCFP, eGFP, eYFP, and mKate2 transgenic filaments are shown. The excitation wavelengths of eCFP, eGFP, eYFP, and mKate2 filaments are 415 nm, 470 nm, 470 nm, and 530 nm, respectively.

[0078] Reference Figure 6This illustration shows a schematic diagram of the fabrication of a silk protein regeneration and edible PUF device using fluorescent filaments (i.e., eCFP, eGFP, eYFP, and mKate2 filaments) and natural white silk, according to the contents of this application. The construction of the plasmid vector DNA for silkworm transgenesis is discussed first. Using germline transformation (i.e., piggyBac transposons), the transition vectors pBac-3xP3-DsRed2-pFibH-(eCFP, eGFP, or eYFP) and pBac-3xP3-eGFP-pFibH-mKate2 are constructed as piggyBac-derived vectors, and the vector DNA with the helper vector is injected into the forembryo layer embryo. (Refer to...) Figure 2The diagram shows the transformation vector p3xP3-DsRed2-FibH-(eCFP, eGFP, eYFP, or mKate2) used to generate fluorescent filaments in transgenic silkworms. In the case of mKate2, eGFP is used instead of DsRed2. The nucleotide sequences of pFibH-NTR and CTR were obtained from GenBank accession number AF226688. pFibH: filamentin heavy chain promoter domain (1124bp), NTR1: N-terminal region 1 (142bp), intron: first intron (871bp), NTR2: N-terminal region 2 (417bp), CTR: C-terminal region (179bp), PolyA: poly(A) signal region (301bp), eCFP: enhanced cyan fluorescent protein, eGFP: enhanced green fluorescent protein, eYFP: enhanced yellow fluorescent protein, mKate2: monomeric far-red fluorescent protein, DsRed2: red fluorescent protein (a mutant form of DsRed from Discosoma sp.), ITR: inverted repeat sequence of piggyBac arm, 3xP3: 3xP3 promoter, and SV40: SV40 polyadenylation signal sequence. Restriction enzyme sites used to construct the recombinant vector are indicated by arrows. To construct the plasmid, DsRed2 (eGFP of mKate2) cDNA was amplified by polymerase chain reaction (PCR) using specific primers with NheI / AflII sites from pDsRed2-C1 (NheI-DsRed2-F: 5'-GCTAGCATGGCCTCCTCCGAGAAC-3', and DsRed2-AflII-R: 5'-CTTAAGCTACAGGAACAGGTGGTGGCG-3'; Clontech, Mountain View, California, USA) as a marker, and then cloned into the pGEM-T Easy Vector System (Promega, Co.), named pGEMT-DsRed2. The DsRed2 gene was excised from pGEM-DsRed2 digested with the restriction enzyme NheI / AflII and replaced with the eGFP gene from pBac-3xP3-eGFP to form pBac-3xP3-DsRed2.To obtain the filamentin promoter, a DNA fragment containing the N-terminal region (1430 bp) of an intron (972 bp) of the filamentin H gene (GenBank accession number AF226688, nt. 61312-63870) and a promoter domain (1124 bp) was amplified by PCR using genomic DNA from the silkworm (Bombyx mori) and primers (pFibHN-F: 5'-GGCGCGCCGTGCGTGATCAGGAAAAAT-3' and pFibHN-R: 5'-TGCACCGACTGCAGCACTAGTGCTGAA-3'). This fragment was then cloned into the pGEM-T Easy Vector System. The resulting DNA fragment was named pGEMT-pFibH-NTR. A DNA fragment containing a 180 bp 3' end sequence of the H-chain gen open reading frame (ORF) and a 300 bp 3' region of the fibroin H gen (GenBank accession number AF226688, nt.79021-80009) was amplified by PCR using genomic DNA from the silkworm (Bombyx mori) and primers (pFibHC-F: 5'-AGCGTCAGTTACGGAGCTGGCAGGGGA-3', and then cloned into the pGEM-TEasy Vector System. This fragment was named pGEMT-CTR. Fragments were prepared by digesting pGEMT-pFibH-NTR with AscI / SalI and pGEMT-CTR with SalI / SacI, respectively. These two fragments were cloned using the pBluescriptII SK(-) vector (Stratagene) digested with ApaI / SalI to generate pFibHNC-null. The fluorescent genes (eCFP, eGFP, eYFP, and mKate2) were synthesized and purchased from BIONEER (Korea). They have NotI and SbfI restriction sites at the N-terminus and C-terminus, respectively. The fluorescent genes were digested with NotI / SbfI and subcloned into pFibHNC-null to generate pFibHNC-eCFP, pFibHNC-eGFP, pFibHNC-eYFP, and pFibHNC-mKate2, respectively. The resulting vectors were named pBac-3xP3-eCFP-FibH, pBac-3xP3-DsRed2-FibH-eGFP, pBac-3xP3-DsRed2-pFibH-eYFP, and pBac-3xP3-eGFP-FibH-mKate2, respectively.

[0079] To avoid heat-induced denaturation of fluorescent proteins in the silk, the fluorescent silk was dissolved at low temperature. Sericin was removed from the transgenic silk cocoons to minimize heat-induced denaturation of fluorescent proteins (i.e., eCFP, eGFP, eYFP, and mKate2). The cocoons were treated several times with a mixed solution of Na₂CO₃ (0.2%) at temperatures below 50°C, followed by several rinses with warm deionized water (≈35°C). During degumming, an additional low-pressure treatment (620 mmHg) was applied to completely remove the sericin. After degumming, the sericin-free cocoons were dried in darkness. For white silk, conventional degumming methods were used during boiling. It should be noted that Na₂CO₃ (also known as soda crystals) is an inactive ingredient in FDA-approved pharmaceutical products. Furthermore, it is well known that natural silk also contains carbon (C), oxygen (O), and sodium (Na).

[0080] After removing the sericin, the fluorescent filaments were cut into small pieces smaller than 2 mm to 5 mm and then dissolved in a 9.5 M LiBr aqueous solution at 45 °C and 400 rpm for four hours with stirring. The dissolved solution was filtered through a miracloth and dialyzed against deionized water using a cellulose semipermeable tube for about two days at room temperature to remove salts (i.e., LiBr). For eCFP, eGFP, eYFP, and mKate2 fluorescent filaments, a concentration of approximately 5% (wv) was obtained after centrifugation at approximately 4 °C at 9000 rpm for about 20 minutes. -1 The regenerated filament protein solutions were prepared. The regenerated solutions were freeze-dried at approximately -18°C for seven days. The freeze-dried filaments were mechanically ground into granular particles using a mortar and pestle. Fluorescent filament particles ranging in size from 90 μm to 106 μm were selected by agitating them through a stack of two standard test sieves with opening sizes of 90 μm and 106 μm. Similarly, a white filament protein solution was prepared by stirring at 400 rpm for four hours at 60°C in a LiBr (9.5 M) solution to obtain 4% (wv) filament protein. -1 The concentration of ) was determined. It was filtered through a miracloth and dialyzed in deionized water with a cellulose semipermeable tube for about two days at room temperature, and then centrifuged at about 9000 rpm for about 20 minutes at about 4°C.

[0081] To fabricate an edible PUF device, fluorescent filament microparticles were mixed in a microcentrifuge tube at a ratio of 1:1:1:1 (eCFP, eGFP, eYFP, and mKate2 filaments) and then hand-shaken. Mechanical shaking dispersed the microparticle mixture through a 106 μm sieve onto the surface of a 35 mm diameter polystyrene culture dish. Subsequently, 4 mL of a natural white filament protein solution was poured onto the plastic culture dish and cast in the dark for three days under ambient conditions (25 ± 2 °C and 40% to 50% relative humidity) to obtain a filament film approximately 150 μm thick, comprising four different fluorescent filament microparticles. This film was then processed using a 7 × 7 mm... 2 Edible PUF is prepared by stamping a square area of ​​silk film (see discussion below). Figure 8 ).

[0082] As readily available common light sources for photoexcitation, ultraviolet, blue, and green light-emitting diodes (LEDs) with emission wavelengths of 415 nm (FWHM = 14 nm), 470 nm (FWHM = 25 nm), and 530 nm (FWHM = 33 nm), purchased from Thorlabs Inc. (Newton, NJ, USA), were used. Bandpass filters (FB410-10, FB470-10, and FB530-10; Thorlabs Inc.) at 410 nm, 470 nm, and 530 nm were placed between the light source and the PUF device. For the 415 nm, 470 nm, and 530 nm LEDs, the optical power was maintained at 1 mW mm² at the PUF surface. -2 3mW mm -2 and 10mW mm -2 To image the PUF, a charge-coupled device (CCD) camera (Princeton Instruments PIXIS 1024B) with a conventional zoom lens (Navitar, Rochester, NY, MVL7000) was used via a 7nm FWHM liquid crystal tunable filter (VariSpec VIS-07-20; PerkinElmer, Waltham, MA, USA). As a result, the following group of excitation and emission bands were selected such that λ ex =415nm and λ em =460nm; λ ex =470nm and λ em =510nm; λ ex =470nm and λ em =560nm; λ ex =530nm and λ em =630nm, and they are optimized for eCFP, eGFP, eYFP and mKate filaments respectively.

[0083] According to one embodiment of this application, during the regeneration process for manufacturing colored filament particles, an edible fluorescent dye (Table 2) can be mixed with a dissolved silk protein solution or an edible polymer solution. Specifically, in addition to genetically modified fluorescent filaments, the edible fluorescent dye can be used as a color source to achieve the colored filament particles. The dye is readily soluble in water and binds to the silk protein or polymer during the regeneration process.

[0084] Reference Figure 7 A bar graph showing the size distribution of fluorescent filament particles for edible PUF according to the present application is presented. Particle size was characterized by SEM images. Gaussian fitting returned a mean of 99.3 μm and a standard deviation (SD) of 7.9 μm. (Refer to...) Figure 8 Photographs of 30 different edible PUF devices used to characterize the overall performance of PUF, according to the contents of this application, are provided. Each device measures 7×7mm. 2 . Reference Figure 9 The figure shows a series of fluorescence intensities versus time (seconds), depicting the time-photostability of eCFP, eGFP, and eYFP filaments excited at 440 nm and mKate2 filaments excited at 514 nm. Emission intensities for eCFP, eGFP, and eYFP filaments were measured over 1000 seconds using a 458 nm low-pass filter, and for mKate2 filaments using a 550 nm low-pass filter. (Reference) Figure 10a and Figure 10b Two photographs are shown, illustrating the scalability of the proposed edible PUF for mass production (Fig. 10a) and its flexibility for easy integration with pharmaceuticals. Figure 10b ).

[0085] Reference Figure 11 This application provides a schematic diagram of an exemplary customized imaging setup for obtaining fluorescence images of an edible PUF device. Ultraviolet, blue, and green light-emitting diodes (LEDs) with center wavelengths of 415 nm, 470 nm, and 530 nm are used as readily available common light sources for photoexcitation. Fluorescence images are acquired using liquid crystal tunable filters with emission wavelengths of 460 nm, 510 nm, 560 nm, and 630 nm at acquisition times of 60 seconds, 5 seconds, 40 seconds, and 30 seconds, respectively. Groups of different combinations of excitation and emission wavelengths are used as challenges to support the parameters of challenge-response pairs in an edible PUF. Considering that some high-end smartphones already have multiple light sources (e.g., flash LEDs), it is envisioned that multiple different colored LEDs can be easily embedded into smartphones as manufacturers realize various direct applications (including the proposed dose-based certification).

[0086] Reference Figure 12 A graph of probability versus normalized Hamming distance (HD) is provided to illustrate intra-device (reproducibility) and inter-device (uniqueness) variability at a cutoff threshold of 0.1808. The obtained false positive and false negative rates are 9.6394 × 10⁻⁶. -13 and 3.0982×10 -12 . Reference Figure 13 The image shows a complex photograph of the drug to illustrate the feasibility of dosage-based (or mid-dosage) certification using edible PUF according to the contents of this application. (See also:) Figure 14 A series of photographs are shown to depict the solubility of an edible PUF composed of silk proteins and fluorescent proteins according to the contents of this application. A natural silk protein membrane embedded with eCFP, eGFP, eYFP and mKate2 silk microparticles (loaded with methylene blue for easy observation) was completely dissolved in deionized water within 240 minutes at room temperature.

[0087] A prominent application of the reported edible PUF is dosage certification to prevent patients from taking counterfeit pharmaceutical products. (See reference) Figure 15 An exemplary schematic diagram illustrating such a certification process is shown. Flexible, edible PUF (see...) Figure 10b This material can adhere to the surface of medicines in solid oral dosage forms (including pills, tablets, and capsules). Each medicine has a unique challenge-response pair, and the end user can verify its authenticity using a smartphone camera or a custom reader and access a registered digital key in a database (e.g., the cloud), where each verification is guided by a trusted authority comparing the digital identity. In fact, edible PUFs possess a self-disappearing characteristic. Due to their decomposition properties and proteolytic activity (i.e., enzymatic degradation), silk fibroin (i.e., fibroin) readily dissolves in aqueous solutions without any special treatment. When reported edible PUFs are loaded with a blue dye (i.e., methylene blue) for easy visual inspection, they completely dissolve in deionized water after 240 minutes (see [link to relevant documentation]). Figure 14 This also supports oral administration. In other words, the end user (i.e., the patient) can take the medication without removing the PUF from the surface.

[0088] It should be recognized that, in addition to the materials discussed herein, other materials, including edible polymers, edible proteins, and edible dyes, can also be used in the PUF. For example, one or more of the following edible polymers can be used in the PUF of this application: edible polymers selected from the group consisting of starch, cellulose derivatives, chitosan, pectin, alginate, gum, carrageenan, and combinations thereof. Additionally, one or more of the following edible polymers can be used in the PUF of this application: gelatin, collagen, albumin, milk protein, and combinations thereof. Additionally, one or more of the following edible polymers can be used in the PUF of this application: zein, soybean, wheat gluten, lectins, and combinations thereof. Additionally, one or more of the following edible polymers can be used in the PUF of this application: fatty acids, triglycerides, phospholipids, and combinations thereof. Additionally, one or more of the following edible fluorescent proteins can be used in the PUF of this application: red fluorescent protein (DsRed), orange fluorescent protein (mKO), and combinations thereof. In addition, one or more of the following edible fluorescent dyes may be used in the PUF of this application: Brilliant Blue FCF, Indigo, Fast Green FCF, Erythrosine, Allura Red AC, Tartrazine, Sunset Yellow FCF, and combinations thereof, as provided in Table 2 below.

[0089] Table 2 lists food colorings approved by the U.S. Food and Drug Administration (FDA). The FD&C stands for the Federal Food, Drug, and Cosmetic Act, a law passed by the U.S. Congress in 1938.

[0090]

[0091]

[0092] It should be recognized that, according to this application, the particles are i) applied to a matrix or ii) applied directly to a drug. According to this application, the matrix can be made of edible filaments or edible polymers. According to this application, the particles can be one or more of edible filaments (e.g., edible fluorescent filaments), edible dyes (e.g., edible fluorescent dyes), and edible polymers. These particles can be cast into random patterns on the matrix or directly onto the drug. Alternatively, these particles can be sprayed onto the matrix or directly onto the drug to generate random patterns. Once the particles are applied, an image and an encryption key representing the original authentication pattern can be obtained from these particles. This encryption key is then stored in a secure database awaiting authentication by the end user. The image can be obtained at the drug source using a single image capture device representing the XY encryption key (i.e., a two-dimensional image) or using more than one image capture device (e.g., stereoscopic photography) representing the XYZ encryption key (i.e., a three-dimensional image of the drug with curvature in the Z direction).

[0093] According to another embodiment, spray deposition can be used as an additional manufacturing process to directly incorporate anti-counterfeiting measures, resulting in pharmaceutical products with embedded security keys. To support non-clonability (i.e., asymmetry), it is crucial to include the source of entropy (physical disorder) in a scalable manner. In other words, the spray system acts as a scalable entropy source of randomness. Such spray deposition of filamentary particles exhibits unpredictable variations due to the random nature of atomization and impact dynamics. Even using the same spray process, it is practically impossible to replicate or clone previously generated patterns. Therefore, this manufacturing method makes PUF printing more cost-effective and scalable.

[0094] To this end, commercially available cold spray systems are used to deposit solid microparticles at low density onto the surface of a pharmaceutical product in a scalable manner. During cold spraying, the solid microparticles (powder) are accelerated in a supersonic gas jet. During collision with the matrix, the microparticles undergo plastic deformation and adhere to the surface. Microparticles are supplied from the diffuser section of the nozzle and accelerated to the target surface by a driving gas. In doing so, the microparticles are impregnated onto the surface of the pharmaceutical product and thus fixed in place. Once fixed, these microparticles can be photochallenged to generate a response in the form of an encryption key, which can be recorded in a remote database for later verification. This verification is based on the subsequent application of light at different wavelengths, which generates an encrypted pattern that can be checked against the encryption key.

[0095] Those skilled in the art will recognize that various modifications can be made to the specific implementation described above. The implementation should not be limited to the specific constraints described. Other implementations are also possible. sequence list <110> Purdue Research Foundation Kim, Young L Leem, Jung Woo <120> Edible, non-clonable functional components <130> PRF-68821-02 <150> 62915666 <151> 2019-10-16 <150> 62915667 <151> 2019-10-16 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 717 <212> PRT <213> Mulberry silkworm <400> 1 Asp Ala Ser Gly Ala Val Ile Glu Glu Gln Ile Thr Thr Lys Lys Asn 1 5 10 15 His Gly Ile Leu Gly Lys Asn Glu Lys Thr Phe Val Ile Thr Thr Asp 20 25 30 Ser Asp Gly Asn Glu Ser Ile Val Glu Glu Asp Val Leu Met Lys Met 35 40 45 Val Ser Glu Leu Ile Lys Glu Asn Met His Met Lys Leu Tyr Met Glu 50 55 60 Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly 65 �0 75 80 Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ala Val Glu Gly Gly Pro 85 90 95 Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr Gly Ser 100 105 110 Lys Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp 115 120 125 Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly 130 135 140 Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly 145 150 155 160 Trp Glu Ala Ser Thr Glu Thr Leu Tyr Pro Ala Asp Gly Gly Leu Glu 165 170 175 Gly Arg Met Pro Gly Val Tyr Tyr Val Asp Arg Arg Glu Ala Asp Lys 180 185 190 Glu Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp 195 200 205 Leu Pro Ser Lys Leu Gly His Arg Pro Gln Gln Val Asp Ser Val Ser 210 215 220 Tyr Gly Ala Gly Arg Gly Tyr Gly Gln Gly Ala Gly Ser Ala Ala Ser 225 230 235 240 Ser Val Ser Ser Ala Ser Ser Arg Ser Tyr Asp Tyr Ser Arg Arg Lys 245 250 255 Asn Cys Gly Ile Pro Arg Met Arg Val Lys Thr Phe Val Ile Leu Cys 260 265 270 Cys Ala Leu Gln Tyr Val Ala Tyr Thr Asn Ala Asn Ile Asn Asp Phe 275 280 285 Asp Glu Asp Tyr Phe Gly Ser Asp Val Thr Val Gln Ser Ser Asn Thr 290 295 300 Thr Asp Glu Ile Ile Arg Asp Ala Ser Gly Ala Val Ile Glu Glu Gln 305 310 315 320 Ile Thr Thr Lys Lys Met Gln Arg Lys Asn Lys Asn His Gly Ile Leu 325 330 335 Gly Lys Asn Glu Lys Met Ile Lys Thr Phe Val Ile Thr Thr Asp Ser 340 345 350 Asp Gly Asn Glu Ser Ile Val Glu Glu Asp Val Leu Met Lys Thr Leu 355 360 365 Ser Asp Gly Thr Val Ala Gln Ser Tyr Val Ala Ala Asp Ala Gly Ala 370 375 380 Tyr Ser Gln Ser Gly Pro Tyr Val Ser Asn Ser Gly Tyr Ser Thr His 385 390 395 400 Gln Gly Tyr Thr Ser Asp Phe Ser Thr Ser Ala Ala Val Gly Ala Gly 405 410 415 Ser Ser Gly Arg Met Val Ser Glu Leu Ile Lys Glu Asn Met His Met 420 425 430 Lys Leu Tyr Met Glu Gly Thr Val Asn Asn His His Phe Lys Cys Thr 435 440 445 Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile 450 455 460 Lys Ala Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala 465 470 475 480 Thr Ser Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly 485 490 495 Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu 500 505 510 Arg Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp 515 520 525 Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly 530 535 540 Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly 545 550 555 560 Trp Glu Ala Ser Thr Glu Thr Leu Tyr Pro Ala Asp Gly Gly Leu Glu 565 570 575 Gly Arg Ala Asp Met Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile 580 585 590 Cys Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu 595 600 605 Lys Met Pro Gly Val Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys 610 615 620 Glu Ala Asp Lys Glu Thr Tyr Val Glu Gln His Glu Val Ala Val Ala 625 630 635 640 Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Arg Pro Gln Gln Val 645 650 655 Asp Ser Val Ser Tyr Gly Ala Gly Arg Gly Tyr Gly Gln Gly Ala Gly 660 665 670 Ser Ala Ala Ser Ser Val Ser Ser Ala Ser Ser Arg Ser Tyr Asp Tyr 675 680 685 Ser Arg Arg Asn Val Arg Lys Asn Cys Gly Ile Pro Arg Arg Gln Leu 690 695 700 Val Val Lys Phe Arg Ala Leu Pro Cys Val Asn Cys Asn 705 710 715

Claims

1. A method for generating physically unclonable functional components for drug certification, comprising: Generate edible, physically unclonable functional components - PUF; The edible PUF is attached to the drug in a random distribution; as well as The encryption key is generated based on the attached randomly distributed edible PUF. The edible PUF is based on randomly distributed fluorescent filament proteins, which are selected from the group including enhanced cyan fluorescent protein, enhanced green fluorescent protein, enhanced yellow fluorescent protein, and far-red fluorescent protein.

2. The method according to claim 1, wherein, The edible PUF includes silk protein.

3. The method according to claim 1, wherein, The edible PUF comprises edible polymers.

4. The method according to claim 1, wherein, The edible PUF is based on randomly distributed edible fluorescent proteins.

5. The method according to claim 1, wherein, The edible PUF is based on randomly distributed edible fluorescent dyes.

6. The method according to claim 1, wherein, The edible PUF is based on randomly distributed edible polymer particles containing edible fluorescent dyes.

7. The method according to claim 1, wherein, The fluorescent protein-expressing silk can be produced by genetically engineered domesticated silkworms.

8. The method according to claim 7, wherein, The transgene is expressed via germline transformation using gene splicing.

9. The method according to claim 8, wherein, The gene splicing is based on piggyBac.

10. The method according to claim 3, wherein the edible polymer is selected from the group consisting of starch, cellulose derivatives, chitosan, pectin, alginate, gum, carrageenan, and combinations thereof.

11. The method of claim 3, wherein the edible polymer is selected from the group consisting of gelatin, collagen, albumin, milk protein, and combinations thereof.

12. The method according to claim 3, wherein the edible polymer is selected from the group consisting of zein, soybean, wheat gluten, lectins, and combinations thereof.

13. The method of claim 3, wherein the edible polymer is selected from the group consisting of fatty acids, triglycerides, phospholipids, and combinations thereof.

14. The method of claim 4, wherein the edible fluorescent protein is selected from the group consisting of red fluorescent protein, orange fluorescent protein, and combinations thereof.

15. The method according to claim 5, wherein the edible fluorescent dye is selected from the group consisting of Brilliant Blue, Indigo, Fast Green, Erythrosine, Allura Red, Tartrazine, Sunset Yellow, and combinations thereof.

Images