FIELD

[0001] Disclosed embodiments relate to thermal fiber drawing (TFD)-related processing and products therefrom including biosensors.

BACKGROUND

[0002] The efficacy of monodisperse polymer micro- and nanoparticles in chemical analysis and catalysis and photonics is well-established. In biomedical applications such as medical diagnostics, bioimaging, and drug delivery, polymeric particles are typically utilized in two distinct modalities. The particle surfaces may serve as loci for chemical or biological interactions through surface functionalization and control over their hydroscopic characteristics or the particle volume may alternatively be exploited to carry cargo via impregnation or encapsulation for drug delivery.

[0003] Applications of polymer nanoparticles in particular are steadily gaining importance in medicine because of their longer half-life and the versatility by which their composition, size, shape, and physicochemical properties may be tuned via a variety of processing approaches. Recently, there have been successes based on the use of polymer particles in specific biomedical applications including, for example, delivery of dendrimer prodrug using collagen peptides to metastatic tumors, and targeting of breast and prostate tumors for delivery of toxins using surface-conjugated biodegradable polymers.

[0004] However, there remain limitations in tumor localization of nanoparticles mainly due to inefficient accumulation (< 10%) in the target while the majority of the particles end up in the liver or spleen that need to be addressed by extending further control over particle design to ensure the realization of the desired behavior and biodistribution. For example, no single nanoparticle size can reach different areas of tumors at different stages of tumor development, which suggests the benefit of particle fabrication processes that are not limited to narrow size spans. The varying sizes and distinct modalities needed for disparate biomedical applications in turn necessitate different materials-specific fabrication pathways for producing such diverse structures, which typically precludes utilization of the same polymer for multiple purposes.

[0005] The widespread adoption of polymer particles in these and future applications could be enhanced by developing a scalable process that bridges the micro- and nano-scales and is compatible with a variety of polymers. A further challenge is to develop a process that is sufficiently versatile to produce a multiplicity of particle architectures optimized for specific applications using the same polymer.

[0006] Known bottom-up synthetic approaches produce nanoparticles that typically have considerable size dispersion, cannot reach the micro-scale, and are sensitively tuned to specific chemical building blocks. Top-down approaches for fabricating polymeric particles, such as emulsification, the use of non-wetting templates, and microfluidics-based approaches exploit polymers in a low- viscosity state or in solution and rely on prefabricated devices to impart form and size to the particles, which are subsequently solidified. In general, the process kinetics in both bottom-up and known top-down nanoparticle fabrication strategies limit each approach to a narrow set of materials, sizes, and structures.

SUMMARY

[0007] This Summary is provided to introduce a brief selection of disclosed concepts in a simplified form that are further described below in the Detailed Description including the drawings provided. This Summary is not intended to limit the claimed subject matter's scope.

[0008] Disclosed embodiments include scalable top-down processes that produce uniformly sized spherical polymer particles with a size continuously tunable from micro- to nano-scale without the need for any premade device in which the particles are formed. In lieu of prefabricated templates or microfluidic channels filled with co-flowing fluids, a thermal treatment of a core/cladding polymer fiber is used to controllably induce at the interfaces Plateau-Rayleigh capillary instability (PRI) which exploits the natural tendency of low-viscosity cylinders to break up into a plurality of substantially equally sized spherical polymer particles.

[0009] PRI has been found to result in the initially intact (continuous) cylindrically shaped biocompatible polymer core breaking up into a necklace (a plurality in a line) of uniformly sized spherical polymeric particles along the whole fiber length which is held stationary (embedded) in a cladding material. The embedded spherical polymer particles can be subsequently released from the cladding using an appropriate solvent for dissolving the cladding material to form a plurality of spherical polymer particles. The scalability of the process stems from the ability to stack a high density (plurality) of cores inside a single fiber. The separate spherical polymer particles are functionalized with at least one material that adds functionality to form functionalized particles.

[0010] In one embodiment, the separate spherical polymer particles are functionalized to form functionalized particles using a selected biological material that can comprise a polymer, protein, or an oligonucleotide material. Oligonucleotides are relatively short, single- stranded deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) molecules, and their variants. The oligonucleotide can thus comprise nucleic acids such as DNA, RNA, peptide nucleic acid (PNA), or ap tamers (both DNA and RNA).

[0011] Alternatively, a biological material (e.g., polymer, a protein including an antibody, an oligonucleotide, or a small molecule inhibitor including synthetic inhibitor compounds) can be encapsulated within the separate spherical polymer particles either by placing the desired material in a hollow center inside of a scaled-up macroscopic preform having a cladding, that can then be thermally drawn into an extended fiber, or within a hollow-core pre-drawn fiber. In either case, the fiber is heated to provide PRI thereby resulting in polymer shells (capsules) encapsulating the biological material of interest. These filled capsules may be released by dissolving the cladding in a suitable solvent. The filled capsules may also be surface-functionalized as described above to provide multi-functional particles.

[0012] The above-described features combined in disclosed in-fiber fabrication approaches, size control, scalability, compositional and structural control, enable disclosed functionalized particles to be useful for biomedical applications. For example, as biosensors for recognition of specific cell types (e.g., cancer cells) through protein binding separation of specific cells from a sample mixture. Another example involves delivering biological materials (e.g., DNA, shRNA, siRNA proteins, and therapeutic small molecules) on the surface of or within disclosed functionalized particles to a patient.

[0013] Functionalized particles can be used to deliver biological materials (e.g.,

DNA, RNA, protein synthetic compounds or small molecule inhibitors) through intravenous, subcutaneous, intramuscular, intranasal, oral or topical delivery route. The particles can be surface functionalized, for example, through coupling of peptides to the polymers using copper-free click chemistry or EDC and sulfo-NHS based coupling method. Nucleotide aptamers can be conjugated with the polymer particles using chemical or an ultraviolet cross- linking method.

[0014] It is described herein steps for achieving these purposes via three example demonstrations of biomedical applications using disclosed functionalized particles being: (a) protein binding to the spherical polymer particle surfaces for biosensing (surface functionalization), (b) quantitative control over differential surface protein binding so that variable amounts of a protein bound on the surface can be quantitatively recognized by another protein or antibodies, and (c) encapsulation of a biological material in a hollow polymeric shell (volume functionalization), using collagen as a model (example) biological polymer material. As known in the art, collagen is a polymeric material generally defined as a group of naturally occurring triple helix proteins which are found in a variety of animals and humans.

[0015] All three example demonstrations described herein were carried out using the same example biocompatible polymer formed into two different particle structures, (i) a solid spherical polymer particle having surface functionalization of its outer surface and (ii) a hollow spherical polymer particle having volumetric cargo encapsulation, produced with essentially the same fabrication strategy. Biological polymers or materials other than collagen may also be used, and other bio-compatible polymers may be used for the shell. It was also confirmed that both surface and volume functionalization may be combined in the same particle to provide a multi-functional particle, a result expected to have wide implications for site-specific drug delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a flow chart that shows steps in an example method for forming functionalized particles, according to an example embodiment.

[0017] FIG. 2A is a schematic cross-sectional view of three (3) example functionalized particles having a protein, inhibitor or oligonucleotide coating on a spherical polymer particle, according to an example embodiment.

[0018] FIG. 2B is a schematic cross-sectional view of three (3) example functionalized particles having a positively charged coating on the protein, inhibitor or oligonucleotide coating, according to an example embodiment.

[0019] FIG. 3A depicts a plurality of polymer microcapsules having a protein filling held stationary in a cladding material, according to an example embodiment.

[0020] FIG. 3B depicts a plurality of the filled microcapsules after the cladding material was released, according to an example embodiment. [0021] FIG. 3C is a depiction of an example disclosed multi-functionalized particles including a first protein, inhibitor or oligonucleotide coating the polymer particle, wherein the particle has a hollow center including a second protein, inhibitor or oligonucleotide within the hollow center, according to an example embodiment.

DETAILED DESCRIPTION

[0022] Disclosed embodiments are described with reference to the attached figures, wherein like reference numerals, are used throughout the figures to designate similar or equivalent elements. The figures are not drawn to scale and they are provided merely to illustrate aspects disclosed herein. Several disclosed aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the embodiments disclosed herein.

[0023] One having ordinary skill in the relevant art, however, will readily recognize that the disclosed embodiments can be practiced without one or more of the specific details or with other methods. In other instances, well-known structures or operations are not shown in detail to avoid obscuring aspects disclosed herein. Disclosed embodiments are not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with this Disclosure.

[0024] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of this 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. Moreover, all ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein. For example, a range of "less than 10" can include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all sub-ranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 5.

[0025] Disclosed embodiments include in-fiber particle fabrication methods that may be divided into three stages, (I) macroscopic preform preparation including a polymer core surrounded by a cladding material, (II) thermal fiber drawing of the preform, and (III) then thermally (heating) induced in-fiber emulsification of the drawn (extended) preform via the PRI. The structure of the resulting particles is recognized to be determined in the preform- fabrication stage (I) while its size is recognized to be proportional to the fiber core diameter set in the fiber-drawing stage (II). A feature of this methodology is that the core at the macroscopic (e.g., centimeter-scale) preform stage (I) may be readily shaped to produce new particle structures, an aspect used as described herein as a core having a hollow center that allows cargo (e.g. biocompatible polymer material) encapsulation.

[0026] In stage I a preform comprising an example 700^m-diameter cylindrical cyclic - olefin polymer (COP) core surrounded by an example 35-mm-diameter polysulfone (PSU) cladding is provided. The preform is thermally drawn in stage II, such as into hundreds of meters of uniform 1-mm-diameter fiber with a 20^m-diameter core (a drawn fiber). In stage III, the drawn fiber is thermally treated under otherwise ambient conditions, reducing the materials' viscosity and inducing the PRI. The PRI process is seeded by thermodynamic fluctuations that arise at the core/cladding interface upon heating.

[0027] The classic Tomotika model of the PRI predicts that the fluctuation wavelength with the shortest growth time dominates and its amplitude increases without bound, leading to the breakup of the COP or other core into a necklace (a large number) of spherical particles held in the PSU (or other) cladding. This fluctuation wavelength is determined by the interface surface energy, materials' viscosity, and core diameter. After breakup, the COP particles can be extracted and separated by selectively dissolving the cladding (e.g., using dimethylacetamide (DM AC)). This process/method of particle formation is disclosed in related Pub. PCT application No. 20130202888 that also includes inventor Abouraddy, which is hereby incorporated by reference into this application.

[0028] FIG. 1 is a flow chart that shows steps in an example method 100 for forming functionalized particles, according to an example embodiment. Step 101 comprises providing a multi-material fiber having at least one core comprising a first polymer material and an outer first cladding layer comprising a second material different from the first material outside the core. Step 102 comprises thermal fiber drawing (TFD) the multi-material fiber to increase its length to form an extended multi-material fiber having an extended core.

[0029] Step 103 comprises thermally treating the extended multi-material fiber under conditions that cause a breaking up of the extended core to form a plurality of polymer particles embedded in the outer first cladding layer, that are generally spherical in shape. Step 104 comprises removing the outer first cladding layer to separate the plurality of polymer particles embedded in the outer first cladding layer to form a plurality of separate polymer particles.

[0030] Step 105 comprises particle functionalizing including at least one of (i) forming a first protein, inhibitor or oligonucleotide coating on the plurality of core polymer particles, and (ii) wherein the core has a hollow center providing a second protein, inhibitor or oligonucleotide within the hollow center before TFD (step 102) or after TFD and before thermally treating (step 103), wherein the thermally treating encapsulates the second protein, inhibitor or oligonucleotide in the first polymer material. As noted above, the oligonucleotide can comprise nucleic acids such as DNA, RNA, PNA or aptamers (both DNA and RNA). The inhibitor is generally a small molecule inhibitor. An "inhibitor" as used herein refers to a protein synthesis inhibitor that is a substance that stops or slows the growth or proliferation of cells by disrupting the processes at the ribosome level that lead directly to the generation of new proteins. The inhibitor can comprise a synthetic compound, such as a synthetic antibody.

[0031] One can continuously tune the size (e.g., diameter) of the spherical polymer particles by drawing a uniform fiber using a different core fiber diameter (or more generally cross sectional area). As a result, the spherical polymer particle size produced is tunable continuously over several orders of magnitude in linear dimension. Particle sizes with diameters extending three orders of magnitude from ~ 50 μιη down to ~ 50 nm have been verified. The scalability of the process (i.e., the efficiency of particle production) may be increased, while retaining the size-tunability, by increasing the number of identical cores assembled within the cladding in the preform. For example, a 14-core fiber (20^m-diameter each) and a 1,000-core fiber (500-nm-diameter each) where a single thermal treatment after preform drawing leads to the simultaneous break up of all of the cores via the PRI process. The limit on the particle-production rate can thus be set by the fiber-drawing speed, the fiber outer diameter (or more generally cross sectional area), and the fiber-filling ratio (ratio of core-to-fiber areas).

[0032] A disclosed general approach involves placing the desired material in the core within a sacrificial thermoplastic polymer cladding matrix chosen such that they may be thermally co-drawn. A feature of this physical process is that it depends on the rheology and not the chemistry of the materials involved as long as they are immiscible. This was confirmed by reproducing the above results using other polymer pairs, such as polycarbonate (PC, core) and COP (cladding), resulting in PC particles, in addition to the use of combinations of inorganic glasses and polymers in conjunction with this fluid instability. Extracting the resulting spherical particles by selective dissolution of the cladding, on the other hand, does depend on the chemistry of the fiber materials, which can place restrictions on the potential polymer core-cladding pairings.

[0033] This potential constraint can be removed by separating the core and cladding polymers with an inorganic buffer layer thermally compatible with the polymer added in the preform, in which case the core and cladding may be prepared from the same polymer. This design was realized by using polyethersulfone (PES) in both the core fiber and the cladding separated by a buffer layer of a thermally compatible chalcogenide glass As ₂ Se _3. The PRI yields particles having a core (polymer) - shell (glass) structure that are released by dissolving the polymer cladding (e.g., using DMAC), and the polymer particles can be extracted by subsequent removal of the As ₂ Se ₃ shell, such as using NaOH in deionized water.

[0034] Applying disclosed TFD and PRI-driven-breakup-based fabrication methods using the same example biocompatible polymer COP, two distinct polymer particle structures tuned to two different classes of biological applications were produced. The first class of particle structures suited for certain biosensor applications is solid polymer particles having surface bound through adsorption, covalent bonding or cross cross-linking fluorescent proteins and antibodies (large Y-shaped proteins) or oligonucleotides for biosensing formed by adding fluorescent proteins, antibodies or oligonucleotides after separate spherical particle formation.

[0035] FIG. 2A is a schematic cross-sectional view of three (3) example functionalized particles 210a, 210b and 210c each having a protein, inhibitor or oligonucleotide coating 225 on outer surfaces of a spherical polymer particles 220, according to an example embodiment. Although the coating 225 is shown being continuous, the coating can also be discontinuous. The spherical polymer particles 220 comprise a first polymer having a molecularly smooth surface that provides a root mean square (rms) roughness < 2 nm, typically < 1 nm. The spherical polymer particles 220 generally have a median size from 5 nm to 1 mm, and are monodisperse providing a standard deviation in particle size < 10 % of the median size. The functionalized particles 210a, 210b and 210c are shown as each having the same dimensions to depict their monodisperse feature.

[0036] FIG. 2B is a schematic cross-sectional view of three (3) example functionalized particles 230a, 230b and 230c having a positively charged coating 235 on the protein, inhibitor or oligonucleotide coating 225 on a spherical polymer particle 220, according to an example embodiment. The functionalized particles 230a, 230b and 230c are again shown as each having the same dimensions to depict their monodisperse feature.

[0037] In another embodiment described below relative to FIG. 3C, the multi- functionalized particles include at least one protein, inhibitor or oligonucleotide coating on outer surfaces of a spherical polymer particles having a hollow center that is at least partially filled with a second protein, inhibitor or oligonucleotide. The second protein, inhibitor or oligonucleotide is encapsulated in the first polymer material of the spherical polymer particles.

[0038] Controllable protein-coating of the solid spherical polymer particles while maintaining biological specificity has been demonstrated, allowing for binding with antibodies that recognize them. Using a similar method that fills the hollow center of a core before TFD, or post-TFD but generally before core particle formation, a second class of particle structures and applications is described for encapsulation of selected materials inside a polymeric shell (or capsule), demonstrated here using collagen as a model biological polymer material that happens to be a protein.

[0039] It is evidenced herein that the surfaces of the spherical polymer particles produced by disclosed PRI-based processing are suitable for biosensing applications by coating the solid COP (or other polymer) particles after PRI and removal from the fiber cladding (e.g., PSU cladding), such as with antibodies conjugated with fluorophores (fluorescent antibodies ("FA's")). Through adsorption onto the surface of polymer particles it is recognized one can directly monitor optically the presence of bound (attached) proteins in a sample solution using two readily distinguishable FA's yellow-fluorescing FA1 (Cy3 conjugated anti-rabbit antibodies produced in goat) and red-fluorescing FA2 (Alexa Fluor- 647 conjugated anti-mouse antibodies produced in goat. A fluorophore is a fluorescent chemical compound that absorbs light energy of a specific excitation wavelength and re-emits light at a longer wavelength.

[0040] It was next verified that the particle-bound proteins retain their native conformation through optical detection of specific protein-protein interactions. Polymer particles were coated with mouse serum proteins to provide functionalized particles and the fluorescence emitted by two fluorescent antibodies incubated with the disclosed functionalized particles were monitored, FA2 and compared to one that does not include FA1. Lack of fluorescence from FA1 indicates that mouse serum proteins remain bound to the particles preventing binding of FA1 directly to the particles.

[0041] Moreover, protein-binding from a sample may be promoted through adding a positive charge to the polymer particle surface, such as for example by coating it with poly-1- lysine, a positively charged synthetic amino acid chain. This enhancement was confirmed by detecting stronger fluorescence from bound antibodies and also by observing Forster resonant energy transfer (FRET) between two tandem layers of particle-bound fluorescent antibodies: from FA1 to FA3 (Alexa Fluor 647 conjugated anti-goat antibodies produced in donkey). Antibodies FA3 were chosen since they recognize FA1 and the emitted fluorescence from FA1 overlaps with the FA3 excitation band, thereby allowing for FRET transfer from FA1 to FA3.

[0042] In addition to uniformly binding proteins to the surface of polymer core particles, evidence is provided for quantitative control over the relative composition of the immobilized proteins on core particles using two solutions comprising a mixture of two proteins: cofilin and bovine serum albumin (BSA). Both protein solutions were prepared with the same total amount of proteins but with two different relative concentrations: the first solution is 0.1 μg/ml Cofilin + 200 μg/ml BSA, while the second one was 10 μg/ml Cofilin + 190 μg/ml BSA. It was determined that the relative composition of the particle-bound proteins corresponds to that in the solutions using two methods.

[0043] Firstly, the particle-bound proteins were dissociated by boiling in a denaturing buffer and quantified the amount of protein from the two sample populations after electrophoretically separating the proteins based on their sizes in two sodium dodecyl sulfate (SDS) polyacrylamide gels. One gel was stained to identify BSA and confirmed that the numbers of particles in the two samples were approximately the same. The second gel was subjected to western blotting and confirmed that proportionate amounts of cofilin were retrieved from the particles. Secondly, the amount of particle-bound cofilin was monitored directly by incubation with two FA's: A cofilin-specific FA whose fluorescence strength corresponded to the different amounts of bound cofilin, and a non-specific FA used as a negative control. These results thus confirm that the particles can be coated with a controlled amount of a protein, which can be retrieved for subsequent characterization and quantification.

[0044] One unique feature of disclosed polymer particle fabrication strategies that highlights its versatility was then demonstrated. By modifying the macroscopic preform structure, micro-encapsulation of biological materials inside a polymeric shell of the same polymer utilized in the solid particles employed in the protein-binding experiments was established. Collagen was used as the encapsulant which may be useful for cosmetics and dental applications (e.g., for topical application on the skin and as dental implants, respectively) which serves as a model material for other potential biological materials or drugs.

[0045] Like many biological materials, collagen is a recognized to be a globular solution lacking uniform fluid consistency, making it incompatible with fiber drawing and also precludes the use of traditional microfluidics-based multiple-emulsion approaches. This limitation has been found to be removed by injecting the desired encapsulant inside a drawn hollow fiber with a COP lining surrounded by a PSU cladding. Collagen was injected into the 50^m-diameter core having a hollow center and then subjected to heating to thermally induce the PRI, which results in the core breaking up into a necklace of collagen-filled COP microcapsules held stationary in the PSU cladding.

[0046] FIG. 3A depicts a necklace 300 including a plurality of polymer microcapsules

310a, 310b, 310c each having a polymer shell 220' defining a hollow center having a biological or chemical agent filling material 315 comprising a protein, inhibitor or oligonucleotide in the hollow center shown as collagen held stationary in a cladding material 320 (e.g., PSU).

[0047] FIG. 3B depicts a plurality of the filled microcapsules 340a, 340b, 340c and

340d after the cladding material 320 was released on a substrate surface 330 such as a glass slide for example, such as using DMAC to dissolve a cladding material 320 comprising PSU. It was confirmed that the filled microcapsules 340a, 340b, 340c and 340d contain protein (collagen) therein by dissolving the polymer shell 220' comprising COP and then utilizing a Coomassie Brilliant Blue dye Bradford protein binding assay with the recovered the protein (e.g., collagen) filling cargo.

[0048] As described above, the two particle functionalization modalities disclosed above being outer surface binding (surface functionalization) and filled capsules (volume functionalization) can be combined. Multiple hollow centers may be embedded within a polymer shell such that each is filled with a different biological or chemical agent. The PRI- driven-breakup will then produce a spherical polymeric particle that encapsulates these agents in isolated enclaves. In this arrangement, the different and potentially incompatible encapsulants do not come into contact at any point during the process. Consequently, each particle may be considered a multi-compartment micro-reactor that produces localized biological or chemical interactions using prescribed ratios of reactants upon delivery of the cargo.

[0049] FIG. 3C is a depiction of a disclosed multi-functionalized particle 350 including an outer biological or chemical agent coating 360 comprising a protein, inhibitor or oligonucleotide coating the polymer shell 220' , wherein the polymer shell 220 'has a hollow center including a second protein, inhibitor or oligonucleotide within, according to an example embodiment. The hollow center can be filled by a variety of methods, such as using a syringe between the TFD and thermally treating steps described above. In one particular embodiment the multi-functionalized particle 350 comprises fluorescent antibodies bound to a collagen-filled COP microcapsule.

[0050] These disclosed size- and structure-tunable spherical polymeric particles fabricated using the scalable in-fiber fluid-instability mediated process are thus compatible with protein-binding and biosensing through protein-protein and protein-nucleic acid interactions. It is expected that this methodology and structure therefrom will have broad applications in development of diagnostic tools and cell-specific targeting of biomaterials.

[0051] A significant recognized task is to identify polymers that may be processed at lower temperatures to reduce damage or modification to encapsulated or impregnated biological materials, thereby paving the way to therapeutic applications in drug delivery. Combining these results on the precise structuring of multi-material particles using disclosed in-fiber approaches with the biomedical capabilities described herein offer potential for digital design of a new class of nano-engineered theranostic tools where the chemical, hydroscopic, optical, and biomedical properties can all be tuned simultaneously within each structured particle through design of the macroscopic preform from which the fiber is drawn. Advantages of using disclosed functionalized particles for medical applications are enabled by tunable particle size, which is beneficial for selective recognition of diseased tissues or cells, internalization of particles and targeted delivery of therapeutics or diagnostic molecules.

EXAMPLES

[0052] Disclosed embodiments are further illustrated by the following specific

Examples, which should not be construed as limiting the scope or content of this Disclosure in any way.

Methods

[0053] Solid-core fiber fabrication. A single-core fiber was constructed in five steps.

(1) A 6-mm-diameter cylindrical rod of COP (Zeon Chemicals) was extruded at 240 °C through a circular die under vacuum from as-purchased millimeter-sized pellets. (2) 100-μιη- thick PSU films (Ajedium) were rolled around this rod to form the cladding and the resulting 30-mm-diameter cylinder was consolidated into a preform at 200 °C under vacuum. (3) The preform was then thermally drawn into a 3.5-mm-outer-diameter cane (700^m-core- diameter) at 360 °C. (4) PSU films were then rolled around the drawn fiber and the resulting cylinder was consolidated to produce a 35-mm-diameter preform that was drawn into a 1- mm-diameter extended fiber with 20^m-diameter core. The procedure to fabricate the multi- core polymer fiber is similar, with the primary difference being that after step (3), a plurality of (e.g., 14) sections of the cane are stacked inside a PSU tube and consolidated into a preform that is then drawn into a fiber. [0054] Regarding the thermal treatment used applied to the extended COP core/PSU cladding fiber perform to induce PRI, the extended fibers were heated either in a furnace or on a hot plate in an ambient environment. The temperature was in the range 330 °C to °400 C and the heating time was 1 to 5 minutes. The time and temperature to induce PRI used was based on a number of factors (parameters) including the fiber outer diameter, the surrounding environment (open or closed), and the desired breakup speed.

[0055] Particle release. After the in-fiber COP particles were produced, they were released by dissolving the PSU cladding with dimethylacetamide (DMAC) at room temperature for 30 min. After settling in the solution, the particles were gathered after pouring out the solvent (containing dissolved PSU). This process was repeated (3-4 times) until essentially all of the PSU has been removed and the solvent was evaporated, leaving the COP particles on a glass substrate.

[0056] Fabrication of fibers with an inorganic sacrificial layer and particle release.

The fiber core and outer cladding were made of PES with a buffer layer of As ₂ Se ₃ glass (Amorphous Materials Inc.). The preform used to produce the fiber was prepared by multimaterial co-extrusion (see Tao G, Shabahang S, Banaei E-H, Kaufman JJ, Abouraddy AF (2012) Multimaterial preform coextrusion for robust chalcogenide optical fibers and tapers. Opt Lett 37(13):2751-2753) of a 30-mm-diameter cylindrical billet through a 12-mm- diameter circular die. The billet comprised from top to bottom of vertically stacked discs of PES, As ₂ Se ₃ , and PES with heights 30 mm, 20 mm, and 20 mm, respectively. The extruded rod was drawn in two steps into fibers and the PRI-driven breakup resulted in PES- core/As ₂ Se ₃ -shell particles. The PES cladding was dissolved in DMAC, releasing the PES particles. The PES particles were then released from the As ₂ Se ₃ shells by dissolving the As ₂ Se ₃ in NaOH. Method of coating and labeling.

[0057] The extracted COP particles were pelleted by centrifugation to remove the solvent (potentially containing remains of dissolved PSU), were washed twice with 2- Propanol to remove excess DM AC, and were then washed three times with phosphate buffered saline (PBS). The COP particles were divided into four groups. Group- 1 particles were resuspended in PBS and left untreated. These unlabeled COP particles were used as a negative control to confirm the negligible native fluorescence from COP at the wavelengths of interest. Group-2 particles were incubated with (a) FAl (20 μg/ml) for attachment to the particles through adsorption and (b) FA2 (20 μg/ml) for 18 h at 4 °C with end-over-end rotation. Next, the particles were pelleted at 10,000 xg and washed three times with PBS to remove unbound antibodies.

[0058] Group-3 particles were incubated in PBS containing 20% mouse serum

(Sigma- Aldrich) for 18 h at 4 °C with rotation. The particles were washed three times in PBS and subsequently divided into two subgroups to investigate whether antibodies would bind the immobilized proteins. The serum protein-coated particles were incubated with two different sets of antibodies, one that recognizes mouse serum proteins (FA2, 10 μg/ml) and one that does not (FAl, 10 μg/ml). Group-4 particles were coated with positively charged poly-l-lysine to provide a more hydrophilic surface. These particles were acid washed first by incubating with IN HCL at 60 °C for 6 h and washing with diH ₂ 0. Particles were incubated with poly-l-lysine (0.001% in diH ₂ 0; Sigma- Aldrich) for 18 h at 4 C and washed again three times with PBS. Next, particles were incubated with FAl (20 μg/ml) for 18 h at 4 °C with end-over-end rotation. The particles were washed again three times in PBS to remove unbound FAl and then incubated with FA2" (20 μg/ml) for 18 h at 4 °C with rotation. After binding of the secondary incubation the particles were washed again with PBS three times. COP particles bound with bound fluorescent antibodies were mounted on glass slides and visualized in a confocal microscope.

[0059] Quantitative assessment of differential protein-coating of particles. The particles were acid- washed (in IN HC1 for 2 h at 60 °C), pelleted and washed three times with RIPA buffer (50 mM Tris pH 7.5, 2 mM EDTA, 150 mM NaCl, 0.1% NP-40) to remove excess acid. Next, the particles were coated with poly-l-lysine as described above to provide a positive surface charge. The particles were then washed with RIPA buffer and evenly resuspended in RIPA buffer before equal volumes of particle suspensions were transferred into two tubes (a and b) and pelleted. The particles in both tubes were then resuspended and incubated at 4 °C for 12 h with two different protein solutions in RIPA buffer [tube (a): 0.1 μ^πιΐ Cofilin + 200 μ^πιΐ BSA; tube (b): 10 μ^πιΐ Cofilin + 190 μg/ml BSA]. Subsequently, the particles were centrifuged and washed with RIPA buffer three times.

[0060] During the last wash, 10% of the particle suspensions from both samples were transferred to a new microcentrifuge tube for staining with antibodies, while the remaining 90% of both particle suspensions were transferred to new microcentrifuge tubes containing Laemmli sample buffer and boiled for 5 min to elute the bound proteins. The boiled samples in both tubes (a) and (b) were then divided in half and separated on two SDS-PAGE gels. The particles were stuck in the wells but the soluble proteins migrated through the gels. Proteins in one gel were stained with Coomassie Brilliant Blue dye while proteins in the other were electrophoretically transferred to a PVDF membrane containing protein binding sites. The membrane was incubated in dry milk solution to block the unoccupied sites and prevent nonspecific binding of antibodies. The membrane was incubated with anti-cofilin primary antibodies produced in rabbit (Novus Biologicals). The membrane was incubated with HRP conjugated secondary anti-rabbit antibodies produced in goat (Jackson ImmunoResearch) and then with the substrates from Immun-Star WesternC Kit (Bio-Rad). Positive signals in the blot were imaged using ChemiDoc XRS (Bio-Rad).

[0061] The 10% of particles set aside for antibody staining were pelleted and resuspended in RIPA buffer containing 300 ng/ml rabbit anti-Cofilin antibodies, 300 ng/ml mouse anti-a-Tubulin antibodies (Sigma-Aldrich) - to confirm saturated binding of cofilin + BSA mixture to the particles - and 200 μg/ml BSA. The particles were incubated with the antibodies for 12 h at 4 °C before being pelleted and washed three times with RIPA buffer. The particles were then incubated for 4 h at 4 °C in RIPA buffer containing fluorescent secondary antibodies: 500 μg/ml goat anti-mouse conjugated antibodies Alexa Fluor 488 (Invitrogen), or 500 μg/ml goat anti-rabbit conjugated antibodies Alexa Fluor 647 (Invitrogen) + 200 μg/ml BSA. The particles were again washed three times in RIPA buffer before being resuspended in glycerol, mounted on slides, and imaged.

[0062] Fluorescence imaging. All fluorescence images were obtained using a Leica

SP5 confocal fluorescence microscope. Images were adjusted using the Leica LAS AF software.

[0063] Fabrication of hollow-core fibers for capsule production (PSU cladding COP lining). The preform was produced by rolling 75- μιη-thick COP sheets around a 6.5-mm diameter TEFLON (polytetrafluoroethylene) rod to reach a diameter of 9.5 mm. 100- μιη- thick PSU sheets were rolled around this structure to reach a 30-mm diameter. The preform was then consolidated under vacuum at 200 °C for 45 min, after which the TEFLON rod is removed, leaving a hollow-COP core preform. This COP core preform was then drawn into a 5.5 -mm-diameter cane. PSU sheets were rolled around this cane to reach a diameter of 30 mm, which in turn was consolidated and drawn into a 1 -mm-diameter fiber. During the fiber draw, nitrogen gas was flowed through the preform-turn-fiber to maintain constant pressure in the core and prevent the core from collapsing. The pressure was adjusted to produce a 50- μm-diameter core having a hollow center and 10- μιη-thick COP lining.

[0064] Collagen encapsulation and detection after release. A collagen cream

(Matriskin, RL3), containing 90% collagen, was injected into a hollow-core COP-lined fiber section after TFD using a syringe pump (New Era Pump Systems, NE-1000). The fiber section ends were sealed by heating and were then placed in a furnace at 350 °C for 3 min, resulting in collagen encapsulated particles with a COP shell which were released from the fiber cladding PSU matrix using DMAC and placed in a test tube under vacuum to remove any remnant solvent. Chloroform was added to the test tube to dissolve the COP shell and was then evaporated under vacuum to remove it completely, as chloroform can interfere with the protein assay.

[0065] Ultra-pure deionized water was then used to resuspend the precipitated remnants of the COP shells and cores. Next, the suspension was centrifuged at 10,000 μg for 30 s to precipitate any undissolved polymer or particulate materials. To assess the amount of soluble protein (collagen) recovered, Coomassie blue dye Bradford protein binding assay (Bio-Rad) was used. The maximum absorbance of Coomassie dye shifts from 465 nm to 595 nm when bound to protein. The presence of protein in the sample was detected by measuring the absorbance at 595 nm using a spectrophotometer (Biotek Synergy HI). The sample displayed an absorbance of 0.25AU (Absorbance Units) when only the protein assay dye reagent was normalized to 0.00 AU. This result confirms the presence of protein in the COP- encapsulated particles. The absorbance units were used to measure protein concentration using a standard curve of absorbance units versus known amounts of proteins. Protein measurements indicate that about 1.2 μg of protein were recovered from particles extracted from 15 cm of fiber, compared to the theoretical limit of 300 μg (assuming ~ 1 gm/cm ³ density for collagen). [0066] While various disclosed embodiments have been described above, it should be understood that they have been presented by way of example only, and not as a limitation. Numerous changes to the disclosed embodiments can be made in accordance with the Disclosure herein without departing from the spirit or scope of this Disclosure. Thus, the breadth and scope of this Disclosure should not be limited by any of the above-described embodiments. Rather, the scope of this Disclosure should be defined in accordance with the following claims and their equivalents.

[0067] Although disclosed embodiments have been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. While a particular feature may have been disclosed with respect to only one of several implementations, such a feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given application.