WO2019117816A1 - Porous three-dimensional structure comprised of nanofibers and a method of producing the same - Google Patents

Abstract

According to the present disclosure, there is provided a method of producing a porous three-dimensional structure comprised of nanofibers. The method comprises providing a polymer solution comprising a first polymer and a second polymer, electrospinning the polymer solution into a liquid reservoir to form a suspension comprising nanofibers formed from the first polymer and the second polymer, and drying the suspension to form the porous three-dimensional structure. In the preferred embodiment, the first and second polymer are poly(d-lactide) and poly(l-lactide); and the liquid reservoir is water and / or alcohol. The present disclosure also provides a porous three-dimensional structure obtained according to the method disclosed herein; an air filter comprising the porous three-dimensional structure and a tissue scaffold comprising the porous three-dimensional structure.

Description

POROUS THREE-DIMENSIONAL STRUCTURE COMPRISED OF NANOFIBERS AND A METHOD OF PRODUCING THE SAME

Cross-Reference To Related Application

[0001] This application claims the benefit of priority of Singapore Patent Application No. 10201710464S, filed 15 December 2017, the content of it being hereby incorporated by reference in its entirety for all purposes.

Technical Field

[0002] The present disclosure relates to a method of producing a porous three- dimensional structure comprised of nanofibers. The present disclosure also relates to such a porous three-dimensional structure comprised of nanofibers.

Background

[0003] Three-dimensional (3D) structures formed of nanofibers have been used in various applications, for example, (i) to form 3D polymeric scaffolds that may be used as templates for tissue regeneration, which form the basis of tissue engineering, and (ii) as filtration materials.

[0004] In the case of (i), when 3D nanofibrous scaffolds are compared to traditional two-dimensional (2D) culture, a 3D scaffold can provide a better link between single cells and organs. This is because the 3D scaffold offers another dimension for cell-cell interactions, cell migration and cell morphogenesis, which are important in regulating cell cycle and tissue functions. With recent advances in electrospinning technology, electrospun nanofibrous 3D scaffolds were developed to mimic the architecture of natural human tissue at the nanometer scale. The high surface area and their porous structure supported many cell activities, which made electrospun nanofibrous 3D scaffolds potentially useful for tissue engineering applications. However, scalability and control over the production of nanofibrous materials to form 3D macrostructure scaffold remain challenging because electrospun nanofibrous macrostructures tend to be limited to an assembly of conventional 2D membranes or loosely assembled 3D nanofibrous structures that may often be too fragile for handling. A typical thickness of traditionally electrospun 2D mats may he in the range of tens of micrometer. As the size of a rounded cell lies generally in the range of 5-20 pm, the pores in traditionally electrospun 2D mats are then too small to accommodate normal cells and thus obstruct cellular migration, even if the porous structure of the as-spun fibers may be beneficial to local mass transport.

[0005] In the case of ( i i ) . the particulate matters produced by fuel combustion in motor vehicles, biomass burning, etc., especially those submicron- and nanometer sized particles, are very harmful to human health because they may be fine enough to enter the human bloodstream. Conventionally, protection tends to be achieved through the filters in ventilation or air conditioning systems. However, such air filters, for example, high efficiency particulate air (HEPA) filters that are used in commercial air conditioning systems, effectively eliminate particles that are greater than 0.3 pm, but they are unable to effectively eliminate smaller particles. Some air filters with very small pores can achieve higher efficiency for removal of submicron- and nanometer sized particles but they suffer from undesirably high pressure drop across the filter due to their high resistance to air flow, greatly increasing the energy consumption when such filters are used for air filtration.

[0006] Regarding the materials for making the 3D nanofibrous structure, polymer nanofibers may be used over other materials because their surface can be tailored to provide the required chemistry for binding soft organic particles. The structural stability of such 3D filters consisting of polymeric nanofibers tend to be, however, relatively poor. The pore structure of the 3D nanofibrous filters may be adversely altered when the nanofibers are compressed due to pressure drops across the filters.

[0007] To address the above issues, there is a need to provide for a porous three- dimensional structure comprised of nanofibers that ameliorates one or more of the limitations mentioned above. There is also a need to provide for a method of producing such a porous three-dimensional structure comprised of nanofibers. Such porous three-dimensional structure comprised of nanofibers should at least be capable of being used as an air filter and/or a tissue scaffold. Summary

[0008] In one aspect, there is provided for a method of producing a porous three- dimensional structure comprised of nanofibers, the method comprising:

providing a polymer solution comprising a first polymer and a second polymer;

electro spinning the polymer solution into a liquid reservoir to form a suspension comprising nanofibers formed from the first polymer and the second polymer; and

drying the suspension to form the porous three-dimensional structure.

[0009] In another aspect, there is provided for a porous three-dimensional structure obtained according to the method described in the above aspect.

[0010] In another aspect, there is provided for an air filter comprising a porous three- dimensional structure obtained according to the method described in the above aspect.

[0011] In another aspect, there is provided for a tissue scaffold comprising a porous three-dimensional structure obtained according to the method described in the above aspect.

Brief Description of the Drawings

[0012] The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the present disclosure are described with reference to the following drawings, in which:

[0013] FIG. 1A shows the fabrication of 3D nanofibrous macrostructures by stacking multiple layers of 2D electro spun nanofibrous mats.

[0014] FIG. 1B shows the fabrication of 3D nanofibrous macrostructures by folding an electrospun 2D nanofibrous film.

[0015] FIG. 1C shows the fabrication of a 3D nanofibrous macro structure using a collector with pre-determined shape for the collection of electrospun nanofibers.

[0016] FIG. 1D shows the fabrication of 3D nanofibrous macrostructures by the self- assembly of electrospun nanofibers through specific physical or chemical interactions. [0017] FIG. 1E shows the complementary helical structures of PDLA (poly(d- lactide)) and PLLA (poly(l-lactide)).

[0018] FIG. 1F shows the stereocomplex of PDLA and PLLA.

[0019] FIG. 2 shows a general scheme of the present preparation method for a 3D nanofibrous macrostructure.

[0020] FIG. 3A shows a scanning electron micrograph (SEM) image of electropsun PLLA nanofibrous aerogel before tensile test. The scale bar denotes 1 pm.

[0021] FIG. 3B shows a SEM image of electrospun polylactide stereocomplexed (PLA SC) nanofibrous aerogel before tensile test. The scale bar denotes 1 pm.

[0022] FIG. 3C shows a SEM image of electrospun PLA SC-polydopamine aerogel before tensile test. The scale bar denotes 1 pm.

[0023] FIG. 3D shows a SEM image of electropsun PLLA nanofibrous aerogel after tensile test. The scale bar denotes 10 pm.

[0024] FIG. 3E shows a SEM image of electrospun PLA SC nanofibrous aerogel after tensile test. The scale bar denotes 10 pm.

[0025] FIG. 3F shows a SEM image of electrospun PLA SC-polydopamine aerogel after tensile test. The scale bar denotes 1 pm.

[0026] FIG. 4 shows the tensile curves of electrospun PLLA, PLA SC and PLA SC- polydopamine nanofibrous mats.

[0027] FIG. 5 A shows the porous structure of electrospun PLA SC nanofibrous macrostructures with packing density of 0.00873 g/cm³, representing the loose structure in size control of the nanofibrous macrostructure. The scale bar denotes 100 pm.

[0028] FIG. 5B shows the porous structure of electrospun PLA SC nanofibrous macrostructures with packing density of 0.01746 g/cm³, representing the dense structure in size control of the nanofibrous macrostructure. The scale bar denotes 100 pm.

[0029] FIG. 6A shows electrospun PLA SC nanofibrous suspension in ethanol.

[0030] FIG. 6B shows 3D structure of electrospun PLLA nanofibers. Due to poor intramolecular interaction, the designed macrostructure for cylindrical shape was not maintained after post-processing. [0031] FIG. 6C shows 3D PLA SC nanofibrous macro structure with various designed shapes and sizes.

[0032] FIG. 6D shows 3D PLA SC nanofibrous macrostructure with various designed shapes and sizes.

[0033] FIG. 6E shows 3D PLA SC nanofibrous macrostructure with various designed shapes and sizes.

[0034] FIG. 6F shows 3D PLA SC nanofibrous macro structure with various designed shapes and sizes.

[0035] FIG. 7A shows crystallinity behavior and thermal stability analysis of electrospun PLLA (curve a) and PLA SC (curve b) nanofibrous macrostructures by

XRD (X-ray powder diffraction).

[0036] FIG. 7B shows crystallinity behavior and thermal stability analysis of electrospun PLLA and PLA SC nanofibrous macrostructures by DSC (differential scanning calorimetry).

[0037] FIG. 7C shows crystallinity behavior and thermal stability analysis of electrospun PLLA and PLA SC nanofibrous macrostructures by TGA (thermogravimetric analysis).

[0038] FIG. 8A shows a photograph of water droplet on the surface of solvent cast PLLA film.

[0039] FIG. 8B shows a photograph of water droplet on the surface of solvent cast

PLA SC film.

[0040] FIG. 8C shows a photograph of water droplet on the surface of PLA SC- polydopamine film. The PLA SC is solvent cast before a thin layer of polydopamine is deposited on the PLA SC thin film.

[0041] FIG. 8D shows a photograph of water droplet on the surface of electrospun

PLLA nanofibrous macrostructures.

[0042] FIG. 8E shows a photograph of water droplet on the surface of electrospun PLA SC nanofibrous macrostructures.

[0043] FIG. 8F shows a photograph of water droplet on the surface of electrospun PLA SC-polydopamine nanofibrous macrostructures.

[0044] FIG. 9A shows NIH 3T3 fibroblasts cell viability cultured with different nanofibrous macrostructures. [0045] FIG. 9B shows the in vitro proliferation of fibroblasts cells in PLLA and PLLA/PDLA SC scaffolds made based on the present method over 28 days.

[0046] FIG. 9C shows the confocal images of cell growth on PLLA and PLA SC nanofibrous scaffolds on day 4 (left column), day 14 (center column) and day 28 (right column). Cell nuclei were stained blue and F-actin were stained red. The scale bars denote 100 mih.

[0047] FIG. 9D shows the 2D (left column), 3D (center column) and cross-section (right column) images of cell growth on PLLA and PLA SC nanofibrous scaffolds after 28 days. The scale bars denote 100 mih.

[0048] FIG. 10 shows a confocal micrograph showing the phenotype of NIH 3T3 fibroblast cells within different 3D nanofibrous scaffold.

[0049] FIG. 11 shows electro spinning of nanofibers onto a wire mesh in a liquid medium.

[0050] FIG. 12 shows a plot of the tensile curves for PLLA and PLA SC nanofibrous aerogels.

[0051] FIG. 13A shows a photograph of the PLLA (left) and PLA SC (right) aerogels with thickness of about 5 mm for compression tests.

[0052] FIG. 13B shows a plot of the stress-strain curves of PLLA and PLA SC aerogels of FIG. 13A.

[0053] FIG. 14A shows a SEM image of PLLA/PDLA-7/3 nanofibers (the total polymer concentration is fixed at 10 wt% with TBAC concentration of 0.5 wt%) at a magnification of x500. The scale bar denotes 10 pm.

[0054] FIG. 14B shows a SEM image of PLLA/PDLA-7/3 nanofibers (the total polymer concentration is fixed at 10 wt% with TBAC concentration of 0.5 wt%) at a magnification of x2000. The scale bar denotes 10 pm.

[0055] FIG. 14C shows a SEM image of PLLA/PDLA-7/3 nanofibers (the total polymer concentration is fixed at 10 wt% with TBAC concentration of 0.5 wt%) at a magnification of x5000. The scale bar denotes 1 pm.

[0056] FIG. 14D shows a SEM image of PLLA/PDLA-7/3 nanofibers (the total polymer concentration is fixed at 10 wt% with TBAC concentration of 0.5 wt%) at a magnification of x 10,000. The scale bar denotes 1 pm. [0057] FIG. 14E shows a SEM image of PLLA/PDLA-3/7 nanofibers (the total polymer concentration is fixed at 10 wt% with TBAC concentration of 0.5 wt%) at a magnification of x500. The scale bar denotes 10 pm.

[0058] FIG. 14F shows a SEM image of PLLA/PDLA-3/7 nanofibers (the total polymer concentration is fixed at 10 wt% with TBAC concentration of 0.5 wt%) at a magnification of x2000. The scale bar denotes 10 pm.

[0059] FIG. 14G shows a SEM image of PLLA/PDLA-3/7 nanofibers (the total polymer concentration is fixed at 10 wt% with TBAC concentration of 0.5 wt%) at a magnification of x5000. The scale bar denotes 1 pm.

[0060] FIG. 14H shows a SEM image of PLLA/PDLA-3/7 nanofibers (the total polymer concentration is fixed at 10 wt% with TBAC concentration of 0.5 wt%) at a magnification of x 10,000. The scale bar denotes 1 pm.

[0061] FIG. 15 shows a schematic diagram of an air filtration testing system.

[0062] FIG. 16 shows actual setup of the filtration testing system.

[0063] FIG. 17A shows a plot of a typical mass concentration- size distribution curves of model haze particles.

[0064] FIG. 17B shows a plot of the relative change in pressure drop of the 3D PLA SC filter over a duration of 24 hrs at two different relative humidities.

[0065] FIG. 17C shows a plot of the mass size distribution of 0.1 pm, 0.5 pm, 1 pm and 2.5 pm particles generated using a condensation aerosol generator.

[0066] FIG. 18A shows a SEM image of H13 filter at a magnification of xlOO. The scale bar denotes 100 pm.

[0067] FIG. 18B shows a SEM image of H13 filter at high magnification of x600. The scale bar denotes 10 pm.

[0068] FIG. 18C shows a SEM image of a low end HEPA filter (Hl l). The pore size is about 104 pm.

[0069] FIG. 18D shows a SEM image of a high end HEPA filter (H13). The pore size is about 46 pm.

[0070] FIG. 19A shows a SEM image of the cross-sectional morphology of an electrospun 3D PLA SC filter at a magnification of x50. The scale bar denotes 10 pm. The filter is produced by the present method of electro spinning into a liquid reservoir. [0071] FIG. 19B shows a SEM image of the filter of FIG. 19A at a magnification of x500. The scale bar denotes 10 mih.

[0072] FIG. 19C shows a SEM image of the filter of FIG. 19A at a magnification of x2000. The scale bar denotes 10 mih.

[0073] FIG. 19D shows a SEM image of the filter of FIG. 19A at a magnification of cIO,OOO. The scale bar denotes 1 mih.

[0074] FIG. 19E shows a SEM image of the cross-sectional morphology of electrospun 2D PLA SC mat at a magnification of x50. The scale bar denotes 10 mih. The mat is produced by conventional electro spinning onto an aluminum foil.

[0075] FIG. 19F shows a SEM image of the filter of FIG. 19E at a magnification of x500. The scale bar denotes 10 mih.

[0076] FIG. 19G shows a SEM image of the filter of FIG. 19E at a magnification of x2000. The scale bar denotes 10 mih.

[0077] FIG. 19H shows a SEM image of the filter of FIG. 19E at a magnification of x 10,000. The scale bar denotes 1 mih.

Detailed Description

[0078] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0079] Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and/or combinations and/or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.

[0080] The present disclosure describes a method for the facile fabrication of robust three-dimensional nanofibrous macrostructures with pre-designed shapes and tunable pore size via inter-fiber stereocomplexation of polymers, e.g. poly(d-lactide) (PDLA) and poly(l-lactide) (PLLA). The phrases “nanofibrous macrostructures” and “nanofibrous structures” are used interchangeably in the present disclosure to refer to structures comprised of nanofibers.

[0081] The expressions “stereocomplexation of polymers” and “polymer stereocomplex” refer to a stable composite formed from at least two complementing stereoregular polymers, which can interlock each other by strong stereoselective interactions. The stereoselective interactions arise from the network of hydrogens bonds and/or van der Waals interactions that spontaneously form between the nanofibers, when the nanofibers contact each other. The nanofibers are formed from stereoisomers of the polymers. In other words, the polymeric nanofibers are formed from polymer stereoisomers that are cross-linked to one another by hydrogen bonds and/or van der Waals interactions (i.e. inter- fiber cross-linking).

[0082] In the present method, the nanofibrous structures are derived by electro spinning. The electrospun nanofibrous materials can be fabricated into spatially distributed and interconnected scaffolds for tissue growth, vascularization, and diffusion of nutrients. Advantageously, the present method provides for scalable and controllable production of such nanofibrous structures for use as, for example, three-dimensional scaffolds that are mechanically improved for handling.

[0083] The present method allows for the nanofibrous structures to be fabricated with desired shape and pore size from various types of biodegradable polymer, such as but not limited to polylactide. Polylactide, in addition to being biocompatible both in their bulk and degraded forms, allows for the presence of stereocomplexation between PDLA and PLLA, which affords strong physical cross-linking points as inter-fiber junctions, and this facilitates the fabrication of the 3D structure scaffold with desired shapes, e.g. heart, vascular grafts, and cylindrical shapes. The inter- fiber junction may refer to a point in the 3D structure where two nanofibers come into contact and are cross-linked. The dimensions (e.g. length, breath and/or thickness) of the nanofibrous structures (e.g. 100 mm to 300 mm) depend on the size of the mold and this can be further tailored towards specific requirements.

[0084] The strong inter-fiber cross-linking interactions result in mechanically robust and thermally stable 3D nanofibrous structures with tunable packing density and pore size. The fixation of the shape and tuning of the packing density of the nanofibrous macrostructures in desired molds can be simultaneously achieved by varying the content of the stereocomplexed polymer nanofibers.

[0085] In the present method, the stereocomplexation of polymers, e.g. PLLA and PDLA, advantageously allows for formation of physical cross-linkages in electrospun PLA stereocomplexed nanofibers in suspension and/or during a freeze drying process. Due to the cross-linkages resulting from the stereocomplexation of polymers, the present method provides for a strategy to maintain the random morphology of the nanofibrous macro structure under external mechanical forces. The cross-linkages may be formed as strong inter-fiber junctions between the nanofibers. Advantageously, the structures are able to retain their shape without having the structure and the porous morphology mechanically damaged when forces are applied, which in turn permits the ingress of cells and nutrients during three-dimensional cell culture, demonstrating that the resultant scaffold developed through the present method is usable as a three- dimensional bio-implantation scaffold for tissue regeneration.

[0086] In addition, to enhance the surface wettability, cell viability and cell proliferation, the nanofibrous macro structure can be coated with a thin layer of polydopamine under mild conditions without compromising the mechanical strength and thermal stability of the nanofibrous macrostructure.

[0087] Apart from polylactide based polymers, different types of isotactic and syndiotactic polymers, such as poly(methyl mclhacrylalcjs (e.g. isotactic and syndiotactic poly(methyl mclhacrylalcjs), polypeptides (D- and L-amino acids), polyamides (e.g. D- and L-poly(hexamethylene di-0-methyl-tartaramide)s), and other types of enantiomeric helical polymers, can be used instead of PLLA and PDLA.

[0088] With the above in mind, the present disclosure provides for a method of producing a porous three-dimensional structure comprised of nanofibers. The present disclosure also provides for a porous three-dimensional structure obtained according to the present method described herein. The present disclosure further provides for an air filter and a tissue scaffold, both of which comprise the porous three-dimensional structure. Before going into the details of the various embodiments, the definition of certain terms, expressions and phrases are provided as follows.

[0089] The term“nanofiber” used herein refers to a fiber having a diameter of less than 1 mih.

[0090] In the context of the present disclosure, the phrase“organic solvent” refers to a carbon-based solution that is capable of dissolving a polymer. The organic solvent may be polar or non-polar.

[0091] The term“isotactic” used herein refers to a polymer in which all the repeating units have the same stereochemical configuration. Meanwhile, the term“syndiotactic” used herein refers to a polymer in which the repeating units have alternating stereochemical configurations.

[0092] In the context of various embodiments, the term“suspension” generally refers to a mixture in which electrospun nanofibers are not dissolved but are suspended throughout the bulk of a liquid.

[0093] The word“substantially” does not exclude“completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

[0094] In the context of various embodiments, the articles“a”,“an” and“the” as used with regard to a feature or element include a reference to one or more of the features or elements.

[0095] In the context of various embodiments, the term“about” or“approximately” as applied to a numeric value encompasses the exact value and a reasonable variance.

[0096] As used herein, the term“and/or” includes any and all combinations of one or more of the associated listed items.

[0097] Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements. [0098] Having defined the various terms, expressions and phrases as indicated above, the details of the present method, the porous three-dimensional structure comprised of nanofibers, their uses thereof, and the various embodiments are described as follows.

[0099] In the present disclosure, there is provided for a method of producing a porous three-dimensional structure comprised of nanofibers. The method includes providing a polymer solution comprising a first polymer and a second polymer, electro spinning the polymer solution into a liquid reservoir to form a suspension comprising nanofibers formed from the first polymer and the second polymer, and drying the suspension to form the porous three-dimensional structure.

[00100] When the electrospun nanofibers are formed in the liquid reservoir, the nanofibers may begin stereocomplexation. That is to say, the nanofibers may start cross-linking with each other in the suspension (i.e. inter-fiber cross-linking). Such inter-fiber cross-linking may arise due to hydrogen bonding and/or van der Waals interaction between the first polymer and the second polymer. Internal cross-linking (e.g. hydrogen bonding) within each of the nanofibers (intra-fiber) may also occur in the suspension. The stereocomplexation (i.e. cross-linkages) may occur at junctions where a nanofiber physically contacts another. Meanwhile, occurrence of internal cross-linking within individual nanofibers does not require such physical contact. In various embodiments, the nanofibers may be cross-linked between each of the nanofibers, and each of the nanofibers may be internally cross-linked.

[00101] Advantageously, the stereocomplexation between the nanofibers helps to maintain the random orientation of the nanofibers and the porous morphology and structure of the three-dimensional structure even after the three-dimensional structure is subjected to forces, e.g. compressive forces. In other words, the present method, which utilizes the stereocomplexation of polymers, provides for a mechanically robust three-dimensional structure, which is potentially usable as a tissue scaffold and/or an air filter.

[00102] Further advantageously, the inter-fiber stereocomplexation enhances the thermal stability of the porous three-dimensional structure, increasing its ability to withstand higher thermal degradation temperature (i.e. the temperature at which the structure begins to degrade). [00103] In the present method, providing the polymer solution includes dissolving the first polymer and the second polymer in a mixture of organic solvents. The mixture of organic solvents may comprise dichloromethane and/or dimethylformamide. The addition of the second organic liquid, such as dimethylformamide, to form the mixture of organic solvents, is to aid in the spinnability of the polymer solution.

[00104] The first polymer and the second polymer may be dissolved in any weight ratio ranging from 3:7 to 7:3. For example, the first polymer and the second polymer may be dissolved in a weight ratio of 5:5, 3:7, 7:3, etc.

[00105] The first polymer may be an isotactic polymer, a syndiotactic polymer, or an enantiomeric polymer. The second polymer may be an isotactic polymer, a syndiotactic polymer, or an enantiomeric polymer. The enantiomeric polymer may be an enantiomeric helical polymer. Each of the first polymer and the second polymer may comprise an isotactic polymer, a syndiotactic polymer, or an enantiomeric polymer, according to various embodiments.

[00106] In various embodiments, each of the first polymer and the second polymer may comprise poly(d-lactide), poly(l-lactide), isotactic poly(methyl methacrylate), syndiotactic poly(methyl methacrylate), polypeptides formed from one or more D- amino acids, polypeptides formed from one or more L-amino acids, D- poly(hexamethylene di-O-methyl-tartaramide), or L-poly(hexamethylene di-O- methyl-tartaramide) .

[00107] Advantageously, in the present method, the polymer solution may be electrospun into a liquid reservoir instead of a conventional collector plate. The liquid reservoir allows for stereocomplexation of the nanofibers to commence, which is difficult and may not occur if the collector plate is used. The liquid reservoir allows for the nanofibers to move about freely in all directions for the stereocomplexation of the nanofibers to take place, facilitating the formation of cross-linked highly porous 3D nanofibrous macrostructures. On the other hand, when a collector plate is used, the nanofibers cannot move once they are electrospun on the collector plate, and this gives rise to a cross-linked compact 2D nanofibrous film instead.

[00108] In various embodiments, the liquid reservoir may comprise water and/or alcohol. The alcohol may comprise any suitable alcohol, such as but not limited to, ethanol or butanol. As PLA is hydrophobic, the use of an alcohol helps the nanofibers to disperse and be suspended in the liquid collector.

[00109] The present method may further comprise transferring the suspension into a mold before drying so as to have the porous three-dimensional structure adapted to the configuration of the mold. Drying of the suspension may be carried out by or may comprise freeze drying of the suspension, subjecting the suspension to air in the presence of an absorbent material, or subjecting the suspension to vacuum. The freeze drying may be carried out by freezing the suspension in refrigerators, freeze-drier, liquid nitrogen, etc. The absorbent material may be made of superabsorbent polymers, which may be polyacrylic acid-based copolymers/blends, cross-linked carboxymethylcellulose, polyvinyl alcohol copolymers, etc. Other suitable absorbent materials that dry the suspension can be used.

[00110] The present method may further comprise coating the nanofibers of the porous three-dimensional structure with polydopamine after removing most of the solvents. In other words, the coating of the nanofibers with polydopamine is not performed after drying the suspension, instead most of the solvent is removed before the nanofiber suspension is placed into a dopamine solution for coating. The nanofiber suspension may then be freeze dried after the coating of polydopamine. This is advantageous as coating after freeze drying may not be feasible, as PLLA and PDLA are hydrophobic and the dopamine coating solution faces difficulty in penetrating into the dried 3D nanofibrous structures formed from the hydrophobic PLLA and PDLA. Based on such considerations, the nanofibers may be suspended in water for coating with polydopamine before freeze drying as an example. The water may be substantially or completely removed of organic solvents. Said differently, the method may further comprise coating the nano fibers in a suspension with poly dopamine before drying. The coating of the nanofibers with polydopamine renders the nanofibers hydrophilic or more hydrophilic, which in turn enhances surface wettability, cell viability and proliferation. The coating of the nanofibers with polydopamine may include contacting the nanofibers with a dopamine solution according to various embodiments. [00111] The present method may further comprise contacting the porous three- dimensional structure with biomolecules to deposit the biomolecules on the nanofibers. This may be carried out to form a tissue scaffold.

[00112] The present disclosure also provides for a porous three-dimensional structure obtained according to the method described above. Embodiments and advantages described in the context of the present method are analogously valid for the porous three-dimensional structure described herein, and vice versa.

[00113] For instance, various embodiments of the first polymer and the second polymer for forming the nanofibers that make up the porous three-dimensional structure have already been described above.

[00114] The porous three-dimensional structure is advantageous as it is mechanically robust and has improved thermal stability due to stereocomplexation of the polymers as already explained above. That is to say, the three-dimensional structure maintains its porous structure and morphology, and the nanofibers that form the three- dimensional structure also maintain their random orientation, even after forces are applied, e.g. tensile or compressive forces.

[00115] In various embodiments, the porous three-dimensional structure may comprise nanofibers which are cross-linked between each of the nanofibers, and each of the nanofibers may be internally cross-linked. The stereocomplexation (i.e. cross- linkages) may occur at junctions where a nanofiber physically contacts another.

[00116] The porous three-dimensional structure may be used as an air filter or tissue scaffold. Accordingly, the present disclosure also relates to an air filter comprising a porous three-dimensional structure obtained according to the method described above, and a tissue scaffold comprising a porous three-dimensional structure obtained according to the method described above.

[00117] Embodiments and advantages described in the context of the present method, and the porous three-dimensional structure, are analogously valid for the air filter and tissue scaffold, and vice versa.

[00118] While the methods described above are illustrated and described as a series of steps or events, it will be appreciated that any ordering of such steps or events are not to be interpreted in a limiting sense. For example, some steps may occur in different orders and/or concurrently with other steps or events apart from those illustrated and/or described herein. In addition, not all illustrated steps may be required to implement one or more aspects or embodiments described herein. Also, one or more of the steps depicted herein may be carried out in one or more separate acts and/or phases.

Examples

[00119] The present disclosure provides for a simple and scalable method of fabricating robust 3D nanofibrous macrostructures with desired shape and pore size, from a widely used biodegradable polymer, such as but not limited to polylactide (PL A). In addition to both of their bulk and degraded forms being biocompatible, the presence of stereocomplexation, for example, between poly(d- lactide) (PDLA) and poly(l-lactide) (PLLA) are designable to afford strong physical cross-linking points as inter-fiber junctions, which facilitate the fabrication of 3D macrostructure scaffolds with desired shapes, e.g. heart, vascular grafts, cylindrical shapes, etc. The obtained nanofibrous macrostructure scaffolds possess appropriate mechanical properties and controllable pore size to permit the ingress of cells and nutrients. Together with the good biocompatibility, the following examples demonstrate that the as-developed PLA stereocomplex (PLA SC) macrostructure, as one example, is potentially usable as a 3D bio implantation scaffold in tissue regeneration.

[00120] Moreover, 3D nanofibrous macrostructure with thicknesses ranging from millimeter to centimeter scale can provide a spatially distributed interconnected scaffold that aids in tissue growth, vascularization, and diffusion of nutrients. Such 3D nanofibrous macrostructures have potential in applications such as advanced tissue engineering, energy harvesting and storage, and filtration.

[00121] The present disclosure also provides for 3D nanofibrous nonwoven macrostructures derived from the present method, which are usable as filtration materials. The present nanofiber-based filtering media can provide very large specific surface area and enhanced adsorption of small particles by surface energy balance, inertial impaction, interception and diffusion. They also exhibit low pressure drop due to the aerodynamic slip on the nanofibers’ surface, reducing energy consumption. 3D nanofibrous filters can further increase deposit areas for the particles and enhance adsorption of ultrafine particles that transport via Brownian motion, thereby giving higher filtration efficiency with respect to ultrafine particles. Advantageously, the increase of filtration efficiency for smaller particles only results in a very small increase of pressure drop owing to different air flow regime of the 3D filters.

[00122] As the present method for deriving the present 3D nanofibrous structures utilizes a stereocomplexation approach, strong inter-fiber junctions are formed between the nanofibers, which significantly improve the mechanical properties (e.g. compression strength) of the 3D nanofibrous macrostructures. The data obtained additionally demonstrate that such 3D nanofibrous macrostructures have the potential for more efficient filtration of ultrafine air-bome particles without suffering a large increase in pressure drop.

[00123] Details of the present method and present porous three-dimensional structure comprised of nanofibers are further discussed, by way of non-limiting examples, as set forth below.

[00124] Example 1A: General Description of Preparation Method

[00125] As shown in the FIG. 2, PDLA/PLLA blend (PLA SC solution) were firstly electrospun into a liquid collector to form nanofibers. Strong inter- fiber junctions could be formed via inter-fiber stereocomplexation simply by freeze drying the nanofiber suspensions in an open mold, while the cross-linked nanofibers also simultaneously took the shape of the mold. The density of the nanofibrous structures could be manipulated by varying initial fiber content in the suspension and adjusting freezing or drying conditions.

[00126] Example 1B: Specific Description of Preparation Method

[00127] A polymer solution for electrospinning was prepared by dissolving 5 wt% of PLLA and 5 wt% of PDLA in DCM (dichlorome thane). DMF (N,N- dimethylformamide) was added to the solution just before electrospinning at a DCM/DMF weight ratio of 7:3. Addition of DMF improves electrospinnability of the polymer solution. The polymer solution was stirred at 60°C in a sealed glass container for 2 hrs before placing into a plastic syringe that was connected to a spinneret. The sealed glass container prevents the solvent, e.g. DCM, from evaporating to a significant extent, which can adversely affect the electrospinning of the polymer solution. Electrospinning of the polymer solution into a container of ethanol was conducted using a self-fabricated electrospinning setup. A syringe pump was used to feed the polymer solution into the spinneret at a constant rate of 1 mL/hr and a high voltage of 15 kV was applied. The distance between the tip of the spinneret and ethanol was maintained at 15 cm. The electrospun nanofibers were collected as a suspension in ethanol. The ethanol was then replaced with deionized (DI) water using a solvent exchange method, i.e. by repeatedly diluting the suspension with DI water and removing most or all of the solvents (to maintain the original suspension volume). After the solvent exchange, excess water was then removed from the nanofiber suspension to reduce the volume of the nanofiber suspension for filling into the mold of choice. The reduced volume of nanofiber suspension was then filled into the mold of choice.

[00128] The nanofibrous suspension was then immediately frozen by placing the mold into liquid nitrogen to fix the shape of the nanofibrous suspension. The fixated shape was then transferred into a freeze dryer to dehydrate the sample at the same time preserving the shape of sample according to the mold, obtaining the 3D nanofibrous macrostructures.

[00129] For the 3D nano-fibrous macrostructures coated with dopamine, the nanofiber suspension in DI water was immersed into a beaker containing 500 mL of dopamine solution with a concentration of 0.3 mg of Tris buffer (10 mM L ¹) to initiate the polymerization process of dopamine. Excess water from the initially prepared nanofiber suspension may be removed to reduce the volume of the nanofiber suspension for mixing with dopamine in the beaker. The polymerization process of dopamine was allowed to proceed for 4 hrs after which the nanofibrous suspension was washed with DI water for 3 times to remove any excess dopamine. Excess water was removed from the suspension (i.e. to reduce volume of the suspension) before it was packed into the mold followed by freezing and lyophilization. Most of the inter-fiber junctions may be formed before the dopamine coating, as stereocomplexation occurs when the electrospun nanofibers are suspended in the liquid medium.

[00130] Example 2: Presence of Strong Inter-fiber Junctions [00131] The inter-fiber junctions of electrospun PLLA and PLA SC nanofibrous macrostructure were investigated by SEM after tensile tests. To facilitate easy sample clamping, PLLA and PLA SC were electrospun into 2D mats for testing. Each fiber mat was cut firstly into a standard dumbbell shape according to measurements stated in ASTM D638-V. These dumbbell shaped samples were then mounted on the clamps attached to INSTRON instrument (Model 5567). Strain rate was set as 5 mm/min until the sample broke with a 10 N load cell.

[00132] PIG. 3A to PIG. 3P show the state of alignment and size of the nanofibers. Prom these micrographs the fiber diameters were measured. The average fiber diameter of PLLA nanofibers was 821 nm, while that of the PLA SC was about 412 nm. The difference in fiber diameters between the PLLA and PLA SC polymers is due to the difference in their solution viscosity. The stereocomplex cross-links in the PLA SC solution raise the solution viscosity and further plays a significant role in obtaining smaller sized fibers. On the other hand, it can be seen that both the PLLA and PLA SC electrospun fiber mat comprise random oriented nanofibers (PIG. 3A and PIG. 3B) and the polydopamine coated fibers (PLA SC -polydopamine) do not alter the morphology in terms of fiber alignment (PIG. 3C). The different surface morphology of PLA SC after coating is due to the relatively compact morphology of the 2D mats, which causes high dopamine concentration in surface region and low concentration inside, and hence more polydopamine coating on surface. However, when tensile forces were applied, the heterotactic PLLA nanofibers were aligned based on the direction in which the forces were applied (PIG. 3D), causing an irreversible change in morphology, whereas the random morphology of the cross-linked PLA SC nanofibers and PLA SC-polydopamine nanofibers were well retained (PIG. 3E and PIG. 3P). This implies that the inter-fiber junctions are formed in the liquid collection process, although further cross-linking during the freeze drying process may also be possible if the nanofibers are not fully covered by polydopamine at shorter coating time. The strong inter-connectivity observed from PLA SC and PLA SC- polydopamine samples also corresponds to the excellent 3D macro structure shape-forming capability. In addition, this observation correlates with the mechanical results. [00133] As presented in FIG. 4, the stress-strain diagrams showed that electrospun PLLA samples possessed ductile behavior with elongation at break of 240%. This is a great increase compared to solvent cast PLLA films (about 12%). The poor inter-fiber junctions in electrospun PLLA samples enable the nanofibers to align along the force applied, leading to a much larger strain at break. In contrast, the high crystallinity of the nanofibers and strong inter-fiber junctions render electrospun PLA SC and PLA SC-polydopamine samples brittle, which can be observed from tensile tests. Both electrospun PLA SC and PLA SC-polydopamine coated samples maintain a high apparent Young's modulus (about 50 MPa), which is sufficient to retain their shape and porous structure without being mechanically damaged when they are used as 3D scaffold for cell culture and in vivo implantation.

[00134] Example 3: Pore Size Control for Scaffold

[00135] The porous structure of the obtained electrospun fibrous macrostructures was observed using SEM and the density of the nanofibrous structures was successfully manipulated by varying initial fiber content in the suspension and in the mold. For example, with the same mold, packing density in the range of 0.00873 to 0.01746 g/cm³ was successfully obtained (also refer to FIG. 5A and FIG. 5B), which demonstrates a controllable pore size manipulation in the nanofibrous macrostructure (dimensions ranging from millimeter to tens of centimeter scale). In tissue engineering, suitable pore size is a consideration for constructing scaffolds. If the pores are too small, cell migration is limited and pore occlusion by the cells may occur. This prevents cellular penetration and extracellular matrix production. In addition, the diffusion of nutrients and removal of waste from the scaffold are also limited by the small pore size, which further result in necrotic regions within the construct. Conversely, if pores are too large, a decrease in specific surface area results, this limits cell attachment. For the present method and present structure, the PLA SC nanofibrous macrostructures can produce tunable pore size for improved cell infiltration into the layers below the surface, which circumvents cell colonization only at the surface of the scaffolds.

[00136] Example 4: Demonstration of Feasibility of Fabricating Different 3D Nanofibrous Macrostructure Scaffold [00137] As shown in FIG. 6A to FIG. 6F, PLA SC nanofibrous macrostructures in various shapes were successfully fabricated by freeze drying the nanofiber suspensions in the designed molds. The dimensions of the nanofibrous macrostructures ranging from 100 to 300 mm were developed as demonstration. It is worth noting that the estimated dimensions (e.g. length, breath, thickness) of the as- fabricated nanofibrous macrostructures depend on the size of the mold and they can be further tailored towards specific requirements.

[00138] Example 5: Crystallinity Behavior and Thermal Stability of the Nanofibrous Macrostructures

[00139] Wide-angle XRD was used to investigate the formation of PLA SC interactions between enantiomeric PDLA and PLLA in nanofibrous macrostructures. The measurements were performed on Bruker D8 Discover GADDS X-ray diffractometer, operating under Cu-Ka (1.5418 A) radiation (40 kV, 40 mA) at room temperature. The samples were scanned from 5 to 40° (20). FIG. 7A depicts the XRD curves of the typical PLA SC nanofibrous macrostructures, together with PLLA samples as control. As shown in FIG. 7A, curve a, which represents the as-prepared PLLA nanofibrous macrostructures, exhibits prominent diffraction peaks at 20 = 16.5° and 19.0°, which are ascribed to the a-form homocrystallities PLLA. On the contrary, for PLA SC nanofibrous macrostructures, additional diffraction peaks were observed at 20 = 12.2°, 21.0° and 24.2°, which are attributed to the planes of [110], [300]/[030] and [006] of PLA SC crystals with a triclinic unit cell of dimensions a = 0.916 nm, b = 0.916 nm, c = 0.870 nm, a = 109.2, b = 109.2, and g = 109.8, where L- lactide and D-lactide segments are packed laterally in parallel fashion by taking 3i helical conformations (FIG. 7A, curve b).

[00140] The formation of stereocomplex interactions was also confirmed by comparing the DSC data of electrospun fibrous macro structures of neat PLLA and the mixture of PDLA and PLLA. The protocol used for DSC measurement is depicted as follows: equilibrating the temperature at 20°C and heating from 20°C to 250°C at l0°C/min. Data were collected from the first heating runs. As shown in FIG. 7B, electrospun PLLA nanofibrous macrostructure exhibits melting peak at about l52°C at the first heating scan, corresponding to the homocrystallites, while the electrospun PLA SC nanofibrous macrostructure shows an additional melting peak at about 206°C at the first heating scan, indicating the formation of SC-crystallites. The increased melting temperature also indicated that the PLA SC nanofibrous macrostructures are thermally stable. The thermal stability was further investigated by thermogravimetric analysis (TGA) using the TA Instruments Q500. During the measurement, samples were heated at lO°C/min to 700°C under nitrogen flow rate of 60 mL/min. As presented in FIG. 7C, the onset of thermal degradation for PLLA and PLA SC nanofibrous samples occurred above 200°C and continued into the region 250-350°C, indicating good thermal stability. In addition, the 3D nanofibrous macrostructures can be annealed or coated with a thin layer of polydopamine to further improve their stability.

[00141] Example 6: Biological Evaluation of Electrospun Nanofibrous Macrostructures as 3D Scaffold

[00142] Before the biological evaluation of the electrospun nanofibrous macrostructures as 3D scaffold, static contact angle was performed to understand the surface wettability behavior. PLLA and a 50/50 PLLA/PDLA mixture were dissolved in the mixed solvent, respectively, and then drop cast onto glass slides for the static contact angle measurement. It is worth noting that PLLA and PDLA chains can start stereocomplexation to form PLA SC in the solution when they are mixed together. This will result in a PLA SC cast film. The contact angle measurement results are shown in FIG. 8A to FIG. 8F. Accordingly, for the solvent cast PLLA and PLA SC films, the contact angle of water droplets were measured to be 80.17° ± 0.40° and 87.65° ± 2.02°, respectively. Surface contact angle of electrospun PLLA (FIG. 8D) and PLA SC (FIG. 8E) nanofibrous macrostructures were dramatically increased by more than 50° as compared to the respective solvent cast films shown in FIG. 8A and FIG. 8B, indicating an enhanced surface hydrophobicity of the electrospun nanofibrous surface. Comparing to the solvent cast films, the porous nature of electrospun nanofibrous macrostructure increased the surface roughness at nanoscale. If a polymer is hydrophobic, increasing its roughness at nanometer scale causes an increase in the hydrophobicity of this polymer sample, i.e. which is the famous lotus effect.

[00143] However, cell growth on highly hydrophobic surfaces is not favorable and such a surface inhibits the growth of cells. Therefore, modification to improve the hydrophilicity of PLA SC nanofibrous macro structure was necessary for better biocompatibility. Polydopamine is an excellent mimic of biological melanins. The hydrophilicity of a polydopamine coating provides desirable surface characteristics without the need for further modification. Previous studies showed that the water contact angles of polydopamine coated substrates have been measured to be nearly uniform 50°-60°, regardless of the underlying material, suggesting that the poly dopamine surface coating strongly affects the surface properties. In the present example, the polydopamine coated PLA SC film showed a water contact angle of 52°. In addition, polydopamine is stable in aqueous environments and offers facile conjugation of nanofibrous macro structure with bioactive molecules such as proteins and growth factors to facilitate cell attachment and growth. The conjugation could be easily realized by Michael addition or Schiff base-type reactions with quinones present in the polymer. In the present example, PLA SC nanofibrous macrostructures coated with a thin layer of polydopamine were also obtained by self-polymerization of dopamine in a basic aqueous suspension containing the PLA SC nanofibers followed by freeze drying. Comparing with the untreated PLA SC nanofibrous macrostructures, the water contact angle recorded on PLA SC-polydopamine macrostructure reduced by 65°, indicating a significantly increased surface wettability (FIG. 8E and FIG. 8F). The cell response to the polydopamine coated PLA SC nanofibrous macrostructures and its use as a 3D cell culture scaffold were further evaluated.

[00144] The cell response to different nanofibrous macrostructures was performed by cell viability assay using the following materials procedure. The NIH 3T3 fibroblasts were provided by American Type Culture Collection (ATCC). The cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated FBS (Invitrogen), 100 mg mL ¹ penicillin, and 100 pg mL ¹ streptomycin (Thermo Scientific) and maintained in a humidified incubator at 37°C with 5% C0₂. Before experiment, the cells were precultured until confluence was reached.

[00145] For cytotoxicity studies, 3-(4,5-Dimethythiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assays were used to assess the metabolic activity of NIH 3T3 fibroblasts. The cells were seeded into the 3D PLLA, PLA SC and PLA SC-polydopamine nanofibrous macrostructures located in a 24-well plates (Costar, IL, USA), respectively, at a density of 2 x 10⁴ cells mL ¹. After 48 hrs incubation, the cell-scaffold composites were washed twice with lx PBS buffer. Freshly prepared MTT (500 pL, 0.5 mg mL ¹) solution in culture medium was then added into each well. The MTT medium solution was carefully removed after 3 hrs incubation at 37°C. DMSO (500 pL) was then added into each well and the plate was gently shaken to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (infiniteM200, Tecan). As shown in FIG. 9A, the cell viability is directly proportional to the absorbance value. The cell viability in PLA SC-polydopamine scaffolds is significantly higher as compared to the cells seeded in the PLLA and PLA SC scaffolds. No significant difference was found for the cell viability of cells cultured in PLLA and PLA SC scaffolds. The results demonstrated that hydrophilic PLA SC-polydopamine scaffold has stronger capability in supporting cell attachment and proliferation.

[00146] Further evaluation of the electrospun nanofibrous macrostructures as 3D scaffold was carried out by using laser scanning confocal microscopy (LSCM). During the cell culture, F-actin staining was carried out to observe the cell phenotype in different 3D scaffolds. After 48 hrs, cell-scaffold composites were initially fixed with 4% paraformaldehyde at room temperature for 15 mins. Then, the cell-scaffold composites were further incubated with Alexa Fluor® 633 Phalloidin and DAPI overnight at 4°C. DAPI is a fluorescent stain that binds strongly to A-T rich regions in DNA present in the cell nucleus. Final washing of the constructs was done with 1 x PBS. To understand the interaction of fibroblasts with PLLA, PLA SC and PLA SC-polydopamine scaffolds, F-actin of NIH 3T3 fibroblast was stained by Alexa Fluor 633 phalloidin and visualized using LSCM. Fluorescence intensity of F-action were analysed using ImageJ. As shown in FIG. 10, the stained F-actin showed a cytoplasmic filamentous distribution and the cells on PLA SC-polydopamine scaffold shown elongated morphology as compared to the cells with limited extensions on PLLA and PLA SC scaffolds. The higher activity of cells cultured in PLA SC-polydopamine scaffold was also confirmed by the fluorescence intensity of F-action. The intensity values are 23944.4 ± 4237.8 for the cells incubated with PLA SC-polydopamine scaffold which is much higher than 14716.4 ± 2356.0 and 13758.7 ± 1019.4 of cells incubated with PLLA and PLA SC scaffolds, respectively. All these results demonstrated that PLA SC-polydopamine scaffold can best support the cell proliferation (FIG. 10).

[00147] Example 7: Advantages and Improvements Over Conventional Methods and Materials

[00148] The present method and product rely on the formation of a polymer stereocomplex. A polymer stereocomplex is a stable composite of two complementing stereoregular polymers, which interlock each other by strong stereoselective interactions (FIG. 1E and FIG. 1F). Using PLA stereocomplex as an example, an intermolecular C_a-H· · · O carbonyl H-bond existed in PLA stereocomplex and this H- bond has larger angle between C_a-H and H- - -0 and shorter H-bond length than those of intramolecular C_a-H- 0 carbonyl H-bond. This unique intermolecular H-bond network leads to stronger thermodynamic stability as well as kinetic stability.

[00149] In the present disclosure, the stereocomplexation capability of poly(d-lactide) (PDLA) and poly(l-lactide) (PLLA) is utilized to physically cross-link electrospun PLLA/PDLA nanofibers, for example, by freeze drying the nanofibers suspended in water. The stereoselective inter-molecular forces between PDLA and PLLA are strong physical interactions. Thus, when PDLA and PLLA chains are present on nanofiber surfaces, they can form inter-fiber stereocomplex spontaneously when the nanofibers are in touch with each other, leading to strong inter- fiber junctions. This gives mechanically robust and thermally stable 3D nanofibrous macrostructures with tunable packing density. The macrostructures can retain their shape and porous morphology without being mechanically damaged when subjected to various forces, and can be used as biocompatible 3D scaffold for tissue engineering.

[00150] The present method is advantageously versatile not only because the polymers can be directly mixed, but also because it allows the simultaneous fixation of the shape and packing density of the 3D nanofibrous macrostructures in a standard freeze drying process. The shape of the macro structure follows the shape of the mold used to hold the nanofiber aqueous suspension, while the packing density and pore size can be easily controlled through adjustment of the initial nanofiber contents in the suspension. [00151] Conventional techniques available for fabrication of polymeric nanofibrous materials are electro spinning, self-assembly, and phase separation. Among them, electro spinning may be the most promising approach and have already demonstrated its potential in tissue engineering applications while nanofibers synthesized by self- assembly and phase separation demonstrated limited success while exploring for their application in tissue engineering. On the other hand, although some systemic and process parameters such as polymer concentration, solvent conductivity, electric potential, distance between capillary and collector, and motion of collector determine the pattern formation during fiber deposition, such techniques are conventionally used to produce nanofiber meshes with limited thickness range of the nanofibrous materials. One simple way of fabricating electrospun 3D nanofibrous macrostructures is through multi-layering electro spinning from which the fiber mats in a thickness range of hundreds of microns can be obtained. However, it took a very long time till a 3D macrostructure was sufficiently formed.

[00152] Nanofibrous macrostructures can also be fabricated by a series of post processing after traditional electro spinning and in such a method, the as-spun fiber mats were peeled off from the collector and then bended/folded or stacked into 3D fibrous structure. However, the macro structure generated by this approach cannot be used as scaffolds directly because they often have a large space or distance between the adjacent fibrous layer surfaces. In this case, cells only attach and stretch on the 2D surface, rather than forming bridges between surfaces due to the large distance. Conventionally, biodegradable polymeric nanofibrous macrostructures are mainly made from this approach. The horizontal laying of most nanofibers with weak inter fiber interactions also renders the macrostructure a relatively dense morphology, which further lead to fragile property with low mechanical strength. This makes it difficult to support the 3D nanofibrous macrostructures with desired scaffold shapes. Chemical cross-linking by reacting with chemical vapour, on the other hand, allows the fabrication of cross-linked nanofibrous macro structures with large pores, but the reaction rates are very low (usually in terms of days).

[00153] Compared to the conventional methods described above (a summarized comparison is shown in table 1 below), the present method allows the fabrication of 3D nanofibrous macrostructures with tunable pore size to fully utilize the unique advantages of polymer nanofibers in tissue engineering. A liquid reservoir collector combined with an electro spinning technique was used to produce electrospun porous structures. In the present method, PLA SC nanofibers are derived by electro spinning into the liquid bath and dispersed to form a relatively fluffy suspension. After a subsequent freezing process in a mold at a low temperature, a foamed 3D porous structure of the desired shape was fabricated. This is a facile fabrication process in which the inter-fiber junctions and desired shapes of the nanofibrous macrostructures are formed simultaneously. In addition to the materials and processing parameters, the pore size of the as-prepared 3D nanofibrous macro structure scaffold can be simply controlled by the amount of fibres used, for example, in the freeze drying process. Furthermore, the diameter of the obtained nanofibers are tuned to closely match that of extracellular matrix fibers, and the highly porous structure with well interconnected pores not only facilitates cell seeding and diffusion but also provides better diffusion of nutrients and waste throughout the scaffolds. The increased surface area generated from the use of a liquid collector is also beneficial for cell attachment, proliferation, migration, and differentiation, all of which are highly desired properties for tissue engineering applications. With special design, the robust nanofibrous macrostructures can also be used as a type of 3D scaffold to fabricate artificial organs such as vascular grafts, cardiac and skeletal muscle regeneration, and to serve as carriers for bioactive factor delivery.

[00154] Table 1 - Comparison of Present Method and Conventional Methods of Producing 3D Fibrous Macrostructures.

[00155] Table 2 below shows a comparison of the present product and commercially available scaffolds.

[00156] Table 2 - Comparison of Product Specifications of Present Product and Commercial Scaffolds.

[00157] Example 8A: Experimental Procedures for Filtration Application

[00158] To verify the inter-fiber cross-linking, the deformation behavior of the 3D nanofibrous macrostructures under compression force was studied. This also explores the potential of these macrostructures for filtration of small air-bome particles, and the following experiments were carried out.

[00159] Example 8B: Fabrication of 3D Nanofibrous Macrostructures of Different Compositions

[00160] The nanofibrous macrostructures composed of PLLA/PDLA stereocomplex (PLA SC) were prepared by first dissolving the blend of PLLA and PDLA in DCM. The total concentration of PLLA and PDLA was kept at 10 wt%, wherein the wt% is based on the total weight of the polymer solution (e.g. includes the polymers, DCM and DMF), while the ratio of PLLA and PDLA used were 3:7, 5:5 and 7:3, respectively. The solutions were left to stir under room temperature for 2 hrs followed by the addition of DMF with the DCM/DMF ratio fixed at 8:2. 10 wt% of pure PLLA solution was also prepared without the addition of PDLA to be used as a reference for comparison. 0.5 wt% (relative to total polymer weight) tetrabutylammonium chloride (TBAC) was added to each solution to improve the conductivity for electro spinning.

[00161] Example 8C: Fabrication of 3D Nanofibers Filters

[00162] The PLA SC solution was electrospun into a very small amount of tertiary butanol on a wire mesh (FIG. 11). The electro spinning was conducted under a working distance of 15 cm and the feeding rate was fixed at 1.0 mL/hr. The working voltage used in the electro spinning process was kept at 17 kV. After electro spinning, the as-spun PLA SC nanofibers were left to freeze in a refrigerator followed by freeze drying to obtain the 3D nanofibrous aerogel. [00163] Example 8D: Characterization of 3D Nanofibrous Filters

[00164] The morphology of the nanofibrous filters was investigated using a field emission scanning electron microscope (FESEM) (JEOL 6340) at an acceleration voltage of 5 kV. The densities of the aerogels were estimated by evaluating the weighed mass and measured dimensions of the cylindrical- shaped aerogels. Three samples were evaluated to derive the average density of the PLA SC aerogel. The porosity of the aerogel is defined as the volume fraction of void:

PorOSlty = l-(paerogel/ppolymer)·

[00165] An Instron 5567 machine with a 10 N load cell was used to perform compression tests on the cylindrical- shaped samples and the compression rate adopted was l.O mm/min.

[00166] In filtration experiments, the particle concentration was measured with an optical particle sizer (OPS 3330, TSI Instruments Ltd) which utilized a laser detector to detect 16 specific ranges of particle sizes between 0.3 pm to 10 pm. Particle sizes between 0.01 pm to 0.42 pm were detected using a condensation particle counter (NanoScan SMPS 3910, TSI Instruments Ltd). The pressure drop values were measured using a differential pressure gauge (Digital Manometer, Bluewind Laboratory Pte Ltd). The wind velocity was measured by the air velocity meter (Airflow instruments velocity meter TA430, TSI Instruments Ltd).

[00167] Example 9: Results and Discussion - Presence of Strong Inter-fiber Junctions

[00168] To confirm the formation of inter-fiber junctions, the tensile test results of PLLA and PLA SC aerogels shown in FIG. 12 are referred to. The stress-strain curves indicate that electrospun PLLA samples possessed ductile behavior with elongation at break of 240%. Due to the poor inter- fiber junctions in electrospun PLLA samples, the nanofibers align along the force applied, giving rise to the ductility and leading to an irreversible change in morphology (FIG. 3A and FIG. 3C) and much larger apparent strain at break. In contrast, the high crystallinity of the nanofibers and strong inter-fiber junctions cause electrospun PLA SC to have brittle behavior. The random morphology of the cross-linked PLA SC nanofibers is well retained after the tensile test (FIG. 3B and FIG. 3D). The PLA SC sample maintains the high apparent Young’s modulus (about 50 MPa), which is sufficient to retain its shape and porous structure without being mechanically damaged when they are used as 3D nanofibrous aerogel for air filtration. The in vitro proliferation of fibroblast cells over 28 days are shown in FIG. 9B. Both PLLA and PLA SC samples performed similarly over the first 7 days but the PLLA sample demonstrated a drop in cell count after 7 days and plateau. Meanwhile, the PLA SC sample showed an increase in cell count over a period of 14 days.

[00169] To further verify the formation of the inter-fiber junctions via stereocomplexation, compression tests were also performed on PLLA aerogel (reference sample) and the PLLA/PDLA- stereocomplex aerogel samples, as shown in FIG. 13 A, and the results are shown in FIG. 13B. It is evident that the stereocomplex performs better than the PLLA samples of similar densities. The stereocomplex is able to withstand stresses almost 2 times that of the PLLA samples with similar apparent densities. This further confirms the presence of cross-links due to formation of the stereocomplex. Hence, the presence of stereocomplex interactions does enhance the single-fiber rigidity as well as induce the formation of inter-fiber junctions, strengthening the overall structure of the 3D nanofibrous structure. The improved resistance of the 3D nanofibrous macrostructures to compression stress is beneficial to their application as air filtration materials.

[00170] In principle, the extent of stereocomplexation is related to the number of available free chain ends on nanofiber surfaces because without free chain ends, the polymer chain segments from neighbouring nanofibers are unable to form entangled helix structure. Theoretically, the lower molecular weight PDLA (shorter chains) has a higher number of free chain ends as compared to the higher molecular weight (high- MW) PDLA (longer chains), assuming both types of PDLA are added in equal weight. Thus, the extent of entanglement and the number of inter-fiber junctions should be distinctly more in the case of the PDLA with a lower molecular weight (low-MW). Hence, if there are more free chain ends available for cross-linking via stereocomplexation, the cross-linking density will increase, which may increase the robustness of the 3D nanofibrous structures but reduce their specific surface area due to fiber merging.

[00171] In order to verify this hypothesis, polymer solutions with different ratios of high-MW PLLA/low-MW PDLA, namely PLLA/PDLA-7/3 and PLLA/PDLA-3/7, were prepared. The total polymer concentration in each solution was fixed at 10 wt%. As shown in FIG. 14A to FIG. 14H, the difference between the bundling of the fibers was distinctive. The PLLA/PDLA-7/3 displayed very small bundles of fibers randomly entangled together, which may be because it comprised of a higher content of high molecular weight PLLA (longer chains) and the density of free chain ends is lower, giving rise to a smaller extent of stereocomplexation and weaker inter-fiber junctions. On the opposite, PLLA/PD LA-3/7 comprised of a higher content of low- MW PDLA (shorter chains) and the density of free chain ends is higher, leading to a greater extent of stereocomplexation and stronger inter-fiber junctions. As a result, larger bundles of several fibers are formed. Therefore, the density of free chain ends does influence the extent of inter-fiber stereocomplexation, which allows control of the morphology of the 3D nanofibrous structures easily. In other words, the cross- linking density can be controlled by varying the composition. For instance, when high-MW PLLA and low-MW PDLA are used, higher PDLA content may result in a higher degree of cross-linking.

[00172] Example 10: Air Filtration Studies - Testing System

[00173] The filtration efficiency and pressure drop of the prepared 3D air filters were tested according to the information provided in the following international standards:

[00174] (i) GB/T 14295 - 1993, Air Filters, 2009-06-01

[00175] (ii) ANSI/ASHRAE Standard 52.2-2012 - Method of Testing General Ventilation Air-Cleaning Devices for Removal Efficiency by Particle Size

[00176] A testing system has been designed, and the schematic diagram showing the air filtration testing system is presented in FIG. 15. The actual setup can be seen in FIG. 16.

[00177] Example 11: Air Filtration Studies - Particle Size Distribution of Model Haze Particles

[00178] For filtration efficiency tests, haze particles in the air were generated by burning incense. The face velocity used was 0.1 m/s and the humidity was 60%. The concentration (dM in FIG. 17A) was controlled by diluting the incense smoke with air in the mixing chamber, down to a hazardous pollution level equivalent to the PM 2.5 value reported during the 2013 haze episode in Singapore (300-400 pm/m³). Two optical particle counters (OPS, TSI, size range 0.3-10 pm and NanoScan SMPS, TSI, size range 0.01-0.42 pm) were used for real-time monitoring of the particle concentrations in the system. The amount of particles in the air flow before and after the air inlet was covered by the filter materials were recorded by the particle counters, and the filtration efficiency was calculated by comparing the number concentration before and after filters.

[00179] Based on the measurement, as seen in FIG. 17A, the incense smoke displayed a wide particle size distribution from 10 nm to around 10 pm, with majority of the particles with diameter less than 1 pm. Furthermore, particles with sizes between 100 nm and 300 nm displayed the highest mass concentration. In summary, the size distribution of the model haze particles is in the range of tens to several hundred nanometers, while most of the particles have sizes of less than 500 nm. Therefore, the size distribution of the haze model PM particles is a good representation of the actual haze that occurred in Southeast Asia. The relative change in pressure drop (DR at time t over initial DR) of the 3D PLA SC filter over a duration of 24 hrs under 60% and 90% relative humidity conditions was also investigated, and the results are plotted in FIG. 17B.

[00180] Example 12A: Testing of Reference Samples Using the Present Air Filtration Setup

[00181] A type of commercially available HEPA filters, H13, supplied by Fushun Shun Hua Auto Parts Manufacturing Co., Ltd, China was used as reference. The morphologies of H13 were studied under SEM, and the results of the H13 morphology are shown in FIG. 18A and 18B. A comparison of the characterization results between H13 (a high end HEPA filter) and Hl l (a low end HEPA filter), both made of glass fibers (p_giass = 2.6 g/cm³), are shown in table 3A below (also see FIG. 18C and 18D). From the SEM images, the average diameter of the fibers was estimated. The densities of the filtration materials were also measured, from which porosity can be approximated. All data are listed in table 3B. Based on the data, it can be observed that the HEPA filter has an average fiber diameter of about a few microns, and a density of hundreds of micrograms per cubic centimetre. Thus, the commercial filter has a relatively low porosity.

[00182] Table 3A: Comparison of Characterization Results of H13 and Hl l Filters.

[00183] Table 3B: Characterization Results of H13 Reference Sample.

Average diameter Density

Sample -3 Porosity

(pm) (mg cm )

H13 2.35+1.82 275.5 89.4%

[00184] In order to verify the effectiveness of the facility used in testing the air filtration materials, the H13 filters were tested, which are for high-efficiency filtration. Based on the specifications provided by the supplier, the theoretical filtration efficiency of H13 for PM particles with size above 300 nm is 99.97% based on the international standard tests mentioned above.

[00185] The measured filtration efficiency and pressure drop using the setup described above are summarized in table 4A. It is shown that a layer of H13 with thickness of 0.45 mm has the lowest efficiency, and the efficiency increases as the number of H13 layers increased. This trend is within expectation because filtration efficiency increases with thickness of the filter. However, the measured filtration efficiencies are slightly lower than the supplier’s data because the model gas contains higher concentration of smaller particles. More importantly, the PM filtration efficiency increases with the particle size, showing that H13 is not very efficient for filtration of ultrafine particles, for example, high efficiency for PM 0.1 can only be achieved at very high pressure drop. Additionally, the pressure drop increases proportionately with the thickness of the filter. Therefore, these results indicate that the testing facility is able to provide reasonable data for estimation of pressure drop and filtration efficiency.

[00186] Table 4A: Pressure Drop and Filtration Efficiencies Results of Different Layers of H13 Filter.

Pressure drop PM 0.1 PM 0.5 PM 1.0

H13

(Pa) filtration filtration filtration efficiency efficiency efficiency

1 layer 48.17+4.25 98.28+0.81 99.50+0.006 99.51+0.05

2 layers 91.25+7.04 99.67+0.08 99.77 99.77

3 Layers 139.00+6.73 99.86+0.15 99.90+0.02 99. 90+0.02

[00187] Example 12B: Testing of Hl l and Hl l + 3D PLA SC Samples Using the Present Air Filtration Setup

[00188] A filtration test was carried out to compare the performance of Hl l samples and Hl l with 3D PLA SC samples. The mass size distribution of various particles are shown in FIG. 17C, while the results are shown in table 4B below.

[00189] Table 4B - Comparison Between Hl l and Hl 1 + 3D PLA SC

Filtration Filtration Filtration Filtration

Sample efficiency efficiency efficiency efficiency Pressure drop (Pa)

(0.1 pm) (0.5 pm) (1.0 pm) (2.5 pm)

H-ll 80.48% 85.13% 95.16% 99.74% 13.20+0.35

H11+

98.56+0.16 99.88+0.02

3D PLA 99.99% 99.99% 63.50+8.22

% %

SC

[00190] Based on the results, it can be observed that Hl l exhibits low filtration efficiency for smaller particles. Meanwhile, the Hl l + PLA SC composite filter has significantly higher filtration efficiency for 0.1 pm and 0.5 pm particles with a moderate increase in pressure drop.

[00191] Example 13: Comparison of Pressure Drop of 2D Nanofibrous Mats with 3D Nanofibrous Filters

[00192] Conventionally, thin 2D nanofibrous mats have been combined with cellulose substrate filtration media for increasing filtration efficiency for submicron dust. In order to compare the 2D electrospun nanofibrous mats with 3D nanofibrous filters, cross-sectional morphologies were examined and the pressure drops of the electrospun 2D and 3D samples collected in the same electro spinning period were measured. As shown in FIG. 19A to FIG. 19H, the SEM images showed that the 3D sample produced by the present liquid collection method is composed of randomly oriented nanofibers that form a very porous and fluffy aerogel, while the 2D mat collected on aluminum foils consists of nanofibers that are more densely packed in planes parallel to the foils, causing the 2D mat filter to be much denser in packing than that of the 3D filter. The 3D filter therefore exhibits much smaller pressure drop.

[00193] As mentioned earlier, conventional HEPA filters mainly filter out particles with size of more than 0.3 mhi, and they are unable to effectively eliminate ultrafine particles unless the pressure drop becomes very high. In contrast, 3D nanofibrous filters can effectively adsorb ultrafine particles that transport with Brownian motion, thereby giving high filtration efficiency for ultrafine particles, whereas they are not suitable for filtration of relatively large particles via inertial impaction and interception mechanisms due to their very high porosity. Thus, by combining a HEPA filter with a very thin layer of 3D nanofibrous filter, a more efficient air filter for effective filtration of both relatively large and ultrafine particles may be achieved. Moreover, the increase of filtration efficiency for smaller particles is expected to give only a very small increase of pressure drop owing to the high porosity and the aerodynamic slip caused by small diameter of the nanofibers. To test this hypothesis, pressure drops of the 2D and 3D nanofibrous samples, each of which was combined with a layer of H13 filter, were measured, respectively. Indeed, the measurement results (table 5 below) revealed that the porous aerogel (the 3D filter) exhibits much smaller pressure drop than the 2D mat. This also confirms that the present fabrication method is effective for fabrication of 3D nanofibrous filters with high porosity.

[00194] Table 5 - Pressure Drop of H13-SC.

Sample Pressure drop

(a) H13-1 layer 48.17+4.25

(b) H13-1 layer + PEA SC (3D) 68.88+8.19

(c) H13-1 layer + PLA SC (2D) 357.04+26.63

[00195] Example 14: Air Filtration Behavior of 3D Nanofibrous Filters

[00196] Following the pressure drop tests, the performance of the combination of a layer of the 3D nanofibrous filter and a layer of commercial H13 filter (H13-SC) was tested with the filters composed of various layers of H13 filtration materials as reference samples. [00197] The filtration efficiencies and pressure drops of H13-SC, H13-1 layer, H13-2 layers and H13-3 layers are summarized in table 6 below. A layer of H13 HEPA filter was unable to achieve 99.9% efficiency for PM 1.0 because the model pollutant air contains more particles with sizes of less than 500 nm. The addition of a thin layer of 3D PLA SC filter proved to be more efficient in removing smaller particles with sizes less than 500 nm as compared to the addition of a second layer of the H13 (high efficiency filter). Furthermore, the pressure drop of the H13-1 layer + 3D PLA SC (H13-SC) filter was significantly lower than that of the H13-2 layers filter, which further confirmed that the high porosity and aerodynamic slip flow gave rise to lower pressure drop. It is also worth noting that H13-SC and H13-3 layer filters have comparable efficiencies for the removal of ultrafine particles. However, the filter with 3 layers of H13 (H13-3 layers) exhibits a pressure drop twice that of the H13-SC filter. This result has verified that the present 3D nanofibrous filters are able to adsorb ultrafine particles more effectively than the conventional HEPA filters and simultaneously reduce pressure drop (save energy).

[00198] Table 6: Filtration Efficiencies of Various H13 Layer Filters for Different Particle Size.

PM 0.1 PM 0.5 PM 1.0

filtration filtration filtration Pressure drop

Sample

efficiency efficiency efficiency (Pa) (%) (%) (%)

H13-1 layer 98.28+0.81 99.50+0.01 99.51+0.05 48.17+4.25 H13-2 layers 99.67+0.08 99.77 99.77 91.25+7.04 H13-3 layers 99.86+0.15 99.90+0.02 99.90+0.02 139.00+6.73 H13-1 layer +

99.83+0.01 99.86+0.01 99.86+0.01 63.67+1.15 PLA SC (3D)

[00199] Example 15: Summarized Discussion of Results for Present Method and Present Nanofibrous Structure for Use as Air Filters

[00200] 3D PLA SC nanofibrous macrostructures with pre-designed shape and dimensions have been successfully fabricated by introducing strong inter- fiber junctions through inter-fiber stereocomplexation. It is observed that stereocomplexation of the chains on surfaces of neighbouring nanofibers causes some nanofibers to merge together, inducing“cross-linking”. This“cross-linking” can be adjusted through certain materials’ parameters, such as the ratio of high-MW PLLA/low-MW PDLA. The formation of inter-fiber junctions, or so-called cross- linking, also make the nanofibrous aerogels more mechanically robust, as reflected by their better compression resistance (higher compression stress at a certain strain), which is useful for air filtration applications.

[00201] A system for measuring pressure drop and air filtration efficiency was set up, and the system was calibrated using commercially available reference materials. To demonstrate the potential of the cross-linked nanofibrous macrostructures as 3D air filters, a combination of a thin layer of PLA SC aerogel with a layer of H13 HEPA filter was tested for capturing PM 0.1, PM 0.5 and PM 1.0 particles in the model pollution air. The results showed that the combination of a thin layer of 3D PLA SC aerogel and a layer of H13 filter exhibits higher filtration efficiency and significantly lower pressure drop than 2 layers of H13. In addition, it shows similar filtration efficiency (about 99.9%) to that of 3 layers of H13 but much lower pressure drop than 3 layers of H13. This type of robust and highly porous aerogels has the potential to combine with various types of commercial air filters to increase the filtration efficiency for ultrafine particles and reduce energy consumption.

[00202] Example 16: Summary

[00203] The present method relates to forming of a three-dimensional nanofibrous macro structure scaffold, the method comprises preparing a solution containing a first polymer and a second polymer both dissolved in a solvent system, wherein the first and the second polymers are capable of forming a polymer stereocomplex with each other, and electro spinning the solution to obtain a network of cross-linked nanofibers comprised of the polymer stereocomplex, thereby forming the scaffold. The polymer stereocomplex may occur within the nanofibers and at nanofibers’ junctions.

[00204] The first and second polymers may be each selected from a group consisting of isotactic polymers, syndiotactic polymers, and enantiomeric helical polymers. The first and second polymers may each comprise poly(d-lactide), poly(l-lactide), isotactic poly(methyl methacrylate) s, syndiotactic poly(methyl methacrylate)s, polypeptides (D- and L-amino acids), or polyamides (D- and L-poly(hexamethylene di-O- methyl- tartaramide)s).

[00205] The present method may further comprise suspending the nanofibers in a liquid medium. The liquid medium may be water, alcohol, or a mixture thereof.

[00206] The present method may further comprise adjusting the concentration of nanofibers in the liquid medium to adjust the packing density and the porosity of the scaffold formed thereof.

[00207] The present method may further comprise transferring the nanofiber suspension into a mold of a desired shape or configuration.

[00208] The present method may further comprise removing the liquid medium from the nanofibers. The removing may comprise freeze drying, vacuum drying or air drying with the assistance of superabsorbent materials.

[00209] The present method may further comprise modifying the surface of nanofibers with bioactive molecules.

[00210] The present disclosure also relates to a three-dimensional nanofibrous macro structure scaffold comprising a network of cross-linked nanofibers, wherein the nanofibers comprise a polymer stereocomplex formed of a first polymer and a second polymer. The nanofibers may comprise electrospun nanofibers. The first and second polymers may be each selected from a group consisting of isotactic polymers, syndiotactic polymers, and enantiomeric helical polymers. The first and second polymers may comprise poly(d-lactide), poly(l-lactide), isotactic poly(methyl methacrylate) s, syndiotactic poly(methyl methacrylate) s, polypeptides (D- and L- amino acids), or polyamides (D- and L-poly(hexamethylene di-O- methyl- taiiaramide)s). The surface of the nanofibers may be modified with bioactive molecules.

[00211] The three-dimensional nanofibrous macrostructure scaffold may be for use in therapy, wherein the scaffold may comprise a network of cross-linked nanofibers, and wherein the nanofibers may comprise a polymer stereocomplex formed of a first polymer and a second polymer. The nanofibers may comprise electrospun nanofibers. The first and second polymers may be each selected from a group consisting of isotactic polymers, syndiotactic polymers, and enantiomeric helical polymers. The first and second polymers may comprise poly(d-lactide), poly(l-lactide), isotactic poly(methyl methacrylate) s, syndiotactic poly(methyl methacrylate)s, polypeptides (D- and L-amino acids), or polyamides (D- and L- poly(hexamethylene di-O-methyl- tartaramide)s). The surface of the nanofibers may be modified with bioactive molecules.

[00212] Example 17: Commercial and Potential Applications

[00213] The present method and product can be used for encapsulation of bioactive molecules and growth factors in the 3D nanofibrous macrostructures to further promote cell proliferation and interaction. In vitro studies and animal trials can also be carried out with the present 3D nanofibrous macrostructures. The present product can be used for air filtration applications. The present 3D nanofibrous structures have stable loose morphology and large surface area, which are good for adsorption of small organic particles, such as haze particles, and hence can be utilized in haze filtration.

[00214] While the invention has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

1. A method of producing a porous three-dimensional structure comprised of nanofibers, the method comprising:

providing a polymer solution comprising a first polymer and a second polymer;

electro spinning the polymer solution into a liquid reservoir to form a suspension comprising nanofibers formed from the first polymer and the second polymer; and

drying the suspension to form the porous three-dimensional structure.

2. The method according to claim 1, wherein the nanofibers are crosslinked between each of the nanofibers, and each of the nanofibers is internally crosslinked.

3. The method according to claim 1 or 2, wherein providing the polymer solution comprises dissolving the first polymer and the second polymer in a mixture of organic solvents.

4. The method according to any one of claims 1 to 3, wherein each of the first polymer and the second polymer comprises an isotactic polymer, a syndiotactic polymer, or an enantiomeric polymer.

5. The method according to any one of claims 1 to 4, wherein each of the first polymer and the second polymer comprises poly(d-lactide), poly(l-lactide), isotactic poly(methyl methacrylate), syndiotactic poly(methyl methacrylate), polypeptides formed from one or more D-amino acids, polypeptides formed from one or more L- amino acids, D-poly(hexamethylene di-O-methyl-tartaramide), or L- poly(hexamethylene di-O-methyl-tartaramide) .

6. The method according to claim 3, wherein the mixture of organic solvents comprises dichloromethane and dimethylformamide.

7. The method according to any one of claims 1 to 6, wherein the liquid reservoir comprises water and/or alcohol.

8. The method according to claim 7, wherein the alcohol comprises ethanol or butanol.

9. The method according to any one of claims 1 to 8, further comprising transferring the suspension into a mold before drying so as to have the porous three- dimensional structure adapted to the configuration of the mold.

10. The method according to any one of claims 1 to 9, wherein drying the suspension comprises:

(i) freeze drying the suspension;

(ii) subjecting the suspension to air in the presence of an absorbent material; or

(iii) subjecting the suspension to vacuum.

11. The method according to any one of claims 1 to 10, further comprising coating the nanofibers in the suspension with polydopamine before drying.

12. The method according to claim 11, wherein coating the nanofibers comprises contacting the nanofibers with a dopamine solution.

13. The method according to any one of claims 1 to 12, further comprising contacting the porous three-dimensional structure with biomolecules to deposit the biomolecules on the nanofibers.

14. A porous three-dimensional structure obtained according to the method of any one of claims 1 to 13.

15. The porous three-dimensional structure according to claim 14, wherein the porous three-dimensional structure comprises nanofibers which are crosslinked between each of the nanofibers, and each of the nanofibers is internally crosslinked

16. An air filter comprising a porous three-dimensional structure obtained according to the method of any one of claims 1 to 13.

17. A tissue scaffold comprising a porous three-dimensional structure obtained according to the method of any one of claims 1 to 13.