Bacterial cellulose derived nanoparticles and uses thereof

Abstract

Various implementations described herein relate to methods and compositions for production of bacterial cellulose nanoparticles and uses thereof. According to some implementations, the size and shape of bacterial cellulose nanoparticles are controlled. The bacterial cellulose nanoparticles are linked to proteins to facilitate drug delivery, in some implementations. Various implementations described herein can be used for research, diagnostic, or therapeutic uses.

Description

BACTERIAL CELLULOSE DERIVED NANOPARTICLES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the priority of U.S. Provisional Patent Application No. 63 / 584,098, filed on September 20, 2023, and which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] This application relates to bacterial cellulose nanoparticles and uses thereof. The disclosed nanoparticles are scalable, sustainable, and can be used for drug delivery.BACKGROUND

[0003] Within the pharmaceutical industry, nanomedicine is a rising sector in which scaling up for manufacturing is a major issue. By 2030, the global nanomedicine market is projected to be a $427-billion-dollar industry driven by continued investment in the development of novel drug delivery systems and an increasing demand for safe and affordable therapeutics (Nanomedicine Market Size Worth $427. 18 Billion By 2030. Nanomedicine Market Growth & Trends). The state- of-the-art materials being used for these applications are synthetic polymers derived from nonrenewable petroleum resources such as poly(glycolic acid) (PGA) and poly(lactic-co-glycolic acid) (PLGA) and lipids (Bhatia, S., Natural Polymer Drug Delivery Systems. 2016; Grund, S., et al., Advanced Engineering Materials, 201 1. 13(3): p. B61 -B87; Ickenstein. and Garidel., Expert Opin Drug Deliv, 2019. 16(11 ): p. 1205-1226). Advantages of synthetic polymers and lipids are the tunability of mechanical properties and degradation kinetics, batch reproducibility, and consistency (Bhatia, supra; Parisa Ghasemiyeh., Research in Pharmaceutical Sciences, 2018. 13(4): p. 303). However, not only are these synthetic feedstocks non-renewable, but they often require use of organic solvents during their synthesis and processing which then need to be disposed of appropriately. These solvent disposal processes create further environmental challenges, particularly in terms of generating waste streams associated with undesirable environmental impacts of the therapeutic products (Savelski, M.J., et al., Clean Technologies and Environmental Policy, 2017. 19(10): p. 2467-2477). The field of sustainable nanomedicine aims to circumvent these harmful effects of traditional nanotherapeutics by eliminating solvent use and adopting low-waste and green engineering methodologies.

[0004] Cellulose, the most abundant natural polymer, has been studied in the nanomedicine industry for biomedical and drug delivery applications (Abeer, M.M., et aL, J Pharm Pharmacol, 2014. 66(8): p. 1047-61). However, most studies utilize plant biomass-sourced cellulose where there is a need for extraction which requires solvent processing and significant energy consumption for mechanical defibrillation and chemical isolation (Moon, R.J., et al., Chem SocRev, 2011 . 40(7): p. 3941 -94). Alternatively, cellulose can be produced as an extracellular product by some bacterial species in specific culturing conditions (Abeer, supra; Wahid, F., et aL, Biotechnol Adv, 2021. 53: p. 107856). The benefit of bacterial cellulose (BC) is that it is the only product of that biosynthesis process and therefore there is no need to extract and chemically treat the resulting biomass to isolate cellulose fibers. BC can be produced in an aqueous and low-cost sugar-based media, known as a kombucha media, from certain bacterial strains co-cultured with yeast species (Yoshinaga, F., et al., Bioscience, Biotechnology, and Biochemistry, 1997. 61 (2): p. 219-224). BC grown in such co-cultures has several advantages: (1) it is produced in mild conditions at room temperature with no pressure, specific gas flows, or sterility required; (2) BC production uses a metabolic, fossil-free process, where the only product of the bacterial cultures is BC, and (3) it is produced at a commercial scale (Yoshinaga, supra; Dudman, W.F., Journal of General Microbiology, 1960. 22(1 ): p. 25-39; Petersen and Gatenholm, Applied Microbiology and Biotechnology, 201 1. 91 (5): p. 1277-1286). For these reasons, BC is an emerging biopolymer platform used in engineering and biomedical applications (Yoshinaga, supra).BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0006] FIG. 1 illustrates an example environment for production of bacterial cellulose nanoparticles (BCNPs).

[0007] FIGs. 2A-2E illustrate BCNP synthesis and preparation for drug loading applications: (2A) growth process of bacterial cellulose (BC) in a kombucha media and agitated for 24 hours; (2B) termination of growth and isolation of BCNPs; (2C) endotoxin removal from BCNPs; (2D) application of surfactant solution and particle size separation; and (2E) drug incorporation process.

[0008] FIG. 3. illustrates microbial co-cultures in a symbiotic culture of bacteria and yeast (SCOBY) starter kit: (Left) in pellicle and (Right) in liquid.

[0009] FIGs. 4A-4E illustrate BC growth at various timepoints: (4A) photographs of the culturing flasks; (4B) STEM image of BC; (4C) scanning electron microscope (SEM) images of BC; (4D) atomic force microscopy (AFM) images of BC; and (4E) Histogram of AFM data.

[0010] FIG. 5. Light microscope images at a 5x optical setting for BCNPs grown for: (Left) 3 days and (Right) 5 days.

[0011] FIGs. 6A-6C illustrate (6A) the growth of BC (grams) wet mass and dry mass; (6B) the absolute growth rate (AGR) of wet and dry BC mass; and (6C) the ratio of dry to wet mass (Dry:Wet Mass) plotted against the ratio of the dry AGR to the wet AGR (Dry:Wet AGR).

[0012] FIGs. 7A-7C illustrate the characterization of BC structure and thermal properties: (7A) x- ray diffraction (XRD), (7B) Fourier transform infrared spectroscopy (FTIR) spectra, and (7C) thermal gravimetric analysis (TGA) and differential TGA (DTGA) profiles.

[0013] FIGs. 8A-8C illustrate (8A) the hydrodynamic size of BCNPs at concentration of 8.35x107± 1 ,18x107particles / mL; (8B) scanning transmission electron microscopy (STEM) of BCNPs at 1 urn scale bar; and (8C) STEM of a BCNP at 100 nm scale bar. FIG. 8D illustrates a STEM image of BCNPs at 100 nm to show the distribution of particle morphology. Scale bar: 100 nm

[0014] FIGs. 9A-9C illustrate (9A) STEM images of 8.34% DL BSA-BCNPs / P80 (scale bar set to 200 nm); (9B) results for cumulative BSA drug release (% w / w) from BCNPs / P80, calculated using equation (6); and (9C) results for cumulative BSA drug release (ug) from BCNPs / P80, calculated using equation (6).

[0015] FIG. 10 illustrates STEM images of BCNPs at two different magnifications.

[0016] FIG. 1 1 illustrates XRD spectra of BC grown for 1 -day, 3-day, and 5-day timepoints and for comparison, cellulose nanocrystals (CNCs). The increase in crystallinity is qualitatively shown over time of growth.

[0017] FIGs. 12A and 12B illustrate FTIR spectra of BC grown for 1 -day, 3-day, and 5-day timepoints and CNCs: (12A) wavenumber range 3600-2800 cm'1and (12B) wavenumber range 1200-900 cm1.DETAILED DESCRIPTION

[0018] Sustainable nanomedicine is an emerging field addressing the challenges of scalability, reproducibility, thermal stability, and excessive waste generation for nanotherapeutic manufacturing. Bacterial cellulose (BC) fibers have the potential to overcome these challenges and be a versatile drug delivery platform. To date, BC has primarily been used in the drug delivery field for transdermal applications, where BC films are prepared for use in antimicrobial wound healing and dressing, and face and eye masks for anti-aging and scarred skin rejuvenation (Mohammadi, S., et al., Int J Biol Macromol, 2023. 242: p. 124955; Mohammadi, S., et aL, Int J Biol Macromol, 2023. 242(Pt 3): p. 124955; Ullah, H., et aL, Curr Pharm Des, 2019. 25(34): p. 3692-3701 ). Spherical BC particles with diameters in the 0.5-1.0 mm range have also previously been reported to enable a controlled drug release and exhibit antibacterial properties for tissue engineering and drug delivery applications (Abeer, supra Yoshinaga, supra; Ullah, supra; Zhang,W., et al., Int J Biol Macromol, 2019. 140: p. 196-205; Hu and Catchmark, Biomacromolecules, 2010. 11 (7): p. 1727-1734; Jovic, M., et al. Startups Developing Nanocellulose Products in 2020. 2020. p. 52). Despite the drug delivery potential demonstrated by BC microparticles, their application for long-circulation times and deep tissue penetration is limited due to their size. For injectable drug delivery systems, a nanoscale BC particle would provide longer circulation times, improved bioavailability and biodistribution, and greater targeted delivery through increased tissue penetration and cell specific uptake. Various implementations of the present disclosure provide methods to produce BC in nanoparticle form and enable BC as a drug delivery platform.

[0019] BC growth occurs as self-assembled cellulose chains extracellularly extruded from certain types of bacteria such as Gluconacetobacter xylinus, formerly known as Acetobacter xylinum, a gram-negative bacterium (Moon, supra). The cellulose chains are arranged in a parallel configurations stabilized by intermolecular bonding (hydrogen bonding, van der Waals interactions) forming elemental fibrils which subsequently bundle to form fibers (Moon, supra). Elemental BC fibrils from Gluconacetobacter xylinus have high crystallinity (up to 80%) with a cross-sectional thickness of 6-10 nm. Elementary fibrils bundle to form larger fibers with reported thicknesses ranging from 30-80 nm and lengths up to several microns (Moon, supra). Depending on the culture conditions, the yield, morphology, structure, crystallinity, and degree of polymerization of the BC fibers can be altered and controlled (Singhsa, P., et aL, Cellulose, 2018. 25(3): p. 1571-1581 ). Incubation in static conditions enables the formation of a layered 3D- network of BC fibers, called a pellicle, in which the interlayer connectivity is facilitated by entangled, randomly oriented BC fibers. Agitated culture conditions, on the other hand, utilize centrifugal forces to bundle the growing cellulose fibers into spherical-like particles. In both static and agitated culture conditions, the high degree of crystallinity, high fiber aspect ratio, and the absence of other polymers as extracellular components of the produced pellicle or spherical particle, serve as advantages for the produced BC as compared to plant-derived cellulose.

[0020] The present disclosure describes development of BC nanoparticles (BCNPs) for sustainable drug delivery applications motivated by the biodegradability and biocompatibility of BC, which are identifiable properties for nanomedicine applications. In addition, formulation of the disclosed BCNPs has a reduced environmental impact, an overall eco-friendly life cycle, and can be implemented following green engineering methodologies. The present disclosure describes use of static and agitated culture conditions to develop BC nanoparticles (BCNPs). Various implementations of the present disclosure provide scalable methods to generate BCNPs that reduce waste production and the use of harsh chemicals. In various implementations, the disclosed BCNPs can be used as a drug delivery platform. To demonstrate the drug loadingcapacity of BCNPs, bovine serum albumin (BSA) was used as a model drug in the present disclosure. BSA is a low cost, biodegradable, and biocompatible protein therapeutic with a high aqueous solubility and can be easily surface modified (Wong, C.Y., et al., Analytical and Bioanalytical Chemistry, 2018. 410(27): p. 6991-7006; Wang and Zhang, Curr Med Chem, 2018. 25(25): p. 2938-2953). The present disclosure describes methods to produce BCNPs, facilitating use of BCNPs as sustainable natural polymer carriers for protein therapeutics. According to various implementations, production of the disclosed BCNPs can be scaled up for manufacturing purposes. Implementations described herein can be used for research, diagnostic, or therapeutic uses.

[0021] Embodiments of the present disclosure will now be described with reference to the accompanying figures.

[0022] FIG. 1 illustrates an example environment 100 for production of bacterial cellulose nanoparticles (BCNPs). In various implementations, the processes described with reference to FIG. 1 are performed by one or more entities, such as a computing device, laboratory equipment (e.g., an orbital shaker, an incubator, a homogenizer, a sonicator, a magnetic stirrer, or the like), or a user (e.g., a trained user, a laboratory technician, a researcher, or the like). “Nanoparticles,” “nanocarriers,” and their equivalents, as used herein, refer to particles with dimensions (e.g., diameter, height, length, width, etc.) less than or equal to 1000 nanometers (nm). Nanoparticles may have various shapes, including spheres, tubes, dendritic shapes, amorphous shapes, or any other shape. Nanoparticles may be hollow, solid, porous, or have a core-shell structure. A core-shell structure refers to a particle with a core component that is encased in a shell component. The core component may include different materials and / or have different properties (e.g., density, molecular weight, porosity, zeta potential, etc.) than the shell component. Polymeric nanoparticles refer to nanoparticles that include one or more polymers, for instance, such as poly (lactide-co-glycolide) (PLGA), polyethylene glycol) (PEG), polylactic acid (PLA), and polyglycolic acid (PGA).

[0023] Polymeric nanoparticles have gained traction in nanomedicine and other fields that use nanotechnology (e.g., environmental testing, cosmetics, etc.). Polymeric nanoparticles provide reduced toxicity, biodegradability, tunability (e.g., size, shape, functionalization, etc.) compared to metallic nanoparticles. However, the synthesis of polymeric nanoparticles requires harsh chemicals. Biologically derived nanoparticles, such as those including polysaccharides, proteins, and peptides, provide a more sustainable solution by enabling the production of nanoparticles without harsh solvents. Biologically derived nanoparticles, such as chitosan nanoparticles and alginate nanoparticles, generally require processing to isolate (e.g., extract, purify, etc.) thepolymer of interest from the source material and to form nanoparticles from the isolated polymer. This processing often generates byproducts, adding to the waste production of the nanoparticle synthesis process.

[0024] These issues can be addressed, for example, by using bacterially-produced source material to generate nanoparticles. These nanoparticles can be produced without generating any byproducts to isolate the source material. Various implementations of the present disclosure provide nanoparticles that are generated under particular agitation conditions, enabling the production of nanoparticles without using harsh solvents or other reagents that may be discarded, for instance, as biohazardous waste. Various implementations of the present disclosure provide bacterial cellulose nanoparticles (BCNPs) that are produced without using cellulose as a source ingredient. Accordingly, the disclosed nanoparticles do not require processing to isolate cellulose from source materials (e.g., wood, fibrous plants, etc.). The disclosed nanoparticles are safe for in vivo use and tunable to facilitate a wide variety of applications. Various implementations of the present disclosure provide sustainable and scalable nanotechnology solutions that reduce waste production and avoid the use of harsh solvents.

[0025] In various implementations, a microbial solution 102 is configured to culture a cellulose- producing microbe. In some examples, the cellulose-producing microbe includes acetic acid bacteria and yeast. For instance, the cellulose-producing microbe includes a symbiotic culture of bacteria and yeast (SCOBY). In some examples, the cellulose-producing microbe includes at least one of acetobacter bacteria, gluconobacter bacteria, or gluconacetobacter bacteria. In some examples, the cellulose-producing microbes include a bacteria that has been genetically engineered to produce cellulose.

[0026] The microbial solution 102, in some examples, includes one or more nutrients configured to facilitate the production of cellulose by the cellulose-producing microbe. For instance, the microbial solution 102 may include at least one carbon-nitrogen source. Examples of carbonnitrogen sources include tea leaves, sucrose, fructose, glucose (e.g., D-glucose), glycerol, mannitol (e.g., D-mannitol), ethanol, peptone, or yeast extract. In some examples, carbonnitrogen sources may include food and food scraps (e.g., fruit peels, vegetable scraps, coffee grounds, nut shells, egg shells, etc.) or other natural sources of carbon and / or nitrogen (e.g., wood chips, garden clippings, etc.). In various cases, the microbial solution 102 includes sugar, such as sucrose, fructose, glucose, glycerol, or mannitol. In some examples, the microbial solution 102 includes acetic acid. For instance, the microbial solution 102 may include acetic acid bacteria and yeast, sugar, and acetic acid.

[0027] In particular implementations of the present disclosure, the microbial solution 102 is spun at a particular rate and for a particular time to generate the nanoparticles 104. For instance, a rotational motion may be applied to a container holding the microbial solution 102. In some examples, the microbial solution 102 is spun by adding a magnetic object (e.g., a magnetic rod) to the container holding the microbial solution 102. A magnetic field configured to rotate the magnetic object may be applied to the container. In some examples, the microbial solution 102 is spun at a rate of 100 to 200 rotations per minute (rpm). In some examples, the microbial solution 102 is spun at a rate of 150 rpm. In various instances, the microbial solution 102 is spun in aerated conditions (e.g., in the presence of oxygen). Spinning (e.g., agitation) the microbial solution 102, in various cases, provides greater aeration of the bacterial cellulose in the microbial solution 102 than, for instance, bacterial cellulose in a static solution. In some examples, the microbial solution 102 is spun for a time of 12 to 36 hours. In some examples, the microbial solution 102 is spun for a time of 24 hours. In various cases, the microbial solution 102 is spun at a particular temperature. For instance, the microbial solution 102 may be spun at a temperature of 20°C to 40°C. In various cases, the microbial solution is spun at a temperature of 25°C to 35°C. Based on spinning the microbial solution 102 at the particular rate and for the particular time, the nanoparticles 104 can be extracted from the microbial solution 102. In some examples, a termination reagent configured to stop the formation of the nanoparticles 104 may be added to the microbial solution 102. Examples of the termination reagent include sodium hydroxide or the like. The byproducts remaining in the microbial solution 102 may include the carbon-nitrogen source (e.g., tea leaves), sugar, and acetic acid. The microbial solution 102 can be replenished to produce additional nanoparticles or disposed of as organic waste.

[0028] According to various implementations, the rate, the time, and the aeration conditions may be determined based on a volume of the microbial solution 102. Various implementations of the present disclosure provide methods for producing the nanoparticles 104 that do not depend as significantly on the volume of the microbial solution 102.

[0029] In particular implementations of the present disclosure, the microbial solution 102 is incubated to produce aggregated fibers 106 of bacterial cellulose. In various cases, the aggregated fibers 106 may include a sheet or a three-dimensional volume. The microbial solution 102, in some examples, is incubated in static conditions (e.g., without movement) to produce the aggregated fibers 106. Accordingly, it may be more efficient, in some cases, to incubate certain volumes (e.g., volumes above a certain threshold) under static conditions than under agitated conditions. The threshold, for instance, may be 25 milliliters (mL), 50 mL, 100 mL, 250 mL, 500 mL, 1 liter, or more than 1 liter. For instance, it may be beneficial (e.g., safer, easier, more efficient,etc.) to incubate a larger volume (e.g., 500 mL) under static conditions than under agitated conditions.

[0030] In various examples, the microbial solution 102 may be incubated at a temperature of 10°C to 40°C. In various examples, the microbial solution 102 may be incubated at room temperature (e.g., 15°C to 25°C). In particular cases, the microbial solution 102 is incubated for a period of time. For instance, the microbial solution 102 may be incubated for 14 days at room temperature. The period of incubation, in some examples, depends on the volume of the microbial solution 102, the container holding the microbial solution 102 during incubation (e.g., container dimensions, container shape, surface area of the microbial solution 102 in the container, airflow into the container, etc.), the incubation temperature, or other factors affecting bacterial cellulose growth. For instance, the period of incubation may be shorter than 14 days at an incubation temperature above room temperature.

[0031] In some implementations, the entity may be provided with aggregated fibers 106. For instance, the entity may be provided with aggregated fibers 106 that are preserved, for instance, by freezing, freeze-drying (e.g., lyophilization), drying, or the like.

[0032] The aggregated fibers 106, in various examples, are fragmented to generate separated fibers 108 of bacterial cellulose. In some examples, the aggregated fibers 106 are broken apart (e.g., manually or by one or more devices configured to separate fibers). For instance, a homogenizer (e.g., a mechanical homogenizer, a high shear homogenizer, a microfluidizer, or the like) may be configured to apply a shear force to the aggregated fibers 106, thereby generating the separated fibers 108. In some examples, a sonicator (e.g., a water bath sonicator, a probe sonicator, an ultraso nicator, or the like) may be configured to use ultrasound waves to fragment the aggregated fibers 106, thereby generating the separated fibers 108. In various cases, a diameter of the separated fibers 108 is less than 1000 nm. For instance, the separated fibers 108 may include individual and / or small aggregates of fibers having diameters less than 1000 nm. Accordingly, the separated fibers 108 may be suitable for nanoparticle formation. In some examples, a length of the separated fibers is less than 1000 nm. In some examples, a length of the separated fibers is greater than 1000 nm.

[0033] In some examples, the aggregated fibers 106 are incubated in one or more solvents. The solvents are, for instance, configured to facilitate the fragmentation of the aggregated fibers 106. In some examples, the solvents are disposed on the surface of the separated fibers 108. Examples of solvents include lithium chloride (LiCI) and / or dimethylacetamide (DMAc). In some examples, the solvents may facilitate an enzymatic process to fragment the aggregated fibers 106.

[0034] In various cases, a solution including the separated fibers 108 is agitated to form nanoparticles 104. In some examples, the solution including the separated fibers 108 may be spun at a particular rate and for a particular time. In various implementations, the rate is 600 to 800 rpm. In some examples, the rate is 650 to 700 rpm. In various implementations, the time is 0.5 to 3 hours. In some examples, the time is 1 to 2.5 hours. In some examples, the separated fibers 108 are exposed to one or more surfactants. Surfactants may include, for instance, pluronic F-127, polysorbate-80, or the like.

[0035] The nanoparticles 104, in various examples, are extracted from the solution. In various cases, the nanoparticles 104 include fibrous nanoparticles that include bacterial cellulose fibers. In some instances, a diameter of the nanoparticles 104 is less than 200 nanometers (nm). A polydispersity index of the nanoparticles 104, in various instances, is 0 to 0.3. In some examples, the polydispersity index of the nanoparticles 104 is 0 to 0.2. The polydispersity, in some examples, is calculated using the following equation where a is the standard deviation of the mean particle diameter and d is the mean particle diameter:Equation 1 : PDI = (^)2(1 )

[0036] (Raval, N., et al., Basic Fundamentals of Drug Delivery. 2019. p. 369-400). In some examples, a zeta potential (also referred to as “surface charge”) of the nanoparticles 104 is negative or neutral at a physiological pH. Physiological pH, in various instances, is 5.2 to 8.0. A negative or neutral zeta potential may facilitate prolonged in vivo circulation half-lives, reduced accumulation in the spleen, liver, and lungs, and cellular uptake for therapeutic, diagnostic, or research purposes. In various examples, the zeta potential of the nanoparticles 104 is positive at a physiological pH. Positively charged nanoparticles may be used in other applications, such as topical therapies (e.g., dermal applications, ophthalmic applications, etc.), cosmetics, and the like. In various cases, the zeta potential of the nanoparticles 104 is -30 millivolts (mV) to +30 mV.

[0037] In various implementations, endotoxins are removed from the extracted nanoparticles 104. In some examples, endotoxins are located in the cell wall of the cellulose-producing bacteria. In some examples, a level of endotoxins in the nanoparticles 104 is reduced to a regulatory level or to a level that is safe for medical use. For instance, the level of endotoxins in the nanoparticles may be 0 to 0.5 endotoxin units (Ell) per milliliter (mL). In various cases, the level of endotoxins is 0.46 EU / mL.

[0038] In various implementations, the nanoparticles 104 are functionalized. For instance, the nanoparticles 104 may be surface functionalized with a hydrophilic group or a hydrophobic group. In various examples, the nanoparticles 104 are functionalized with at least one of: an amine group, an acetyl group, a methyl group, a sulfonic acid group, an ether group, or a carboxylic acid group.In various examples, the nanoparticles 104 are functionalized via acid hydrolysis. Acid hydrolysis, for instance, breaks the glycosidic bonds between the cellulose units of the bacterial cellulose. In some examples, the functionalization facilitates improved activity (e.g., cellular uptake) of the nanoparticles 104. In some examples, the functionalization facilitates nanoparticle conjugation.

[0039] In some examples, the nanoparticles 104 are linked to a targeting moiety configured to bind to a target antigen. For instance, the nanoparticles 104 may be linked to an antibody, an antibody binding domain, a protein, a nucleic acid, or the like. In various implementations, the targeting moiety in configured to bind to a surface protein or a surface receptor on a cell.

[0040] In some examples, the nanoparticles 104 are linked to a detectable label, a drug (e.g., a therapeutic agent), or the like. In various cases, the bacterial cellulose of the nanoparticles 104 forms hydrogen bonds to, for instance, a detectable label. In some instances, the surface functionalization of the nanoparticles 104 may facilitate conjugation of the nanoparticles 104 to the detectable label. Detectable labels can, in various examples, include a chromophore tag, a quantum dot, a fluorescent molecule, an optically detectable particle (e.g., gold nanoparticles, silver nanoparticles, etc.), a radioactive isotope, biotin, a dye, a radionuclide, or the like. Detectable labels, in various examples, can be used in diagnostic assays, diagnostic imaging (e.g., in vivo imaging and ex vivo imaging), and the like. Detectable labels, in some cases, can be used for cell separation, microscopy (e.g., fluorescence microscopy), chromatography, and various other applications. “Drug,” “therapeutic agent,” and its equivalents, as used herein refer to a protein, peptide, nucleic acid molecule, a small molecule, a radioactive isotope, an immunotoxin, or the like that is configured to treat, prevent, or reduce the symptoms of a pathological condition.

[0041] Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0042] An "effective amount" is the amount of a formulation used to result in a desired physiological change in the subject. For example, an effective amount can provide a change in a metric associated with a pathological disease (e.g., a severity or frequency of symptoms or a change in a marker associated with the pathological disease). Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically significant effect in an animal model or in vitro assay relevant to the assessment of a pathological disease’s development or progression.

[0043] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a pathological disease or displays only early signs or symptoms ofa pathological disease such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the pathological disease further. Thus, a prophylactic treatment functions as a preventative treatment against a pathological disease. In particular embodiments, prophylactic treatments reduce or delay physical symptoms associated with a pathological disease.

[0044] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a pathological disease and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the pathological disease. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the pathological disease and / or reduce control or eliminate side effects of the pathological disease.

[0045] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0046] In particular embodiments, therapeutically effective amounts induce a reduced progression of a pathological disease. In particular embodiments, the reduced progression includes reduced or stabilized symptoms, reduced or stabilized levels of one or more markers associated with the pathological disease, or a change in another metric known in the art. For instance, the reduced progression may include a reduction in systemic and / or localized inflammation.

[0047] For administration, therapeutically effective amounts (also referred to herein as “doses” or “dosages”) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of condition, stage of condition, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0048] Useful doses can range from 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pg / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0049] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., hourly, every 2 hours, every 4 hours,every 6 hours, every 12 hours, daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol.

[0050] Various compositions described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, subcutaneous, and / or sublingual administration. Formulations are generally be administered by injection.

[0051] In various implementations of the present disclosure, a composition including the nanoparticles 104 can be administered by, for example, injection, infusion, perfusion, or lavage. Routes of administration can include bolus intravenous, intradermal, intraarterial, intraparenteral, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, and / or subcutaneous administration.

[0052] In some implementations, the nanoparticles 104 are administered with a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety and purity standards as required by United States FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0053] Exemplary generally used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and / or lubricants.

[0054] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers and / or trimethylamine salts.

[0055] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

[0056] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol or mannitol.

[0057] Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers or polysaccharides.

[0058] In particular implementations, treatments described herein (e.g., the nanoparticles 104) can be formulated into a carrier in a therapeutically-effective amount. As described herein, exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), and combinations thereof.

[0059] Compositions including the nanoparticles 104 and / or other components described herein may be administered in a formulation that includes one or more carriers, stabilizers, anesthetics, preservatives, or any combinations thereof.

[0060] In particular implementations, carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In particular implementations, a carrier for infusion includes buffered saline with 5% HSA or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0061] Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0062] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent cell adherence to container walls. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran.

[0063] Where beneficial, formulations can include a local anesthetic such as lidocaine to ease pain at a site of injection.

[0064] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.EXPERIMENTAL EXAMPLE

[0065] In this Experimental Example, BCNPs were fabricated by growth in a kombucha media in agitated and aerated conditions for 24 hours, followed by size separation using centrifugation and polysorbate 80 surfactant solution. According to some implementations, the disclosed particles are 100 nm in diameter, have a slightly negative zeta potential, and are predominantly amorphous morphology. The growth of BC fibers after 1 , 3, and 5 days was investigated, and the BC’s timedependent physiochemical properties were evaluated using X-ray diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis. Implementations of the present disclosure include primarily amorphous BCNPs obtained after culturing for 1 day, with longer culture duration leading to larger BC particles with increasing degrees of crystallinity. Moreover, various implementations of the disclosed BCNPs are thermally stable up to 90°C. The present disclosure describes drug loading capability by incorporating bovine serum albumin (BSA) as a drug into the disclosed BCNPs and quantified sustained release of BSA. These results further motivate the use of BCNPs as a useful nanotherapeutic platform.Materials and MethodsBCNPs preparation, endotoxin removal, and size separation

[0066] BCNPs were prepared in agitated and aerated conditions in a kombucha media on an orbital plate shaker at 150 rpm and 30 °C (FIGs. 2A-2E). The tea media was prepared with 1 L of nitrogen sourced black tea (Lipton Classic - black tea bags, Englewood Cliffs, NJ, USA) steeped for 5 min. in hot deionized (DI) water, 200 g white pure cane sugar (Domino Sugar, Yonkers, NY, USA), and 20 mL apple cider vinegar (Bragg Organic, Santa Barbara, CA, USA). Symbiotic Cultures of Bacteria and Yeast (SCOBY) were purchased from Joshua Tree Kombucha (Joshua Tree, CA, USA), and its contents were separated by membrane (pellicle) and liquid, both containing microbial co-cultures (FIG. 3). 23 mL of tea media and 2 mL SCOBY liquid were added to a 250 mL Erlenmeyer flask and placed on a plate shaker for 24 h. Agitation enables the synthesized cellulose fibers to take a spherical shape, rather than pellicle form (F. Yoshinaga, et al., Biosci., Biotechnol., Biochem., 1997, 61 , 219-224; W. F. Dudman, J. Gen. Microbiol., 1960, 22, 25-39). At 24 h, 0.1 M sodium hydroxide solution (NaOH, Thermo Fisher, Waltham, MA, USA)is titrated into the kombucha media to a slight excess to terminate the growth of cellulose (Yoshinaga, supra; Dudman, supra). The BCNPs were harvested from media with vacuum filtration and cleaned from excess media and NaOH via dialysis tubing made of cellulose ester (MW cutoff: 3K Da, Spectrum Laboratories, Inc., San Francisco, CA, USA) for 24 h. A 100 K centrifugal tube filter centrifuged at 4000 rpm for 25 min was used to separate BCNPs from solution.

[0067] FIGs. 2A-2E illustrate BCNP synthesis and preparation for drug loading applications: (2A) growth process of BC in a kombucha media and agitated for 24 hours; (2B) termination of growth and isolation of BCNPs; (2C) endotoxin removal from BCNPs; (2D) application of surfactant solution and particle size separation; and (2E) drug incorporation process. FIGs. 2A-2E were created with Biorender.com.

[0068] FIG. 3. Microbial co-cultures in SCOBY starter kit: (Left) in pellicle and (Right) in liquid. Higher density of microbial cells in pellicles, yet still exhibited in liquid. Scale bars set at 10 pm. Endotoxin removal and endotoxin quantification assay for BCNPs

[0069] To remove the endotoxins from BCNPs, 25 mL of Triton X-114 (TX-114, Thermo Fisher, Waltham, MA, USA) were added to the BCNPs and incubated overnight at 4 °C on a RotoBot R4045 standardized sized tube rotator (Stellar Scientific, Baltimore, MD, USA). The BCNPs and TX-114 solution were centrifuged at 100,000 ref for 30 min, collected, and washed with 1 .5 mL of DI water three times at 15,000 rpm for 30 min. The collected BCNPs were next washed with 1.5 mL of endotoxin-free ultra-pure water (Sigma-Aldrich, St. Louis, MO, USA) 10 times at 15,000 rpm for 30 min. To quantify the endotoxin units per milliliter (EU / mL), a Pierce™ Chromogenic Endotoxin Quant Kit (Thermo Fisher, Waltham, MA, USA) measured the supernatant after the last wash step. Prior to assay analysis, the supernatant was filtered with a 200 nm syringe filter, dilute 1 :50 in endotoxin free water, and filtered with a 200 nm syringe filter.BCNPs size separation

[0070] To size separate further, 1 mL BCNPs suspended in water were bath sonicated with 500pL of 0.1 M polysorbate 80 surfactant solution (P80, Thermo Fisher, Waltham, MA, USA) and size filtered with a 200 nm syringe filter until further use.BCNPs size and zeta potential characterization

[0071] The particle size was measured by nanoparticle tracking analysis (NTA, NanoSight Malvern PANalytical, Salisbury, UK) at 23.9-24.1 °C and zeta potential (^-potential) was determined using a ^-potential analyzer (NanoSizer Zeta Series, Malvern Instruments, Malvern, UK) at 37 °C. Samples were diluted to a 1 :100 ratio (sample to DI water) from a batchconcentration of 5 mg / mL. To accurately measure the ^-potential, 5 pL of 10 mM sodium chloride (NaCI, Sigma-Aldrich, St. Louis, MO, USA) was added to the dilute samples.Scanning transmission electron microscopy (STEM)

[0072] To prepare BCNPs for STEM, the samples were diluted to 1 :200 ratio (sample to DI water) from batch concentration. BCNPs were adsorbed on a 200-mesh carbon film grid (Fisher Scientific, Waltham, MA, USA), negatively stained with UAR-EMS uranyl acetate replacement stain (Electron Microscopy Sciences, Hatfield, PA, USA), and then washed in DI water. Samples were prepared and imaged on the Tecnai G2 F20 SuperTwin TEM (Field Electron and Ion Company, Hillsboro, OR, USA).BC particle 3-day and 5-day preparation

[0073] BC particles were grown for 3-day (3d) and 5-day (5d) timepoints in a kombucha media and in agitated and aerated conditions, as mentioned in section 2.1 . At the 3d and 5d timepoints, the BC particles were separated from media via gravity filtration and then washed with 0.1 M NaOH and water to terminate the cellulose growth. Particles were placed in dialysis tubing (MW cutoff: 3K Da, Spectrum Laboratories, Inc., San Francisco, CA, USA) for 24 h to fully remove excess media and NaOH, and then were left suspended in water.Light microscopy

[0074] A Motic Panthera TEC-BF light microscope (Motic, Kowloon Bay, Hong Kong) equipped with a 5x objective was used to conduct particle size measurements of the 3d and 5d BC particles (n=20). The Feret's particle diameter was measured for 3d and 5d particles (n=20).BC growth curve measurement

[0075] To measure the mass of BC particles overtime, the collected particles were gravity filtered and weighed before and after air-drying (n=3).Scanning electron microscopy (SEM)

[0076] The particles were air-dried individually on a silicon wafer (University Wafer, Inc., Boca Raton, FL, USA) and placed on an aluminum stub (Ted Pella, Inc., Redding, CA, USA) with a piece of carbon tape (Ted Pella, Inc., Redding, CA, USA). The samples were then sputter coated with platinum on a Coater Leica-ACE600 (Leica Microsystems, Wetzlar, Germany) and imaged on a Thermo-Apreo-S SEM (Thermo Fisher Scientific, Waltham, MA, USA).Atomic force microscopy (AFM)

[0077] Particles were ultrasonicated using a Fisher Scientific Model 505 probe sonicator (Thermo Fisher, Waltham, MA, USA) and diluted to a weight percent of 0.1% with Mili-Q water (EMD Millipore, Burlington, MA). 60 pL of the sample solution were subsequently dropped onto a freshly cleaved mica (Ted Pella, Redding, CA), and naturally dried in a clean and dry environment,facilitating the adsorption of the samples onto the mica substrate. The samples were imaged on a Dimension Icon AFM (Broker Corporation, Billerica, MA) using tapping mode. The Multi75AI-G AFM probe (Budget Sensors, Sofia, Bulgaria) with a force constant of 3.0 N / m and a resonance frequency of 75 kHz was utilized for the imaging. The offline data processing was done with NanoScope Analysis (Broker Corporation, Billerica, MA) BCNPs chemical and physical characterization

[0078] To prepare BCNPs for Fourier Transform Infrared Spectroscopy (FTIR), X-Ray diffraction (XRD), and thermal gravimetric analysis (TGA), the samples were lyophilized using Labconco FreeZone 2.5 Plus 2.5 Liter Cascade Benchtop Freeze Dry System (Labconco Corporation, Kansas City, Missouri, USA). Samples were analyzed using a ThermoNicolet iS10 FT-IR instrument (Thermo Fisher, Waltham, MA, USA) where spectra were collected in the 4000 to 400 cm-1range with 20 scans and 4 crrr1resolution. XRD was analyzed on a D8 Advance XRD (Broker Co., Billerica, MA, USA) using a Cu / Ka radiation source wavelength of 1.5418 A, accelerating voltage of 40 kV, and filament emission of 40 mA. The degree of crystallinity, Xc, was calculated using Segal’s method (Equation 2) (E. J. Foster, et aL, Chem. Soc. Rev., 2018, 47, 2609-2679). Ic is the crystalline peak intensity (at 22.8°) and la is the amorphous peak intensity (at 18°) after the background signal is subtracted. The apparent crystallite size, D, is also calculated for a specific plane orientation (29) using the Scherrer equation (Equation 2) where A is the wavelength of incident radiation, k is a dimensionless shape factor set to 1 , and p is the full width at half maximum intensity of the peak corresponding to the crystal orientation in question (Yoshinaga, supra).Equation 2: 100 (2)Equation 3: (3)

[0079] TGA was performed on a TA Discovery TGA 550 instrument (TA Instruments, New Castle, DE, USA) from 25 to 1000 °C at a heating rate of 10 °C / mL, and under nitrogen atmosphere of 25mL / min to prevent thermoxidative degradation.Drug incorporation with BCNPs

[0080] BSA (Thermo Fisher, Waltham, MA, USA) was the model drug incorporated into BCNPs. 0.25 mg, 0.5 mg, and 1.0 mg BSA solutions were prepared in phosphate buffer solution (PBS, Thermo Fisher) and 1 mL was added to size separated 0.5 mL BCNPs with P80 surfactant (BCNPs / P80-BSA). BCNPs / P80-BSA was incubated for 2 h at ambient temperature on an orbital rotator mixer and collected and washed in DI twice at 15,000 rpm for 25 min via centrifugation. The supernatant was collected from each wash and combined for BSA quantification usingbicinchoninic acid (BCA) protein assay kit (Pierce BCA Protein Kit, Thermo Fisher, Waltham, MA, USA)Bicinchoninic acid assay to quantify BSA in BCNPs

[0081] The supernatant samples from BCNPs / P80-BSA and 0.25 mg, 0.5 mg, and 1.0 mg BSA solutions were added to a 96-well microplate containing the BCA standard solutions and BCA reagents from the BCA assay kit. The BCA assay was measured using Ultraviolet-visible (UV-vis) spectrometry (BioTek Synergy H1 Microplate Reader, Fisher Scientific, Waltham, MA, USA) at 562 nm. A calibration curve was created by measuring the absorbances of BCA standard solutions and known BSA concentrations. The supernatant of non-BSA incorporated BCNPs served as a control. The BSA drug loading (DL) and encapsulation efficiency (EE) of the BCNPs were calculated using the following equations (A. Joseph, et al., Nano Res., 2018, 11 , 5670- 5688):Equation r- ..Equation

[0082] The particle size and ^-potential were measured as described in the Methods. From the NTA results, the polydispersity index (PDI) was calculated using the Equation 1 (Raval, supra). In vitro drug release profile

[0083] BCNPs / P80-BSA were resuspended in 1 mL of phosphate buffer solution (PBS) and placed in dialysis tubing (MW cutoff: 3K Da, Spectrum Laboratories, Inc., San Francisco, CA, USA). The dialysis membrane was submerged in 20 mL of pH 4.5 PBS containing 1% P80 surfactant solution (Thermo Fisher, Waltham, MA, USA)), ratio 100:1 , in a 250 mL Erlenmeyer flask and placed on an incubated plate shaker at 90 rpm and 37 °C (Joseph, supra). At the designated timepoints, the PBS / 1 % P80 solution with BSA was collected and fresh PBS / 1% P80 was replaced for the next time point. BSA released at each timepoint was quantified using a BCA assay measured on UV-Vis spectrometry. The percent of BSA released by BCNPs at each timepoint was calculated using the following equation (Joseph, supra)-.Equation 6: BSA Release (7(6)v 7ResultsBCNPs growth and morphological observations

[0084] BCNPs were prepared by bacteria co-cultured with yeast cells (see Methods). In FIG. 4A photographs of the culture flasks taken every 24 hours until day 5 show that early-stage formation of BC can be visually observed from the 2-day timepoint when distinct particles have started to form. Further aggregation of the observed particles becomes apparent at 4-day and 5-day as macroscopic particles are formed. The average particle diameter was 206.9 ± 56.2 nm, 1.04 ± 0.187 mm and 1.535 ± 0.644 mm for the BC 1 day (1d, FIG. 4B), 3d, and 5d particles respectively (FIG. 5). It is noted that for 1 d the average particle diameter was measured for n = 12 particles where particles that appeared aggregated, such as the three particles in FIG. 4B, were measured individually. For 3d and 5d, the average diameter is measured for n > 20 particles. The micrographs show that all collected particles (1d, 3d, and 5d) include randomly oriented BC fibers that form a porous nanof ibrillar network as an extracellular matrix. The SEM images of the macroscopic 3d and 5d particles show the distinct BC fiber bundles that form large sheets as the particles are dried on the imaging substrate (FIG. 3). The BC fiber bundles have macroscopic length seen in the SEM images. High-magnification AFM images (FIG. 4D) showed findings for the cellulose fiber density and exhibited notable growth from 1 d to 5d. At BC 1 d, only a limited number of individual fibers in the thickness of 3.358 (± 1 .684) nm are present. The fibers are arranged in a mesh-like network (FIG. 4D (left)). The cellulose fiber density becomes more pronounced in the 3d sample, showcasing an average thickness of 7.395 (±3.595) nm. Subsequently, the fiber density of the 5d sample reaches a much higher level, and the average thickness increases further to 12.182 (±4.057) nm.

[0085] Analysis of the measured fibril thicknesses (FIG. 4E) shows that both the average fibril thickness and the variation in fibril thickness increase with time. Therefore, the elemental fibrils available at a given time have increasingly larger thickness distributions as growth time increases. Unlike the mesh-like network observed for 1 d BC, the fibrils observed for 3d (FIG. 4D (middle)) and 5d (FIG. 4D (right)) BC are assembled into fibers of increasing thickness. At both 3d and 5d, fibers can be observed that appear to be directly formed from intertwined elementary fibrils. There are also fibers present at 5d (FIG. 4D (right)), that are formed from bundles of these intermediate fibers, demonstrating the true hierarchical nature of BC. The size of these fiber bundles increases from 15.481 (±2.835) to 19.834 (±2.946) nm between 3d and 5d. These results collectively show that over the 5-days of monitored culture, BC fibrils increase their thickness and intertwine to form thicker and longer fibers which ultimately aggregate to form spherical-like particles. The diameter of these particles increases from a few hundreds of nanometers (1 d) to a few millimeters. It’s possible that for short growth times (1 d) shear forces associated with agitation prevent the formation of organized fibers above a threshold that would promote hierarchical fiber formation, and instead, the distinct mesh-like network morphology is favored. However, given sufficient time (3d and 5d), the BC will preferentially assemble to form the hierarchical fibers of increasing magnitude. Thus, different fiber arrangements are obtained for short (1 d) versus longer (3d-5d) culture durations.

[0086] FIGs. 4A-4E illustrate BC growth at various timepoints: (4A) photographs of the culturing flasks at 24 hour timepoints until 5-day; (4B) STEM image of BC 1 d; (4C) SEM images of BC 3d and 5d; (4D) AFM images of BC 1d, 3d and 5d; and (4E) histogram of AFM data - relative frequency versus fiber diameter (nm) for 1 d, 3d, and 5d.

[0087] FIG. 5 illustrates light microscope images at a 5x optical setting for BC microparticles grown for: (left) 3 days and (right) 5 days. Increased particle size at 5 days of growth compared to 3 days. Scale bars set at 100 pm.

[0088] The BC yield data (wet and dry mass) over the growth period of 5 days are presented in FIGs. 6A-6C. The measured data was fit with a logistic growth model in which biomass (M) is plotted as a function of time (t):Equation

[0089] K is the upper horizontal asymptote (measured maximum value of biomass) and r is the fitted parameter associated to the growth rate. The absolute growth rate (AGR) is calculated using equation 8.

[0090] FIGs. 6A-6C illustrate (6A) the growth of BC (grams) wet mass and dry mass as a function of growth time (days) with logistic growth fit curves and corresponding equations; (6B) the AGR of wet and dry BC mass plotted as a function of time on opposing axes; and (6C) the ratio of dry to wet mass (Dry:Wet Mass) at each recorded time point plotted against the ratio of the dry AGR to the wet AGR (Dry:Wet AGR) at all times 0-6 days.

[0091] While data was only collected at 24-hour increments, the logistic fit extrapolates the mass growth, and thus the AGR, to time = 0. The growth rate parameter (r) observed for dry BC (r=0.97) is greater than that previously reported for BC grown in a static culture (r=0.55) (M. Hornung, et aL, Eng. Life Sci., 2006, 6, 537-545). This is consistent with additional findings that BC production can be increased by agitation for some bacteria species. It should be noted that the logistic fit for dry mass growth projects the maximum value of biomass to be 0.32 g. The mass measurements show that the wet biomass AGR starts from 0.2 and is maximized at 0.6 g / day between days 1 and 2. From that point on it is projected to decrease and level off after day 4. On the other hand, the dry biomass growth rate shows a symmetric curve peaking at 0.2 g / day on day 3. Between day 3 and 4 of growth, the ratio between the AGR of dry and wet mass (dry:wet) surpasses 1.0 and increases nonlinearly until data collection ended (FIG. 6C). The ratio between the dry and wet mass also began to increase significantly between day 2 and the end of the experiment. The water holding capacity of BC is also reflected by the intense fiber network paired with a swellingbehavior from its porosity and high tensile strength that demonstrate BC’s biological and mechanical properties (M. Konopacki, et aL, Biochem. Eng. J., 2022, 182, 108432; M. Badshah, et al., Cellulose, 2017, 24, 5041-5052).Structure and physical properties of BCNPs

[0092] The structural and thermal properties of BCNPs were compared to BC particles grown for 3d and 5d. XRD, FTIR, and TGA were used to identify the crystallinity, chemical bonding, and thermal behavior of the BC particles. XRD patterns of the BCNPs grown for 1d (FIG. 7A) show broad, indistinct peaks at 18-19° and 30-35° 20. The lack of sharp, salient peaks in the diffraction pattern indicates that while some nanocrystalline regimes may exist, the BCNPs are predominantly amorphous (W. Yao, et aL, Cellulose, 2020, 27, 5563-5579; C. Aulin, et aL, Langmuir, 2009, 25, 7675-7685). The formation of nanocrystalline cellulose is more evident for particles grown for 3d and 5d as the characteristic peaks at 15° 29 (corresponding to ("1 " "1 " “0" ) and (110) ip planes) and 22.7° 20 (corresponding to (200) ip plane) are detected (T. G. Fawcett, et aL, Powder Diffr., 2013, 28, 18-31 ; A. D. French, Cellulose, 2014, 21 , 885-896). Cellulose ip diffraction planes are used herein to denote specific peaks although likely both la and ip polymorphs coexist. In the absence of high temperatures, the alkaline conditions used to purify bacteria cellulose particles are not harsh enough to significantly reduce the la content (Moon, supra). The degree of crystallinity calculated for BCNPs, and the BC particles grown for 3d and 5d are 37.8%, 76.6%, and 81.5%, respectively. The crystallite sizes for the (200) crystallographic planes are 3.29 and 4.80 nm respectively for the 3d and 5d particles, indicating that both crystallinity and crystallite size increase with time. The BCNPs do not exhibit crystals with the (200) orientation and so they have been excluded from the crystallite size analysis. However, the broad nature of the peaks in the diffraction pattern for BCNPs suggest crystallite sizes smaller than those observed for 3d and 5d BC particles. Therefore, in this experimental example, primarily amorphous cellulose nanoparticles are obtained after 1d, while the particles harvested after 3d and 5d of culture include nanocrystalline BC microfibers. The fact that predominantly amorphous BCNPs can be obtained is beneficial to their applicability as a drug delivery system, as amorphous carriers promote the bioavailability of the drug, and increase the solubility and the dissolution rate of a drug (Stewart and Grass, Mol. Pharm., 2020, 17, 180-189; Hancock and Zografi, J. Pharm. Sci., 1997, 86, 1-12).

[0093] FIGs. 7A-7C illustrate characterization of BC structure and thermal properties: (7A) XRD showing the 24 hour BCNPs are amorphous, the 3d and 5d particles have small amounts of cellulose I nanocrystals; (7B) FTIR spectra to identify cellulose functional groups; and (7C) TGAand DTGA profiles show the thermal degradation for each sample. All results include the samples BCNPs, BC 3d, and BC 5d.

[0094] The FTIR spectra of the BC samples was assessed (FIG. 7B). The broad peak between 3000-3500 cm'1is attributed to O-H stretching vibrations (A. Vazquez, et al., J. Polym. Environ., 2013, 21 , 545-554 ; C. M. Lee, et al., J. Phys. Chem. B, 2015, 119, 15138-15149). In this region, the BCNPs spectrum is broader and more uniform than the spectra of the 3d and 5d particles. The 3d particle O-H band, while also broader than the 5d, has more salient features. The O-H band of the 5d particles is the narrowest and most defined of the BC samples. These differences in the O-H band suggest that the ratio of intermolecular and intramolecular hydrogen bonding between surface hydroxyl groups varies between samples. The peak-narrowing as a function of growth time for BC particles may be the result of increasing homogeneity of the physical and chemical environment as fibrillar order increases over time (Foster, supra; Joseph, supra). The XRD spectra support this concept as crystallinity was found to increase with growth time (FIG. 7A). Peaks between 2920-2922 cm'1represent C-H stretching and vibrations, while 2897 cm-1 represents asymmetric -CH2 stretching and 2855 cm-1represents symmetric -CH3 stretching (V. V. Revin, et aL, Polymers, 2021 , 13, 1422; E. Erbas Kiziltas, et aL, Carbohydr. Polym., 2015, 129, 148-155). 1440 cm1identifies -CH2 bending and 1300 cm1identifies C-H bending (N. Halib, Sains Malays., 2012, 41 , 211 ). The peaks at 1 163 cm1, 1030-1060 cm-1and 1105 cm-1correspond to C-O-C stretch of the p (1 -4) glycosidic bonds, C-0 stretch and C-O-C in-plane stretch of the glucose ring in cellulose, respectively (Halib, supra; O. Asanarong, et aL, Eur. Polym. J., 2021 , 143, 110224). These signature peaks are observed clearly in the 5d particles, and are broader, yet still distinguishable in the 3d particles. The spectra of the 24 hour BCNPs shows the peaks at 1030-1060 cm1and 1105 cm1broader than all other samples, while the 1160 cm-1peak appears as a broad shoulder downshifted to 1140 cm-1. The increasing intensity, area, and sharpness of C-0 and glycosidic bond peaks with longer culture times suggests that the cellulose fibers have a higher number of those skeletal bonds (i.e. higher molecular weight). Lastly, the broad peak around 1650 cm-1is associated with the O-H vibrations of the adsorbed water (Cichosz and Masek, Materials, 2020, 13, 4573).Table 1: The degree of crystallinity (Xc) and crystallite size (D) for BCNPs and BC particles cultured for 3d and 5d as calculated from XRD spectra. Additionally, the range of temperatures in which peak degradation (DTG) occurs as well as the temperatures corresponding to 5% (T5%) and 10% (T10%) mass loss from TGA measurements for each material are presented.

[0095] The TGA data for BC allows assessment of the thermal degradation behavior of materials (FIG. 7C). The BCNPs show a 10% mass loss immediately upon heating, which is associated with water evaporation. The 3d and 5d BC particles have lower amount of withheld water (5%) that evaporates more gradually from room temperature to 100°C. Across all studied samples, there is no thermal degradation observed before 90°C in this experimental example. The thermal decomposition of cellulose gives rise to the mass loss steps observed between 190-400°C for the BC particles grown for 3 and 5d and 90-350°C for the BCNPs (Jiang and Hsieh, Carbohydr. Polym., 2013, 95, 32-40). Differences in the crystal size, molecular weight, and molecular weight dispersity of cellulose give rise to the large differences in the thermal degradation profiles (O. M. Vanderfleet, et al., Cellulose, 2019, 26, 507-528). The thermal stability is indicated by the initiation of thermal decomposition and not the temperature range that the decomposition spans, or the temperature at which the maximum decomposition rate occurs. The T5% (temperature at which 5% of the sample mass has been lost) for 3d BC and 5d BC are 80°C and 180°C respectively, while T10% is 230°C for 3d BC and 245°C for 5d BC (Table 1 ). Therefore, in this experimental example, the thermal stability of 5d BC particles is higher than that of 3d BC. In addition, the maximum degradation rates (DTG peaks) are achieved between 290°C and 330°C for the BC particles. The DTG peaks for BCNPs appear at 140°C, 230°C and 290°C. Lastly, the molecular weight distribution can be associated with the slope of the weight loss curve or the intensity of the DTG peak, as a uniformity in molecular weight causes a larger share of the molecules to degrade at a similar temperature. Large slope or high DTG peak intensity indicate tight molecular weight distribution or relative homogeneity in molecular weight. Based on these results, in this experimental example, the BC particles have more uniform molecular weight dispersity than the BCNPs. The higher molecular weight (as inferred from the FTIR spectra), tighter molecular weight distribution, and similar crystal size (as shown from XRD analysis) of the BC 3d and 5d particles justify their higher thermal stability, tighter decomposition temperature window, and higher temperatures of maximum degradation rate compared to the BCNPs. The shorter cellulose chains for BCNPs result in a larger number of chain-ends per sample mass, compared to highermolecular weight 3d and 5d BC particles. Chain-ends serve as thermal degradation initiation points, justifying the lower thermal stability of BCNPs (K. Watanabe, et al., Cellulose, 1998, 5, 187-200). Thus, the mass loss profiles, confirm that BCNPs are the particles with the smallest crystal size and lowest molecular weight, while culturing for longer times leads to cellulose fibers with higher molecular weight. Moreover, the TGA confirmed that the thermal stability of the BC samples is above biological temperatures which is a consideration for shipping and storage conditions in biomedical materials.BSA drug loading and encapsulation efficiency in BCNPs

[0096] To prepare the BCNPs for the drug loading process, the collected nanoparticles were sonicated with P80 surfactant solution (see Materials). The measured BCNPs concentration was 8.35x107± 1 ,18x107particles / mL with an average particle size of 103.0 ± 1 .5 nm (FIG. 8A, n=5). The NTA data report the size of BCNPs formed by using P80 as a surfactant to prevent the BCNP aggregation compared to the STEM image (FIG. 4B) of BCNPs prepared without size separation and P80 surfactant solution. To assess the long-term stability of BCNPs, the particle size was measured after storage in room temperature for 2 weeks, 5 months, and 7 months (Table 2). The stored particles have negligible changes in their size up to 5 months of storage, without requiring temperature-controlled storage conditions... • or si BCNP Concentration Particle Size ~ STD .... .Preparation BCNPs , . , .rPDI(particles / mL) (nm)Fresh 8.35* 107± 1.18* 107103.0 ± 1.5 0.00022 weeks 9.00 - 108± 4.24* 10795.2 ± 4.0 0.00185 months 4.37* 108± 7.52* 107105.8 ± 4.4 0.00177 months 3.13xlOE± 8.93x lO7147.4 ± 6.3 0.0018Table 2. Results for particle size and concentration were measured on NTA; n=5 for BCNPs prepared at different times. All values reported as an average ± standard deviation (STD). The polydispersity index (PDI) was calculated from NTA results using equation (1). Preparation BCNPs corresponds to the time frame prior to measurement.

[0097] FIG. 8A illustrates the hydrodynamic size of BCNPs at concentration of 8.35x107±1 .18x107particles / mL and measured on nanoparticle tracking analysis. The red region indicates peak intensity and standard deviation. FIG. 8B illustrates STEM of BCNPs at 1 urn scale bar; and FIG. 8C illustrates STEM of a BCNP at 100 nm scale bar. FIG. 8D illustrates a STEM image of BCNPs at 100 nm to show the distribution of particle morphology. Scale bar: 100 nm.

[0098] To observe if the fibrillar morphology of BCNPs is maintained after treatment with the surfactant solution, STEM was conducted. The collected images of BCNPs (FIGs. 8B-8D) reveal that the fibrillar structure of the nanoparticles was retained.

[0099] Another desirable property for nanoparticles in biomedical applications is a near-neutral or slightly negative surface charge as it minimizes ionically-driven cellular membrane disruption (Clogston and Path, in Methods in Molecular Biology, 201 1 , pp. 63-70). To achieve a desired surface charge, this is dependent on the therapeutic application where the administration route and target would be known. Here, the average (-potential of the BCNPs treated with the surfactant solution was measured to be slightly negative, -14.1 ± 4.2 mV (n=3). The endotoxin units per milliliter (EU / mL) of the post processed BCNPs and prior to drug incorporation were 0.46 EU / mL (n=3). This was measured below the FDA regulation limit of 0.50 EU / mL for medical devices containing bacteriostatic water for injection (U.S. Food & Drug Administration, Bacterial Endotoxins / Pyrogens, 1985, 9; U. S. Food & Drug Administration, Guidance for Industry: Pyrogen and Endotoxins Testing, 2012, 1 1 ).

[0100] Given the nanoscale size, surface charge, high surface area, and nanofibrillar morphology of the amorphous BCNPs, the applicability of the disclosed BCNPs as a natural polymer platform for drug delivery applications was evaluated (Yoshinaga, supra; S. Gorgieva, Processes, 2020, 8, 624; C. A. Rosas-Casarez, Int. J. Adv. Comput. Sci. Appl., 2014, 4, 226). Different concentrations of BSA were incorporated into BCNPs / P80 at three relative targeted drug loadings (DL). High encapsulation efficiency (EE) around 90% was achieved for all target loadings. Incorporating BSA into the BCNP / P80 increased the particle size by 2.5-fold, to 250 nm, with similar or more negative (-potentials compared to non-BSA containing BCNPs / P80 (Table 3).BCNP Mass of Target , Particle Size PDI ("-potential ±Concentration BSA DL ± STD (nm) STD (mV)5 0.25 5 4.31±0.0I 89.99±0.31 2563X8.3 0.0010 -11.9±8.75 0.50 10 8.34±0.03 90.99±0.36 255.0±5.4 0.0004 -26.2±4.75 1.0 20 15.46±0.02 91.42±0.12 231.7^3.5 0.0002 - 18.7±9.4Table 3. Results for BSA drug loading (DL) and encapsulation efficiency (EE) of BCNPs / P80 were calculated using equation (3) and equation (4). BCA assay was used in technical triplicates to quantify BSA concentration with UV-Vis spectroscopy (n=3). All values reported as an average ± standard deviation (STD). The polydispersity index (PDI) was calculated from NTA results using equation (1).BSA drug release profile from BCNPs

[0101] The STEM images of the 8.34% DL sample showed similar particle shape to non-DLBCNPs / P80 and a slight increase in size due to the BSA incorporation (FIG. 9A). The drug release assay showed a burst release of BSA at 2 hours, with a significant burst release of 8.9%for the 4.31% DL samples. Data is reported in both in % w / w (FIGs. 9A, 9B) and in pg (FIG. 90). In fact, within the first 4 h, the 4.31% DL samples release 30% of the total amount of BSA that they ultimately release over the entire 168 hour period. After that initial burst release, these samples demonstrate a sustained release profile. At the end of the 168 h testing period, the 4.31% DL samples released cumulatively 55.3% of BSA, which corresponds to 126.2 pg in total, out of 229.5 pg initially loaded. The 8.34% DL samples showed a low release with a sustained profile. After the initial burst release of 3.9%, an average of 2.5% (11 .3 pg) of BSA were released every 24 h compared to the 4.31% DL samples where the average release was of 5.5% (12.6 pg). Cumulatively, the 8.34% DL samples released 24.7% of BSA, corresponding to 113 pg out of 459.1 pg initially loaded, which is 45% less than the 4.31% DL samples. The 15.46% DL samples showed small quantitative changes in the release at later time points, indicated by a flattened curve (FIG. 9B). Over the 168 hour period, these samples had a cumulative release of 8.7%. Notably, only 11 .6% of this total release was observed within the first 48 hours. Subsequently, an average of 10% of the total release, which corresponds to 10.2 pg was released every 24 hours thereafter. At the end of the release study window, the BSA remaining associated with BCNPs is 44.7 ± 18.5, 75.3 ± 10.5 and 91 .4 ± 8.1 % w / w for the 4.76, 8.34 and 15.46% DL, respectively. Therefore, the highest cumulative release and lowest amount of bound BSA was found to be in the 4.31 % DL samples, indicating that the majority of BSA incorporated was surface associated. The 8.34% and 15.46% DL samples may have provided a higher BSA concentration gradient driving the BSA into the material core, seen similarly in a BC-based microsphere study (Zhang, supra). As the BSA-BCNP association is stronger, these samples have a lower release, compared to the sample with minimum BSA loading

[0102] FIGs. 9A-9C illustrate (9A) STEM images of 8.34% DL BSA-BCNPs / P80 (scale bar set to 200 nm); (9B) results for cumulative BSA drug release (% w / w) from BCNPs / P80, calculated using equation (6); and (9C) results for cumulative BSA drug release (ug) from BCNPs / P80, calculated using equation (6).

[0103] Drug loading of BSA into BCNPs / P80 showed a strong binding affinity (Yoshinaga, supra) with a sustained release profile. Physiologically relevant conditions were used to simulate BSA release. This experimental example aligns with prior studies loading BSA into BC microparticles, prepared using a bottom-up approach via the culturing of porous BC-based spheres and adsorbing BSA (W. Zhang, et al., Int. J. Biol. Macromol. , 2019, 140, 196-205). BC- BSA microparticle studies by Lin et al. (Int. J. Biol. Macromol., 2015, 73, 264-269) and Zhang et al, supra showed a strong electrostatic interaction between BC and BSA, where BSA adsorbingat the inner pore surface of the BC sphere was defined by a first-order release model, indicating a controlled drug release of BSA. BSA is one representative drug that can be incorporated into BC for therapeutic delivery, but many other drugs of interest have been incorporated into macroscale or micro-BC platforms (Tan and Ho, Drug Discovery Today, 2018, 23, 1108-11 14; Caraceni and Bonavita, Blood Transfus., 2013, s25). A recent study loaded a BC film with ibuprofen and propranolol hydrochloride, a low and highly soluble drug, respectively (C.Jantarat, et al., RSC Adv., 2021 , 1 1 , 37354-37365). Both drugs showed immediate release with freeze-dried and air-dried BC film preparations, yet the freeze-drying method showed a sustained release for soluble drugs while air drying was optimal for DL at low concentrations (Jantarat, supra). Another study used a top-down approach to mill a BC film into microparticles to drug load cioxacillin and cefuroxime sodium salts, adequate model drugs for antimicrobial activity in wound healing and facial scrubs (H. Ullah, et aL, Curr. Pharm. Des., 2019, 25, 3692- 3701 ). All methods showed an immediate drug release of 85% in the initial 30 min while demonstrating good antibacterial activity (Ullah, supra). Additional examples of alternative therapeutics include benzalkonium chloride, an antimicrobial surfactant used in BC wound dressing, curcumin / polysorbate 20 used in cellulose nanoparticles, and cioxacillin and cefuroxime sodium salts in BC particles for wound dressing and medicated cosmetics (Ullah, supra-, R. K. S. Bhavana, et aL, Cellul. Chem. Technol., 2016, 50, 223; Y. C. Ching, et aL, Cellulose, 2019, 26, 5467-5481 ). Surf ace chemistry modifications can further expand drug incorporation applications by altering the ^-potential, pH, and solubility to promote drug-BC incorporation, or to conjugate a drug to the BC backbone.

[0104] FIG. 10 illustrates STEM images of BCNPs at 2 different magnifications to show the distribution of particle morphologies.

[0105] FIG. 1 1 illustrates XRD spectra of BC grown for 1 -day, 3-day, and 5-day timepoints and for comparison, cellulose nanocrystals (CNCs). The increase in crystallinity is qualitatively shown over time of growth.

[0106] FIGs. 12A and 12B illustrate FTIR spectra of BC grown for 1 -day, 3-day, and 5-day timepoints and CNCs: (12A) wavenumber range 3600-2800 cm1and (12B) wavenumber range 1200-900 cm1.Conclusion

[0107] This experimental example describes the preparation and characterization of BCNPs as a drug delivery platform. The facile fabrication and minimal processing steps required are useful for repeatability and scalability of the material. The use of the disclosed BCNPs serves as a sustainable alternative to the existing synthetic polymers and lipids used in drug delivery in thatBCNPs are (1 ) bioderived, without the use of petroleum, (2) the only product of the culturing media, and (3) processed using minimal solvent.

[0108] This experimental example shows that the disclosed BCNPs produced after 24 hours of bacterial culture and size separated with P80 surfactant solution have an average size of 103 nm, slightly negative zeta potential, and include predominantly amorphous and entangled cellulose nanofibrils. Longer culture duration produces large BC particles that reach a diameter of 1000 - 1500 pm in this experimental example. These particles include longer and larger cellulose fibers which themselves are bundled nanofibrils that have a higher crystallinity and MW compared to the nanoparticles harvested after 24 hours. Nanoparticles can diffuse and distribute more readily in tissue compared to bulk BC; therefore, the disclosed BCNPs offer numerous advantages over BC particles and BC pellicles for drug delivery. TGA and FTIR data showed BCNPs are thermally stable until 90 °C and are chemically stable at room temperature in this experimental example, thereby avoiding the cold-chain storage and distribution required for the manufacturing of other nanomedicine platforms. BCNPs are biodegradable by nature and can be produced as an alternative and tunable drug delivery therapeutic to treat disease while having a reduced environmental impact through use of green engineering.EXAMPLE CLAUSES1 . A synthetic nanoparticle including bacterial cellulose, wherein the synthetic nanoparticle has a diameter of 200 nanometers (nm) or less.2. The synthetic nanoparticle of clause 1 , wherein a zeta potential of the synthetic nanoparticle is negative or neutral at a physiological pH.3. The synthetic nanoparticle of clause 1 or 2, wherein a zeta potential of the synthetic nanoparticle is positive at a physiological pH.4. The synthetic nanoparticle of any of clauses 1 -3, wherein a zeta potential of the synthetic nanoparticle is -30 to +30 millivolts (mV).5. The synthetic nanoparticle of any of clauses 1-4, wherein a surface of the synthetic nanoparticle is functionalized with a hydrophilic group or a hydrophobic group.6. The synthetic nanoparticle of any of clauses 1-5, wherein a surface of the synthetic nanoparticle is functionalized with at least one of: an amine group, an acetyl group, a methyl group, a sulfonic acid group, an ether group, or a carboxylic acid group.7. The synthetic nanoparticle of any of clauses 1 -6, wherein a polydispersity index of the synthetic nanoparticle is 0 to 0.3.8. The synthetic nanoparticle of any of clauses 1 -7, wherein a polydispersity index of the synthetic nanoparticle is 0 to 0.2.9. The synthetic nanoparticle of any of clauses 1 -8, wherein the synthetic nanoparticle is a fibrous nanoparticle including bacterial cellulose fibers.10. The synthetic nanoparticle of any of clauses 1 -9, wherein the synthetic nanoparticle is linked to a radioactive isotope, an immunotoxin, a drug, or a detectable label.11. A composition including the synthetic nanoparticle of any of clauses 1 -10 and a pharmaceutically acceptable carrier, wherein the composition has 0 to 0.5 endotoxin units (Ell) per milliliter (mL).12. The composition of clause 1 1 , wherein the composition has 0.46 EU / mL.13. A solution including sugar, acetic acid, a microbial culture producing cellulose, and nanoparticles including the cellulose.14. The solution of clause 13, wherein the sugar includes at least one of: sucrose, fructose, glucose, glycerol, or mannitol.15. The solution of clause 13 or 14, wherein the microbial culture includes acetic acid bacteria and yeast.16. The solution of any of clauses 13-15, wherein the microbial culture includes a symbiotic culture of bacteria and yeast (SCOBY).17. The solution of any of clauses 13-16, wherein the microbial culture includes at least one of acetobacter bacteria, gluconobacter bacteria, or gluconacetobacter bacteria.18. The solution of any of clauses 13-17, further including a carbon-nitrogen source.19. The solution of any of clauses 13-18, further including tea leaves.20. A method for synthetic nanoparticle formation, the method including: receiving aggregated bacterial cellulose fibers; separating the aggregated bacterial cellulose fibers; spinning a solution including the separated bacterial cellulose fibers at a rate and a time until synthetic nanoparticles form; and extracting the formed synthetic nanoparticles from the solution.21 . The method of clause 20, wherein the separating includes: incubating the aggregated bacterial cellulose fibers in one or more solvents; and / or fragmenting the aggregated bacterial cellulose fibers.22. The method of clause 21 , wherein the one or more solvents include lithium chloride (LiCI) and / or dimethylacetamide (DMAc).23. The method of clause 21 or 22, wherein fragmenting includes: applying a shear force to the aggregated bacterial cellulose fibers; and / or applying ultrasonic waves to the aggregated bacterial cellulose fibers.24. The method of any of clauses 20-23, wherein the aggregated bacterial cellulose fibers are freeze-dried.25. The method of any of clauses 20-24, wherein the rate includes 600 to 800 rotations per minute (rpm).26. The method of any of clauses 20-25, wherein the rate includes 650 to 700 rpm.27. The method of any of clauses 20-26, wherein the time includes 0.5 to 3 hours.28. The method of any of clauses 20-27, wherein the time includes 1 to 2.5 hours.29. The method of any of clauses 20-28, wherein the formed synthetic nanoparticles have a diameter of 200 nm or less.30. The method of any of clauses 20-29, wherein the formed synthetic nanoparticles are fibrous nanoparticles including bacterial cellulose fibers.31. The method of any of clauses 20-30, further including removing endotoxins from the solution.32. The method of any of clauses 20-31 , further including generating the aggregated bacterial cellulose fibers by culturing a media including a microbe forming cellulose and a carbon-nitrogen source.33. The method of clause 32, wherein the method does not use cellulose as a source reagent.34. The method of clause 32 or 33, wherein the microbe includes at least one of: acetic acid bacteria and yeast, a SCOBY, acetobacter bacteria, gluconobacter bacteria, or gluconacetobacter bacteria.35. The method of any of clauses 32-34, wherein the carbon-nitrogen source includes at least one of: tea leaves, sucrose, fructose, glucose, glycerol, or mannitol, peptone, or yeast extract.36. The method of any of clauses 20-35, further including: functionalizing a surface of the extracted synthetic nanoparticles.37. The method of clause 36, wherein the surface of the extracted synthetic nanoparticle is functionalized with at least one of: an amine group, an acetyl group, a methyl group, a sulfonic acid group, an ether group, or a carboxylic acid group.38. The method of any of clauses 20-37, further including: conjugating, to a surface of the extracted synthetic nanoparticles, a radioactive isotope, an immunotoxin, a drug, or a detectable label.39. The method of clause 38, wherein the radioactive isotope, the immunotoxin, the drug, or the detectable label is configured to hydrogen bond to the surface of the extracted synthetic nanoparticles.40. The method of any of clauses 20-39, wherein the method does not generate byproducts.41. A method for synthetic nanoparticle formation, the method including: spinning a media including sugar, acetic acid, and a microbial culture in aerated conditions at a rate and for a time until synthetic nanoparticles form; adding a reagent to the media to stop the formation of the synthetic nanoparticles; and extracting the formed synthetic nanoparticles from the media.42. The method of clause 41 , wherein the rate includes 100 to 200 rpm.43. The method of clause 41 or 42, wherein the rate includes 150 rpm.44. The method of any of clauses 41 -43, wherein the rate and the aeration conditions vary based on a volume of the media.45. The method of any of clauses 41-44, wherein the media is a tea.46. The method of any of clauses 41 -45, wherein the microbial culture includes a symbiotic culture of bacteria and yeast (SCOBY).47. The method of any of clauses 41-46, wherein the time includes 12 to 36 hours.48. The method of any of clauses 41-47, wherein the time includes 24 hours.49. The method of any of clauses 41 -48, wherein the formed synthetic nanoparticles have a diameter of 200 nm or less.50. The method of any of clauses 41 -49, wherein the formed synthetic nanoparticles are fibrous nanoparticles including bacterial cellulose fibers.51 . The method of any of clauses 41-50, wherein the method does not use solvents.52. The method of any of clauses 41 -51 , wherein the method does not use cellulose as a source reagent.53. The method of any of clauses 41-52, wherein the method does not generate byproducts.54. The method of any of clauses 41 -53, further including removing endotoxins from the extracted synthetic nanoparticles.55. The method of any of clauses 41-54, further including functionalizing a surface of the extracted synthetic nanoparticles.56. The method of clause 55, wherein the surface of the extracted synthetic nanoparticle is functionalized with at least one of an amine group, an acetyl group, a methyl group, a sulfonic acid group, an ether group, or a carboxylic acid group.57. The method of any of clauses 41 -56, further including: conjugating, to a surface of the extracted synthetic nanoparticles, a radioactive isotope, an immunotoxin, a drug, or a detectable label.58. The method of clause 57, wherein the radioactive isotope, the immunotoxin, the drug, or the detectable label is configured to hydrogen bond to the surface of the extracted synthetic nanoparticles.CLOSING PARAGRAPHS

[0109] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be used for realizing implementations of the disclosure in diverse forms thereof.

[0110] The term “tea” and its equivalents, as used herein, refer to an infusion of tea leaf or another part of a plant in water, including infusions of coffee beans, chamomile, fruit, and the leaves, blossoms, or other parts of other plants. The term “tea leaf” and its equivalents, as used herein, refer to the leaves of Camellia sinensis or rooibos obtained from Aspalathus linearis. The variants of Camellia sinensis include Camellia sinensis var. sinensis, Camellia sinensis var. assamica, Camellia sinensis var. pubilimba, or Camellia sinensis var. dehungensis (in T, Bartholomew B. "18. Theaceae". Flora of China. 12). In particular embodiments, tea varieties include black tea, green tea, white tea, oolong tea, pu-erh tea, and purple tea which are all made from the Camellia sinensis tea plant.

[0111] As will be understood by one of ordinary skill in the art, each implementation disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the implementation to the specified elements, steps, ingredients or components and to those that do not materially affect the implementation. As used herein, the term “based on” is equivalent to “based at least partly on,” unless otherwise specified.

[0112] Unless otherwise indicated, all numbers expressing quantities, properties, conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the artwhen used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0113] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0114] The terms “a,” “an,” “the” and similar referents used in the context of describing implementations (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate implementations of the disclosure and does not pose a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of implementations of the disclosure.

[0115] Groupings of alternative elements or implementations disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0116] Certain implementations are described herein, including the best mode known for carrying out implementations of the disclosure. Of course, variations on these described implementationswill become apparent to those of ordinary skill in the art upon reading the foregoing description. It is expected that skilled artisans to employ such variations as appropriate, and it is intended for implementations to be practiced otherwise than specifically described herein. Accordingly, the scope of this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by implementations of the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS1 . A synthetic nanoparticle comprising bacterial cellulose, wherein the synthetic nanoparticle has a diameter of 200 nanometers (nm) or less.

2. The synthetic nanoparticle of claim 1 , wherein a zeta potential of the synthetic nanoparticle is negative or neutral at a physiological pH.

3. The synthetic nanoparticle of claim 1 , wherein a zeta potential of the synthetic nanoparticle is positive at a physiological pH.

4. The synthetic nanoparticle of claim 1 , wherein a zeta potential of the synthetic nanoparticle is -30 to +30 millivolts (mV).

5. The synthetic nanoparticle of claim 1 , wherein a surface of the synthetic nanoparticle is functionalized with a hydrophilic group or a hydrophobic group.

6. The synthetic nanoparticle of claim 1 , wherein a surface of the synthetic nanoparticle is functionalized with at least one of: an amine group, an acetyl group, a methyl group, a sulfonic acid group, an ether group, or a carboxylic acid group.

7. The synthetic nanoparticle of claim 1 , wherein a polydispersity index of the synthetic nanoparticle is 0 to 0.3.

8. The synthetic nanoparticle of claim 1 , wherein a polydispersity index of the synthetic nanoparticle is 0 to 0.2.

9. The synthetic nanoparticle of claim 1 , wherein the synthetic nanoparticle is a fibrous nanoparticle comprising bacterial cellulose fibers.

10. The synthetic nanoparticle of claim 1 , wherein the synthetic nanoparticle is linked to a radioactive isotope, an immunotoxin, a drug, or a detectable label.

11. A composition comprising the synthetic nanoparticle of claim 1 and a pharmaceutically acceptable carrier, wherein the composition has 0 to 0.5 endotoxin units (Ell) per milliliter (mL).

12. The composition of claim 11 , wherein the composition has 0.46 EU / mL.

13. A solution comprising sugar, acetic acid, a microbial culture producing cellulose, and nanoparticles comprising the cellulose.

14. The solution of claim 13, wherein the sugar comprises at least one of: sucrose, fructose, glucose, glycerol, or mannitol.

15. The solution of claim 13, wherein the microbial culture comprises acetic acid bacteria and yeast.

16. The solution of claim 13, wherein the microbial culture comprises a symbiotic culture of bacteria and yeast (SCOBY).

17. The solution of claim 13, wherein the microbial culture comprises at least one of acetobacter bacteria, gluconobacter bacteria, or gluconacetobacter bacteria.

18. The solution of claim 13, further comprising a carbon-nitrogen source.

19. The solution of claim 13, further comprising tea leaves.

20. A method for synthetic nanoparticle formation, the method comprising: receiving aggregated bacterial cellulose fibers; separating the aggregated bacterial cellulose fibers; spinning a solution comprising the separated bacterial cellulose fibers at a rate and a time until synthetic nanoparticles form; and extracting the formed synthetic nanoparticles from the solution.21 . The method of claim 20, wherein the separating comprises: incubating the aggregated bacterial cellulose fibers in one or more solvents; and / or fragmenting the aggregated bacterial cellulose fibers.

22. The method of claim 21 , wherein the one or more solvents comprise lithium chloride (LiCI) and / or dimethylacetamide (DMAc).

23. The method of claim 21 , wherein fragmenting comprises: applying a shear force to the aggregated bacterial cellulose fibers; and / or applying ultrasonic waves to the aggregated bacterial cellulose fibers.

24. The method of claim 20, wherein the aggregated bacterial cellulose fibers are freeze- dried.

25. The method of claim 20, wherein the rate comprises 600 to 800 rotations per minute (rpm).

26. The method of claim 20, wherein the rate comprises 650 to 700 rpm.

27. The method of claim 20, wherein the time comprises 0.5 to 3 hours.

28. The method of claim 20, wherein the time comprises 1 to 2.5 hours.

29. The method of claim 20, wherein the formed synthetic nanoparticles have a diameter of200 nm or less.

30. The method of claim 20, wherein the formed synthetic nanoparticles are fibrous nanoparticles comprising bacterial cellulose fibers.31 . The method of claim 20, further comprising removing endotoxins from the solution.

32. The method of claim 20, further comprising generating the aggregated bacterial cellulose fibers by culturing a media comprising a microbe forming cellulose and a carbon-nitrogen source.

33. The method of claim 32, wherein the method does not use cellulose as a source reagent.

34. The method of claim 32, wherein the microbe comprises at least one of: acetic acid bacteria and yeast, a SCOBY, acetobacter bacteria, gluconobacter bacteria, or gluconacetobacter bacteria.

35. The method of claim 32, wherein the carbon-nitrogen source comprises at least one of: tea leaves, sucrose, fructose, glucose, glycerol, or mannitol, peptone, or yeast extract.

36. The method of claim 20, further comprising: functionalizing a surface of the extracted synthetic nanoparticles.

37. The method of claim 36, wherein the surface of the extracted synthetic nanoparticle is functionalized with at least one of: an amine group, an acetyl group, a methyl group, a sulfonic acid group, an ether group, or a carboxylic acid group.

38. The method of claim 20, further comprising: conjugating, to a surface of the extracted synthetic nanoparticles, a radioactive isotope, an immunotoxin, a drug, or a detectable label.

39. The method of claim 38, wherein the radioactive isotope, the immunotoxin, the drug, or the detectable label is configured to hydrogen bond to the surface of the extracted synthetic nanoparticles.

40. The method of claim 20, wherein the method does not generate byproducts.41 . A method for synthetic nanoparticle formation, the method comprising: spinning a media comprising sugar, acetic acid, and a microbial culture in aerated conditions at a rate and for a time until synthetic nanoparticles form; adding a reagent to the media to stop the formation of the synthetic nanoparticles; and extracting the formed synthetic nanoparticles from the media.

42. The method of claim 41 , wherein the rate comprises 100 to 200 rpm.

43. The method of claim 41 , wherein the rate comprises 150 rpm.

44. The method of claim 41 , wherein the rate and the aeration conditions vary based on a volume of the media.

45. The method of claim 41 , wherein the media is a tea.

46. The method of claim 41 , wherein the microbial culture comprises a symbiotic culture of bacteria and yeast (SCOBY).

47. The method of claim 41 , wherein the time comprises 12 to 36 hours.

48. The method of claim 41 , wherein the time comprises 24 hours.

49. The method of claim 41 , wherein the formed synthetic nanoparticles have a diameter of200 nm or less.

50. The method of claim 41 , wherein the formed synthetic nanoparticles are fibrous nanoparticles comprising bacterial cellulose fibers.51 . The method of claim 41 , wherein the method does not use solvents.

52. The method of claim 41 , wherein the method does not use cellulose as a source reagent.

53. The method of claim 41 , wherein the method does not generate byproducts.

54. The method of claim 41 , further comprising removing endotoxins from the extracted synthetic nanoparticles.

55. The method of claim 41 , further comprising functionalizing a surface of the extracted synthetic nanoparticles.

56. The method of claim 55, wherein the surface of the extracted synthetic nanoparticle is functionalized with at least one of an amine group, an acetyl group, a methyl group, a sulfonic acid group, an ether group, or a carboxylic acid group.

57. The method of claim 41 , further comprising: conjugating, to a surface of the extracted synthetic nanoparticles, a radioactive isotope, an immunotoxin, a drug, or a detectable label.

58. The method of claim 57, wherein the radioactive isotope, the immunotoxin, the drug, or the detectable label is configured to hydrogen bond to the surface of the extracted synthetic nanoparticles.

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