PHBHX NANOYARNS

Description

RELATED APPLICATIONS

The present application claims priority to and the benefit of U.S. patent application No. 63/591,558, “PHBHX Nanoyarns,” filed Oct. 19, 2023. All foregoing applications are incorporated herein by reference in their entireties for any and all purposes.

TECHNICAL FIELD

The present disclosure relates to the field of polymer science and also to the field of fibrous textiles.

BACKGROUND

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) is a copolymer of 3-hydroxybutyrate and (R)-3-hydroxyhexanoate (PHBHX) is a residentially-compostable, natural polymer derived from bacteria. Existing approaches for producing nanoyarns from copolymers of polyhydroxybutyrate (PHB) are, however, limited and can require utilization of a textile core yarn such as PVA or PLLA. Accordingly, there is a long-felt need in the field for improved methods of forming PHBHX nanoyarns.

SUMMARY

In meeting the described long-felt needs, the present disclosure provides a nanoyarn, comprising poly(3-hydroxy butyrate-co-3-hydroxyhexanoate) (PHBHX), (i) the nanoyarn being essentially free of a core material, (ii) the nanoyarn being essentially free of a co-polymer, or both (i) and (ii).

Also provided is an article, the article comprising a nanoyam according to the present disclosure.

Further provided is a method, comprising forming a nanoyarn according to the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. Like numerals having different letter suffixes may represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. In the drawings:

FIGS. 1A and 1B provide exemplary images of the disclosed technology.

FIG. 2. Schematic of an exemplary nanoyarn fabrication set-up. NH: Needle height or the distance from the tip of the needle to the funnel, WH: winder to funnel height or the distance from the surface of the funnel to the surface of the winder, ND: Needle distance or the distance.

FIGS. 3A-3C. Twist angle (FIG. 3A), fiber diameter (FIG. 3B) and modulus (FIG. 3C) of PAN nanoyarns with increasing funnel speed (squares=modulus; triangles=ultimate strength).

FIGS. 4A-4D. (FIG. 4A) Average Nanofiber diameter with increasing needle distance, (FIG. 4B) schematic representation of fiber deposition, (FIG. 4C) images of indicating location of fiber deposition (FIG. 4D) SEM images of nanofibers.

FIGS. 5A and 5B provide SEM images of PEO nanoyarn at angle 43 (FIG. 5A) and angle 23 (FIG. 5B).

FIGS. 6A-6B. (FIG. 6A) Average Nanoyarn and nanofiber diameter with increasing winder to funnel distance. (FIG. 6B) Twist angle of nanoyarns at increasing winder to funnel distance.

FIGS. 7A-7C provide SEM images of PHBHx at varying polymer concentrations-4 wt % (FIG. 7A), 8 wt % (FIG. 7B), and 12 wt % (FIG. 7C).

FIGS. 8A-8D. SEM images of nanoyarn and nanofiber using solvent composition (FIG. 8A) 8:2 chloroform:acetic acid and (FIG. 8B) 9:1 chloroform:acetic acid and (FIG. 8C) chloroform. (FIG. 8D) image of 9:1 chloroform:acetic acid nanoyarn.

FIGS. 9A-9E. FTIR spectra of (FIG. 9A) PHBHx powder, (FIG. 9B) hydroxy butyric acid, (FIG. 9C) PHBHx nanoyarn from 100% chloroform solvent, (FIG. 9D) from 9:1 chloroform:acetic acid solvent and (FIG. 9E) 8:2 chloroform:acetic acid solvent.

FIG. 10 Average nanoyarn diameter with increasing winder speed at 10 cm, 12 cm and 14 cm winder height.

FIGS. 11A-11B. Stress-strain curve of PHBHx nanoyam from 9:1 chloroform:acetic acid solvent (FIG. 11A). table of properties calculated from curve (FIG. 11B).

FIGS. 12A-12C. NanoCT images of PHBHx nanoyarn (FIG. 12A) 3D image (FIG. 12B) 2D cross-section in the axial direction and (FIG. 12C) 2D cross-section in the horizontal direction.

FIG. 13. Nanoyarn diameter (a) and Nanofiber diameters (b) of solution blend composite nanoyarns for 12w/v and 16w/v PCL

FIG. 14. First heat of composite PCL:PHBHx nanoyarns.

FIG. 15. Second heat of composite PCL:PHBHx nanoyarns.

FIG. 16. Exemplary data for PHBHX nanoyarns.

FIG. 17. Exemplary data for PHBHX nanoyarns.

FIG. 18. Exemplary data for PHBHX nanoyarns.

FIG. 19. Exemplary data for PHBHX nanoyarns.

FIG. 20. Exemplary fabrication systems for forming composite nanoyarns.

FIG. 21. Exemplary data for composite nanoyarns according to the present disclosure.

FIG. 22. Exemplary data for composite nanovarns according to the present disclosure.

FIG. 23. Exemplary fabrication systems for forming composite nanoyarns.

FIG. 24. Exemplary fabrication systems for forming composite nanoyarns.

FIG. 25. Exemplary data for composite nanoyarns according to the present disclosure.

FIG. 26. Exemplary data for composite nanoyarns according to the present disclosure.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

As used in the specification and in the claims, the term “comprising” can include the embodiments “consisting of” and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having.” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as “consisting of” and “consisting essentially of” the enumerated ingredients/steps, which allows the presence of only the named ingredients/steps, along with any impurities that might result therefrom, and excludes other ingredients/steps.

As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated+10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value: they are sufficiently imprecise to include values approximating these ranges and/or values.

As used herein, approximating language can be applied to modify any quantitative representation that can vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language can correspond to the precision of an instrument for measuring the value. The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” can refer to plus or minus 10% of the indicated number. For example, “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean from 0.9-1.1. Other meanings of “about” can be apparent from the context, such as rounding off, so, for example “about 1” can also mean from 0.5 to 1.4.

Further, the term “comprising” should be understood as having its open-ended meaning of “including.” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprises components A and B can be a composition that includes A, B, and other components, but can also be a composition made of A and B only. Any documents cited herein are incorporated by reference in their entireties for any and all purposes.

Any embodiment or aspect provided herein is illustrative only and does not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more embodiments or aspects can be combined with any part or parts of any one or more other embodiments or aspects.

Poly(3-hydroxy butyrate-co-3-hydroxyhexanoate) (PHBHX) is a bio-based, biodegradable and compostable aliphatic polyester part of a larger family of biobased plastics known as polyhydroxyalkanoates (PHAs). This group of materials provides an advantage over currently accepted materials, as PHAs are the only biobased polymer that is completely synthesized by a biological process that involves conversion of carbon sources directly into PHAs through microbial fermentation. These are nontoxic, biocompatible, biodegradable thermoplastics that have a high degree of polymerization, highly crystalline, optically active, insoluble in water and have piezoelectric properties. Although the present technology is illustrated by reference to examples that involve PHBHX, other polymers can be used with PHBHX or even in place of PHBHX. For example, one can replace PHBHX with one or more other PHAs.

One advantage of PHBHx over currently available bio-based and biodegradable materials is its ability to fully degrade in the body and in marine and soil environments as well as the ability to tune the degradation time based on the manufacturing technique of PHAs. For example, it has been shown that PHB with a longer side-chain displayed higher degradability. Additionally, it is compostable and can degrade under aerobic and anerobic conditions. These properties in combination with it being biocompatible makes PHBHx a sought-out material for tissue engineering applications. The biocompatibility of PHBHx comes from its degradation products being 3-hydroxy butyric acid which is made in the liver during the breakdown to long chain fatty acid. The degradation mechanism is acid hydrolysis of the ester bond. Five different types of PHBs when co-cultured with cells showed high levels of adhesion and activity. Comparing the tissue response, PHBHx demonstrated lower degree of fibrosis and no inflammatory cells when compared to PLA. This can be attributed to an influx of calcium ions into the cytoplasm due to the suppression of fibroblast apoptosis and necrosis by the PHAs. The influx of calcium ions is a key signaling pathway for the activation of cell division.

PHBHX molecular structure is a copolymer of 3-hydroxy butyrate and (R)-3-hydroxyhexanoate which is a hydroxyalkoanoate with a side chain consisting of 3 carbon atoms, shown below: The presence of this medium chain length side group acts as a defect and disrupts the regularity of the polymer chain resulting in a polymer with less crystallinity and lower melting point. This molecular structure is closely resembles that of linear low-density polyethylene which is a copolymer of ethylene with a small amount of either 1-butene or 1-hexene.

Biosynthetic pathways can be used to produce (PHBHx) copolymer. The process begins when two molecules of acetyl-CoA combine to form acetoacetyl-CoA, catalyzed by phaA thiolase. This intermediate is then reduced to 3-hydroxybutyryl-CoA by phaB reductase. Alongside these reactions, fatty acid biosynthesis (phaG) and fatty acid degradation (phaJ, OAR, MFP) pathways contribute additional 3-hydroxyacyl-CoA molecules. The final step involves phaC PHA synthase, which links 3HB-CoA and 3HA-CoA units to produce the PHBHx copolymer.

The following examples and description are illustrative only and do not limit the scope of the present disclosure or the appended claims.

Materials and Methods

Material Preparation: Polyethylene oxide (PEO), 600,000 g/mol, was procured from Thermo Scientific. Spinning solutions were prepared by mixing PEO in distilled water at 6% (w/v) and the solution was placed on a rotating mixer for 24 h.

Electric Field Simulations:Based on the finite element method, Ansoft Maxwell (ANSYS Inc., USA) software was used to plot electric fields. Values for voltage, geometric dimensions and material properties were based on the experimental system.

Experimental System: FIG. 2 shows the schematic of an example conjugated electrospinning system. Nanofibers were produced using 20-gauge blunt needles, an applied voltage of +6 kV, syringe pump rate of 0.5 mL/h, and a funnel speed of 300 rpm. The vertical distance of the needles from the funnel (NH) was fixed at 6 cm, but the needle distance (ND) was varied with spacing of 11.5, 20.5 or 30 cm. The winder to funnel height (WH) was also varied at spacing of 10, 12 or 14 cm. The needle angle (a) was set to three different values, either 43°, 33° or 23° and the winder speed was 7 cm/min. The relative humidity was maintained within the range of 30-39% inside the nanoyarn unit enclosure.

Scanning Electron Microscopy:

Scanning electron microscopy (SEM) images of electrospun samples were taken on Zeiss Supra 50VP after sputter coating the sample with platinum/palladium for 40 mA and 30 s, to improve sample conductivity. Mean nanoyarn and nanofiber diameters (n=18, n=100) were measured using Image JR software. 1.52n (National Institute of Health, USA).

Funnel Speed and Winder Speed

Definitions

Funnel Speed refers to the rotations per minute of the rotating funnel or collector on which the fibers deposit.

Winder Speed refers to the speed of the rotating mandrel in cm/min where twisted nanofiber yarns are collected.

Twist angle is the angle that the individual nanofibers make with the axis of the yarn.

Funnel Speed and Winder Speed have been studied in literature regarding their impact on fiber morphology. Generally, an increase in funnel speed increases the twist angle contributing to an increase in the mechanical properties. As winding speed increases there is a decrease in the twist angle. As the winding speed increase, the twisting angle decreases because the displacement of fibers in a single rotation was greater. In contrast, the twisting angle increased with increasing rotating speed. Generally, when referring to twist angle, it is represented as a function of the ratio between the winding speed to the rotating collector speed. The greatest twist angle is obtained from a larger ratio. In terms of mechanical properties, an increase in twist angle increases tensile strength, Young's modulus, and strain.

There is, however, a twist angle above which there is a decrease in tensile strength and Young's modulus, but the strain continues to increase. Hearle's equation describes this relationship. The improved modulus as the twist angle increases can be explained by the increase in lateral force holding fibers together. Thus, more fibers can contribute to the overall Young's modulus of the yarn. At the same time, as the twist angle increases, the angle that the fibers make with the yarn axis increases. Thus, above a certain twist angle, the fibers are oriented perpendicular to the direction of the applied force, limiting the force that the fibers can withstand before failure.

Needle Distance

Needle Distance is the horizontal distance between the two oppositely charged needles. In traditional electrospinning with a multiple needle system, the goal of the added needles is to increase the production of fibers. However, in the nanoyarn system the presence of the second needle is required to facility yarn production in addition to increasing the fiber production. The second needle carries an opposite charge thus at the onset of electrospinning the two oppositely charged needles produce nanofibers that are attracted to each other.

In the dual needle system reported here, the impact of the needle distance on the fiber morphology is due the changes in the electric field and the rotating funnel. As the needle distance increased the nanofiber diameter decreased (FIG. 4A). This is due to the location of the deposition of fibers. At 11 cm, the deposition of fibers occurred at the center of the funnel where the fiber experiences the least of the rotational force from the rotating funnel while at 30 cm the fibers experience the maximum rotational force from the rotating funnel (FIGS. 4B-D). This is in contradiction to the literature because the range of needle distances investigated in literature is narrow in comparison to what was investigated in this thesis. It can be inferred that in the small needle distance range, electric field is the dominating feature while at the greater needle distances the rotating funnel has a greater impact than electric field.

Needle Angle

Under the applied voltage the copper funnel located in the middle of the two nozzles would have charges that were opposite those of the nearby charged nozzles. Therefore, induction fields were created between both edges of the funnel and their nearby charged nozzles. As a result of the electrostatic induction effect, the charged jets ejected from the oppositely charged nozzles would be attracted toward the side of the inductive funnel with opposing charges. Even in the absence of a collector, fiber deposition still occurs because the jets are oppositely charged and a self-bundling effect occurs.

When the angle of the needle is completely horizontal, the un-grounded funnel is no longer an anchor point. Rather, the oppositely charged jets are attracted to each other creating a self-bundling effect. When a ground glass rod is used to draw the fibers down, we end up with a highly aligned nanofiber. This self-bundling effects has been demonstrated in traditional electrospinning, AC electrospinning and in other nanoyarn set-ups. At a lower needle angle the self-bundling effect dominates leading to highly aligned nanoyarns without a twist. When the angle of the needle is lower, the electric field lines are not targeted towards the funnel rather they are directed towards the other needle. Fiber collection and neutralization occurs mid-point between the two needles and a conglomeration of fibers is formed at a distance below surface of the funnel. As demonstrated in FIG. 5B, a needle angle of 23 demonstrates highly aligned fibers a result of the self-bundling effect.

Winder to Funnel Height:

Definition: Winder to funnel height is the distance between the rotating mandrel and the surface of the funnel.

During the nanoyarn process, the polymer jet coming from the two needles congregates at the funnel and when drawn to the winder, the fibers form a cone of aligned fibers and a twist is introduced into the yarn at the apex of the cone and collect on the winder. The height of the fibrous cone is constant within standard deviation while the distance from the apex of the cone to the winder changes with winder to funnel height. It can be inferred that the change in winder height would impact fiber density and overall diameter of the nanoyarn which makes it a useful parameter to study. As presented in FIG. 6A, as the winder to funnel height increases the distance from the apex of the cone of fibers to the winder increases. The data suggests that above 12 cm the distance there is more axial stress applied in the region from the apex of the funnel to the surface of the winder leading to smaller diameter yarns.

An analysis of the impact of various parameters on fiber morphology in a nanoyarn system was performed. The typical electrospinning parameters and other parameters such funnel speed, winder speed, needle distance, winder-to-funnel height, and needle angle impact nanoyarn and nanofiber diameter as well as nanoyarn twist angle. Needle angle is useful to impart a twist in the nanoyarns and the winder to funnel distance is important for the consistent nanoyarn formation. As winder to funnel height increased, the standard deviation of the nanoyarn diameter increased. At a winder to funnel height of 10 cm the diameter of the nanoyarn varied the least. For 6 wt % PEO in DI water, we found the optimal continuous nanoyarn production to be at a needle distance of 20.5 cm, needle angle at 43 degrees and winder to funnel height at 10 cm at a funnel speed of 300 RPM, and winder speed of 7 cm/min. These findings are applicable to other polymer systems:expanding the use and production of nanoyarns for other application.

PHBHx Nanoyams

Materials & Methods

Material Preparation: Poly-3-hydroxy butyrate with 5.8 ml % 3-hydroxyhexanoate (PHBHx) (M_w˜ 461,387 g/mol) was provided by the University of Delaware. Chloroform and glacial acetic acid were procured from VWR. Spinning solution were prepared by mixing 26.2% w/v PHBHx in chloroform and acetic solvent (9:1). The solutions were kept on a rotator for 24 h before utilization.

Experimental Set-up: A conjugated electrospinning set-up as defined in chapter 2.2 was used to create the nanofiber yarns. Briefly, the electrospinning setup consisted of two high voltage power supplies (Gamma High Voltage Research, Ormond Beach, FL), two syringe pumps (Harvard Apparatus. Plymouth Meeting. PA), and a copper funnel. Solutions were loaded into two 5 mL syringes fitted with blunt 20-gauge needles. The syringe pumps were both set to a rate of 0.2 mL/h and the voltages were set to ±9 kV. Funnel speed=300 RPM, needle distance=30 cm, Needle angle=30-40°. Distance between the needle tip to collector=6 cm, winding rate=16-20 cm/min and winder to funnel height=10-14 cm.

FTIR. Fourier Transform infrared spectroscopy was performed on all samples using a Bruker FTIR at a resolution of 2 cm⁻¹. Samples were placed directly onto the ATR crystal and secured. Spectra were collected in the range of 4000 to 500 cm⁻¹ at an interval of 2 cm⁻¹ with a total of 64 scans per sample.

SEM. Scanning electron microscopy (SEM) imaging was used to analyze the diameters of the nanoyarns. Samples were sputter coated with platinum/palladium at 40 mA for 30 s (Cressington Scientific 108 Auto, Watford, UK) and then imaged with field emission scanning electron microscopy (FESEM Zeiss VP5 Supra). For each solvent system three nanoyarn samples were collected. Six images of each nanoyarn sample and three images inspecting the nanofibers were taken. ImageJ® was used to analyze the diameters of the nanoyarns.

Mechanical Data: Tensile Testing was conducted on a Shimadzu AGS-X Universal tensile tester using a IN load cell at strain rate of 300 mm/min and all the samples were pretensioned to 0.007N. A sample size of N=3 was used.

NanoCT: Zeiss Xradia 620 Versa X-ray microscope (pixel size 1.61−0.48 μm) was used to inspect the inner morphology of the nanoyarns. Additionally, ImageJ was used to process the images to quantify pore volume. A gaussian blur and median filter at a pixel size of 2 was used prior to implementing a threshold to select and quantify the pore region.

Solvent Composition

Polyhydroxy butyrate (PHB) has shown high solubility in mostly halogenated compounds, dimethylformamide and select esters and alcohols, However, practically PHB and its copolymers have only been electrospun into nanofibers using primarily chloroform, dichloromethane, dimethylformamide, hexafluoro-2-propanol and formic acid.

Nanofiber diameter is affected by polymer concentration, solvent composition, and collector type. With an increase in polymer concentration there will be an increase in the nanofiber diameter.

Here, we illustrate the disclosed technology with poly-3-hydroxy butyrate with 5.8 mol % 3-hydroxyhexanoate (PHBHx) (M_w˜ 461,387 g/mol) in chloroform and acetic acid. Initially, the polymer concentration that produce beadless, uniform nanofibers was investigated. Polymer concentration between 4 wt %-15 wt % were investigated. It was found that 8 wt % and above there is sufficient polymer entanglement to produce continuous nanofibers (FIG. 10).

Based on the Hansen solubility parameters PHB is partially soluble in acetic acid. Additionally, acetic acid is generally regarded as safe. For this reason, acetic acid was chosen for fiber production. From Table 5 below it is seen that the addition of acetic acid reduced the pH of the electrospinning solution but there was negligible change in conductivity. As the amount of acetic acid increased, the average nanoyarn diameter increased due to increased fiber production. Similarly, with the addition of 1 mL of acetic acid, the average nanofiber diameter decreased from 7.213±0.1 to 4.961±0.76. However, there is an increase in nanofiber diameter with the addition of 2 ml of acetic acid. With the addition of a small amount of acetic acid, the evaporation time of the solution increased allowing the fiber to be in the whipping state longer before drying.

Chloroform is a volatile solvent that causes issues with needle clogging interrupting the continuous nanoyarn production. Adding acid to solvent is one method to reduce the volatility of the solvent. The addition of acetic acid contributed to better manufacturing as there were lower occurrences of needle clogging.

TABLE 5
Solution properties of the varying solvent composition

	Solvent Mixture	pH

	8:2 chloroform:acetic acid	2.09
	9:1 chloroform:acetic acid	2.35
	100% chloroform	4.10

TABLE 6
Average nanoyarn diameter, nanofiber diameter,
and twist angle of the three solvent mixtures

	Average	Average
	Nanoyarn	Nanofiber	Twist
	Diameter	Diameter	angle
Solvent mixture	(μm)	(μm)	(°)
8:2 chloroform:acetic acid	816 ± 0.17	6.408 ± 1.84	66.8 ± 28.3
9:1 chloroform:acetic acid	390 ± 0.06	4.961 ± 0.32	53.7 ± 17.9
100% chloroform	270 ± 0.09	7.213 ± 0.10	52.86 ± 14.8

For the 8:2 chloroform:acetic acid composition, the twist angle was on average 66.8 and a median of 70.45 degrees with a very large standard deviation. There are regions of the yarn without a discernable twist in the nanoyarn (Table 6). Both 9:1 chloroform:acetic acid composition and the 100% chloroform composition have twist angle of approximately 53 degrees. However, there is much larger difference in the nanoyarn diameter. For further experimentation, the 9:1 solvent composition was selected as the fiber diameters are comparable to traditional textile yarns and medical sutures. The diameter of typical textile yarns ranges between 300-600 μm and medial sutures ranges between 30-700 μm. The 9:1 solvent composition is applicable for these applications in terms of fiber diameter.

Acid Hydrolysis of PHBHx

PHBHx is known to undergo acid hydrolysis. However, the hydrolysis occurs slowly. Thus, it is not expected that the minimal amount of acetic acid added to the solution degraded our polymer prior to spinning based on the pH values measured. Additionally, acid-catalyzed hydrolysis occurs much slowly and is reversible as H+ could attack the hydroxyl groups and carboxylic groups of the degradation products resulting in trans-esterification. The effect of acid hydrolysis of PHBHx presents with a shift of the 1721 cm⁻¹ (C═O) peak and an increase in intensity of the peak at 1055 cm⁻¹ (—OH) and 825 cm⁻¹ (═C—H) due to the degradation products As presented in FIGS. 9A-9E, there is no shift in the location of the 1721 cm⁻¹ peak or an increase in intensity of peaks at 1055 cm⁻¹ and 825 cm⁻¹ indicating no degradation of PHBHx.

Processing Parameters

TABLE 7
Nanofiber diameters of PHBHX nanoyarns at varying
winder to funnel height and winder speed.

	Nanofiber
	Diameter
	(μm)	10 cm	12 cm	14 cm
	16 cm/min	5.0 ± 2	5.3 ± 2	4.5 ± 1
	20 cm/min	10.3 ± 4	7.1 ± 2	7.3 ± 6
	25 cm/min	6.9 ± 3	6.3 ± 2	5 ± 2

For the solvent composition study the parameter used were a winder speed of 16 cm/min and a winder to funnel height of 10 cm with a needle distance of 30 cm. Needle distance could not be modified as a smaller needle distance caused bridging between the two needles. From the SEM in FIGS. 8A-8C, it is evident that not all the nanofibers produced are being twisted into the nanoyarn rather there are a lot of loose nanofibers. Additionally, the large standard deviations on the twist angle of the nanoyarn indicated an unoptimized parameters. In order to optimize the process, various winder speed and winder to funnel height were investigated. For every winder to funnel height (10 cm, 12 cm, 14 cm) a winder speed of 20 cm/min produced highest nanoyarn diameter. Using a winder to funnel of 12 cm produces the highest nanoyarn diameter. This same relationship was seen at a winder speed of 25 cm/min. But for winder speeds 16 cm/min and 20 cm/min with increasing winder to funnel height the nanoyarn diameter decreased (FIG. 10).

Mechanical Properties of PHBHx

FIGS. 12A-12C provide a cross-sectional view of PHBHx nanoyarn and it is evident that the internal structure is very porous. It shows that the inner core is essentially empty. The pore volume of a 1 cm sample is approximately 84%. This large pore volume is what contributes to the sample's tensile strength and elastic modulus. The elongation at break is calculated to be approximately 20% (FIG. 11B).

• • Composite PCL:PHBHx Nanoyarns

Materials and Methods

Materials: Poly-3-hydroxy butyrate with 5.8 mol % 3-hydroxyhexanoate (PHBHx) (M_w˜ 461,387 g/mol) was provided by the University of Delaware. Chloroform and glacial acetic acid were procured from VWR. Spinning solution were prepared by mixing 26.2% w/v PHBHx in chloroform and acetic acid solvent (9:1). The solutions were kept on a rotator for 24 h before utilization.

Polycaprolactone (PCL) (85,000 g/mol) was procured from VWR. A solution of PCL was made using chloroform as a solvent. The solution was kept on a rotator for 24h before utilization. PHBHx and PCL composite solutions were prepared by combining the PHBHx and PCL in volume-to-volume ratios (1:9, 5:5, 9:1).

Fabrication methods: Nanoyarn system defined in Chapter 2 was used to produce the composite nanoyarns however two different methods were used to create composite nanoyarns yarns.

Method 1: the two syringes contained a solution mixture of PCL and PHBHx at varying volume ratios (1:9, 5:5, 9:1). This method created a nanofiber yarn where the individual nanofibers are a composition of PCL and PHBHx (NY_1:9, NY_5:5, NY_9:1). The PHBHx used had a polymer concentration of 26.2 w/v while two different polymer concentrations were investigated (12w/v and 16w/v)

	12NY1:9, 12NY5:5, 12NY9:1	Composite nanoyarns with
		12 w/v PCL and 26.2 w/v PHBHx
	16NY1:9, 16NY5:5, 16NY9:1	Composite nanoyarns with
		16 w/v PCL and 26.2 w/v PHBHx

Method 2: In method two both syringes contained different compositions of solutions. One syringe contained PHBHx while the other syringe contained PCL, which produced nanofiber yarns that contained a mixture of fibers that are 100% PCL and 100% PHBHx (NY_2.1). Alternatively, a nanoyarn was made where one of the syringe contained a solution mixture of 5:5 PCL:PHBHx while the other syringe contained 100% PCL. This produced a nanoyarn containing nanofibers that are a 100% PCL and individual fibers that are a composition of PCL and PHBHx at a ratio of 5:5 (NY_2.2)

SEM. Scanning electron microscopy (SEM) imaging was used to analyze the diameters of the nanoyarns. Samples were sputter coated with platinum/palladium at 40 mA for 30 s (Cressington Scientific 108 Auto, Watford, UK) and then imaged with field emission scanning electron microscopy (FESEM Zeiss VP5 Supra). For each type of nanoyarn, three nanoyarn spools were collected. Six images of each nanoyarn sample and five images inspecting the nanofibers were taken. ImageJ® was used to analyze the diameters of the nanoyarns and nanofibers.

Mechanical Data: Tensile Testing was conducted on a Shimadzu AGS-X Universal tensile tester using a IN load cell at strain rate of 300 mm/min and all the samples were pretensioned to 0.007N. A sample size of N=3 was used.

NanoCT: Zeiss Xradia 620 Versa X-ray microscope (pixel size 1.61-0.48 μm) was used to inspect the inner morphology of the nanoyarns. Additionally, ImageJ was used to process the images to quantify pore volume. A Gaussian blur and median filter at a pixel size of 2 was used before implementing a threshold to select and quantify the pore region.

Linear Density: The linear density of a fiber sample was measured by determining the mass of a 12 cm length sample. The length of fiber was then weighed on a high-precision analytical balance with a sensitivity of 0.0001 grams to minimize measurement error. The mass (m) and the length (L) of the fiber were recorded, and the linear density (denoted as mass per unit length) was calculated using the formula:

Linear ⁢ Density = m L ⁢ g / m

To convert the measured linear density into Tex, the following standard conversion formula was applied:

Tex = 1000 × m L

Pycnometer: The density of the solid material was measured using a pycnometer with water as the displacement liquid. The procedure began by thoroughly cleaning and drying the pycnometer to ensure accurate results. The following steps were performed:

Mass of Empty Pycnometer: The dry, empty 2 ml pycnometer was weighed using an analytical balance with a precision of 0.0001 grams, and the mass was recorded as M_empty

Mass of Pycnometer Filled with Water: The pycnometer was then filled with distilled water up to the calibration mark. The external surface was carefully wiped to remove any excess liquid, and the mass of the water-filled pycnometer was measured and recorded as M_water-filled

Mass of Pycnometer with Solid Sample and Water: The solid sample was introduced into the pycnometer, and water was added to fill the remaining volume. Care was taken to avoid air bubbles. Once filled to the calibration mark, the mass of the pycnometer, the solid sample, and water was recorded as M_sample+water

Calculations: The density of the solid sample was determined using the following formulas:

First, the volume of water displaced by the solid sample was calculated:

V displaced ⁢ water = ( M water - filled - M empty ) - ( M sample + water - M sample ) ρ water

Where ρ_water is the known density of water at the experimental temperature.

The density of the solid sample was then determined by dividing its mass by the displaced water volume:

ρ sample = M sample V displaced ⁢ water

DSC: TA DSC Q2000 was used to investigate the thermal properties of the composite nanoyarns. 3-5 mg of the samples was measured on high precision analytical scale and secured in small DSC pans. A heat-cool-heat cycle was run from a temperature of −30 to 180° C.

TABLE 8
Nanofiber diameter, mass density, linear
density and twist of solution blend.

	Nanofiber		Linear
	Diameter	Density	Density	Twist
	(μm)	(g/ml)	(tex)	Angle

12PCL:PHBHx (1:9)	6.35 ± 2.5	0.13	48.94	63.68 ± 7.4
12PCL:PHBHx (5:5)	6.69 ± 2.9	0.70	63.27	50.89 ± 7.2
12PCL:PHBHx (9:1)	4.51 ± 2.4	0.33	22.90	45.84 ± 5.4
16PCL:PHBHx (1:9)	6.72 ± 2.3	0.17	55.25	47.74 ± 6
16PCL:PHBHx (5:5)	7.11 ± 2.1	1.09	34.79	49.62 ± 6.1
16PCL:PHBHx(9:1)	4.87 ± 4.0	0.10	32.95	44.05 ± 6.2
26.2 w/v PHBHx	7.08 ± 2.1	0.10	137.6	70.23 ± 17.6
12 w/v PCL	4.1 ± 2.1	0.46	29.60	41.8 ± 19.8
16 w/v PCL	8.03 ± 2.7	0.13	66.70	62.3 ± 13.3

Composite nanoyarns 12NY_5:5 and 16NY_5:5 demonstrated the highest volume density at 0.7 g/ml and 1.09 g/ml respectively but the smallest nanoyarn diameters. In terms of linear density, pure PHBHx had the highest linear density but a very low volume density. This indicates that although the individual fibers might be thick and heavy they do not occupy a lot of mass in a fixed volume, or the material is lightweight.

The average nanoyarn diameter of 5:5 PHBHx:PCL for 12w/v was approximately 300 μm and 482 μm for 16w/v while the 1:9 and 9:1 ratios had nanoyarn diameter of 567 μm and 489 μm for 12 w/v PCL and 429 μm and 327 μm for 16w/v respectively. Not only did the 5:5 ratio have the smallest nanoyarn diameter the standard deviation of the yarn diameter was also the smallest of the three ratios investigated. The higher density translates to mechanical properties. The 5:5 ratio had the highest elastic modulus and tensile strength for both 12w/v and 16w/v. However, the higher polymer concentration of PCL had a lower tensile strength and modulus compared to 12w/v. For both polymer concentrations, the ratio containing the higher quantity of PCL demonstrates the highest elongation at break as expected.

There are two principles that could contribute to the enhanced mechanical properties of the composite fibers one is the structure of the nanoyarn and the other is the molecular interaction between PHBHx and PCL. Nanofiber diameter and deposition rate are indicators of changes to the electrospinning process which could indicate changes to the solution properties. The nanofiber diameter of 12w/v PCL was smaller than that of 16w/v PCL which is consistent with literature where an increase in polymer concentration increases fiber diameter. With the addition of 12w/v PCL there was a decrease in the nanofiber diameter compared to pure PHBHx nanofibers. Although there are slight changes to the average nanofiber diameter these changes are not statistically significant. A deposition rate study was conducted to see if the number of fibers depositing changed because of the addition of PCL. As other electrospinning parameters were kept constant, any changes to the deposition would be a result of changes to the solution. The deposition rate for PHBHx was 0.0046 g/min while the 9:1 and 5:5 ratio had a deposition rate of 0.007 but 9:1 had a deposition rate of 0.011 g/min. This increase in deposition might be an indication of reduction in viscosity as a result of the PCL and PHBHx being immiscible. The structure of the nanoyarn provides insights into its mechanical properties. The 5:5 ratio presented 18% pore volume while PHBHx nanoyarn had 80% porosity. This low pore volume is an indication of a compact nanoyarn that would directly contribute to high tensile strength and elastic modulus.

TABLE 9
Mechanical Properties of solution blend
composite nanoyarns from method 1

	Elastic	Tensile	Elongation
	Modulus	Strength	at Break
	(Mpa)	(MPa)	(%)

12 w/v	1:9	19.67 ± 7.83	1.03 ± 0.25	17 ± 1
	5:5	233.81 ± 27.39	10.88 ± 1.86	119 ± 87
	9:1	8.25 ± 4.47	1.33 ± 1.10	295 ± 197
16 w/v	1:9	89.4 ± 25.32	3.8 ± 0.55	50 ± 26.1
	5:5	178.5 ± 48.52	6.7 ± 1.93	49.4 ± 31.5
	9:1	23.3 ± 11.16	2.4 ± 0.66	523.2 ± 93.4

The 12PCL, 100PHBHx nanoyarn utilizing method 2 is comparable to 5:5 PCL:PHBHx as they are both composite yarns with 50% PCL and 50% PHBHx, however the main difference is that the 5:5 PCL:PHBHx is 50% blend in the individual nanofiber while the with method 2, the nanoyarn would be 50:50 blend of pure PCL and PHBHx nanofibers. However, with method 2, the elastic modulus and tensile strength is 20 and 5 times lower respectively. However, the elongation at break is double that of 5:5 solution blend ratio,

TABLE 10
Mechanical properties of nanofiber blend of composite
PCL:PHBHx nanoyarns from method 2 above.

	Elastic	Tensile	Elongation
	Modulus	Strength	at Break
	(MPa)	(MPa)	(%)

12PCL, PCL:PHBHx (5:5)	61.8 ± 12	7.5 ± 3	226.4 ± 154
16PCL, PCL:PHBHx (5:5)	25.0 ± 15	4.4 ± 1	601.7 ± 135
12PCL, 100PHBHx	9.7 ± 6	1.9 ± 1	37.9 ± 12

TABLE 11
Volume Density, Linear Density and Pore
Volume of composite nanoyarns from

	Density	Linear	Pore
	(g/ml)	Density (tex)	Volume (%)

12PCL:PHB (5:5)	0.70	63.27	18 ± 1.20
12PCL, PCL:PHBHx (5:5)	0.22	48.20	33 ± 0.85
12PCL, PHBHx	0.17	54.12	63 ± 2.80
26.2 w/v PHBHx	0.10	137.6	84 ± 1.30

Comparing the 5:5 PCL:PHBHx solution blend from method 1 to the nanofiber blend nanoyarn, the latter nanoyarns had a more compact structure with only 18% pore volume while the nanofiber blended nanoyarn from method 2 had a pore volume of 63%. By adding the solution blend mixture to one syringe while the other syringe contained only PCL, a more compact nanoyarn was created with the pore volume dropping to 33%.

Pure PHBHx shows two melting peaks one at 124° C. and one at 144° C. and no visible T_g however a T_g transition is apparent in the second heat at −0.38° C. There is slight shift in the melting peak after the thermal history has been removed in the second heat. The two melting peaks shifted to a slightly higher temperature to 125° C. and 145° C. The existence of two melting peaks for semi-crystalline polymers has been reported. The presence of the two peaks could be due to the polymorphism presented by some copolymers such as PHBHx as observed in other aliphatic polyesters. Due to the heterogeneous composition of the copolymer itself, different crystalline morphologies can be formed, with different thermal stability. Alternatively, it could be due to the presence of different types of crystals. Crystallization could produce primary crystals with low degree of perfection: these may melt and recrystallize to produce crystals of greater perfection or greater thickness leading to the presence of two melting peaks.

Applicability of composite PCL:PHBHx Nanoyams:

In terms of meeting the benchmarks set, the 5:5 PCL:PHBHx nanoyam have a twist angle that is less than 90 degree which maximizes the overall mechanical properties. This composite nanoyarn did demonstrate the smallest standard deviation for nanoyarn diameter compared to all the composite tested. The yarn diameter is also comparable to common textile and medical suture. The diameters of common textiles are in the range of 0.3-0.6 mm and from the figure below it the composite nanoyarn are comparable to polyglytone 6211. The composite nanoyarn was also comparable to polyglytone in terms of elastic modulus. For textile application, the current nanoyarn still needs to be improved to meet the tensile strength and elastic modulus requirements of common textiles. However, the advantage these nanofiber yarns provide is the ability to imbue the nanoyarn with antimicrobial or drug-eluting properties. With increasing incidence of Surgical Site Infections (SSI) due to wound infections that lead to increased treatment costs, increased hospitalization rates, longer duration of treatment, severe morbidity and high mortality, the ability to easily encapsulate bioactive ingredients such as growth factors, inorganic nanoparticles, antibacterial drugs and herbal extracts to promote wound healing is attractive.

Here, we provide pure PHBHX nanoyarns without the need for a textile core or co-polymer. These PHBHX nanoyarns are composed of 100% biodegradable and biocompatible polymer. Through the disclosed methods, we have a continuous scalable method to produce nanoyarns that can be utilized in biomedical and textile applications. The nanoyarns can be manipulated into various 3D structures for drug delivery and tissue engineering scaffolds. The mechanical properties can, for example, be tuned to mimic native tissue. These nanoyarns can be incorporated into textiles to create fabrics that utilize less polyesters and other polymers, thereby contributing less to textile waste.

In PHBHX, the presence of a medium chain length side group acts as a defect and disrupts the regularity of the polymer chain resulting in a polymer with less crystallinity and a lower melting point. These are nontoxic, biocompatible, biodegradable thermoplastics that have a high degree of polymerization, are highly crystalline, optically active, insoluble in water and have piezoelectric properties. Further, the degradation products of PHBHX are 3-hydroxy butyric acid which is made in the liver during the breakdown to long chain fatty acid. Comparing tissue responses to PLA, PHBHHx demonstrated lower degree of fibrosis and fewer inflammatory cells when compared to PLA. Further, hydroxy butyric acid reduces the amount of apoptosis and cell death.

Non-limiting Example

Nanofiber yarns composed of Poly(3-hydroxy butyrate-co-3-hydroxyhexanoate) (PHBHx) were formed from a solution of 26.2w/v PHBHx in chloroform and acetic acid utilizing a conjugated electrospinning set-up. As an illustrative example, continuous nanoyarns were produced utilizing various solvent combinations, for example, 100% chloroform, and 9:1 and 8:2 chloroform:acetic acid. As an illustrative example, nanoyarns that were approximately 50 cm in length were manufactured.

Aspects

The following Aspects are illustrative only and do not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more Aspects can be combined with any part or parts of any one or more other Aspects.

Aspect 1. A nanoyarn, comprising poly(3-hydroxy butyrate-co-3-hydroxy hexanoate) (PHBHX), (i) the nanoyarn being essentially free of a core material, (ii) the nanoyarn being essentially free of a co-polymer, or both (i) and (ii).

A nanoyarn can have a diameter of, for example, from about 25 to about 1000 μm, e.g., from about 25 to about 1000 μm, from about 50 to about 800 μm, from about 100 to about 700 μm, from about 200 to about 500 μm, or even from about 300 to about 400 μm. As described, a nanoyarn can comprise a plurality of fibers.

A fiber of the disclosed nanoyarns can have a diameter of, for example, about 100 nm to about 8250 nm, including all intermediate values and sub-ranges. For example, a fiber can have a diameter of from about 100 nm to about 8250 nm, from about 200 nm to about 1900 nm, about 1300 nm to about 1800 nm, about 4000 nm to about 7000 nm, or about 5000 nm to about 8250 nm. The diameter can be, for example, from 100 to about 8250 nm, from about 500 to about 7000 nm, from about 1000 to about 5000 nm, from about 1500 to about 4500 nm, or even from about 1000 to about 3000 nm.

Nanoyarns can exhibit a twist angle of from about 40 to about 70 degrees, for example from about 40 to about 70 degrees, from about 50 to about 60 degrees, or even from about 51 to about 59 degrees. In some embodiments, the twist angle can be from about 1 to about 50 degrees, although this is not a requirement.

Aspect 2. The nanoyarn of Aspect 1, wherein the nanoyarn is free of a core material and free of a co-polymer.

Aspect 3. The nanoyarn of any one of Aspects 1-2, wherein the nanoyam has a length of up to about 75 cm. It should be understood, however, that nanoyarns according to the present disclosure can have a length in excess of 75 cm. For example, a nanoyarn can have a length of up to 75 cm, up to 100 cm, up to 150 cm, up to 200 cm, up to 300 cm, up to 400 cm, up to 500 cm, up to 600 cm, up to 700 cm, up to 800 cm, up to 900 cm, or even up to 1000 cm. Nanoyarns having a length in excess of 900 cm or even in excess of 1000 cm are within the scope of the present disclosure, as are nanoyarns having a length of up to 5000 cm.

Aspect 4. The nanoyarn of Aspect 3, wherein the nanoyarn has a length of up to about 50 cm.

Aspect 5. The nanoyarn of any one of Aspects 1-4, wherein the nanoyarn consists of PHBHX. Such nanoyarns can be free of other materials.

Aspect 6. The nanoyarn of Aspect 1, further comprising a second polymer, the second polymer optionally having a greater ductility than PHBHX. Without being bound to any particular theory or embodiment, one can select a second polymer having a ductility greater than that of PHBHX so as to facilitate processing of the nanoyarn, for example knitting and/or weaving. The second polymer can be, for example, a biocompatible polymer. The second polymer can be, for example, a biodegradable polymer. The second polymer can be one that is immiscible with PHBHX. As but one example, the nanoyarn can be formed from a solution of PHBHX and a solution of the second polymers, which two solutions can be immiscible with one another.

Aspect 7. The nanoyarn of Aspect 6, wherein the second polymer comprises polycaprolactone (PCL). The second polymer can comprise (or, alternatively, be free of) any one or more of polylactic acid (PLA), a polyhydroxyalkanoate (PHA), poly(lactic-co-glycolic acid) (PLGA), polyvinyl alcohol (PVA), poly(ethylene oxide) (PEO), poly(3-hydroxy butyrate) (PHB), (4-hydroxy butyrate) (P4HB), poly(ethylene glycol) diacrylate (PEGDA), poly(2-hydroxyethyl methacry late) (PHEMA), poly(e-caprolactone-co-lactic acid) (PCLA), poly(ethylene glycol)-poly(lactic acid) (PEG-PLA), poly(ethylene glycol)-poly(caprolactone) (PEG-PCL), chitosan, hyaluronic acid, alginate, keratin, chitin, carboxymethylcellulose, cellulose acetate, and soy protein isolate.

Aspect 8. The nanoyarn of Aspect 6, wherein the nanoyarn comprises fibers of PHBHX and comprises fibers of the second polymer. Such a nanoyarn is shown in, for example, FIG. 20 (right panel), which illustrates forming a nanoyarn that comprises two different kinds of fiber.

Aspect 9. The nanoyarn of Aspect 6, wherein the nanoyarn comprises fibers that themselves comprise both PHBHX and the second polymer. Such a nanoyarn is shown in, for example, FIG. 20 (left panel), which illustrates forming a nanoyarn that comprises a fiber that includes both PHBHX and a second polymer.

Aspect 10. The nanoyarn of Aspect 6, wherein the volume ratio of the PHBHX in the nanoyarn to the second polymer in the nanoyarn is from 1:99 to 99:1, optionally from 1:9 to 9:1, optionally from 1:2 to 2:1. The volume ratio can be, for example, 1:99 to 10:90, 10:90 to 20:80, 20:80 to 30:70, 30:70 to 40:60, 40:60 to 50:50, 50:50 to 60:40, 60:40 to 70:30, 70:30 to 80:20, 80:20 to 90:10, and 90:10 to 99:1.

The disclosed nanoyarns have use in a range of applications. As one example, the disclosed materials are useful in medical applications, such as sutures. As another example, the disclosed nanoyarns can be useful in tissue engineering, where the nanoyarn can serve as scaffolding for tissue engineering, providing a structure for cells to grow and form new tissues. The disclosed nanoyarns can also be used in drug delivery systems, e.g., a system configured to gradually release medication over time. The disclosed nanoyarns also have application in textiles, in particular in environmentally friendly textiles that decompose naturally, reducing plastic waste. Likewise, the disclosed nanoyarns can be utilized in food packaging materials that are both biodegradable and safe for food contact, helping to reduce the environmental impact of such packaging.

Aspect 11. An article, the article comprising a nanoyarn according to any one of Aspects 1-10. Such articles can be configured as, for example, textiles, tissue engineering scaffolds, drug delivery articles, and packaging. Without being bound to any particular theory or embodiment, the disclosed articles are particularly suitable for use as food-safe packaging.

Aspect 12. The article of Aspect 11, wherein the article is characterized as a textile. A textile need not be a woven textile.

Aspect 13. The article of Aspect 11, wherein the article is characterized as a tissue engineering scaffold.

Aspect 14. The article of Aspect 11, wherein the article is characterized as at least one of a drug delivery article and a food package.

Aspect 15. The article of Aspect 11, wherein the article is characterized as having a mechanical property essentially identical to that of a native tissue. The article can be constructed—for example, by choice of fiber composition, fiber diameter, and the like-so as to have such a mechanical property.

Aspect 16. A method, comprising forming a nanoyarn according to any one of Aspects 1-10.

Aspect 17. The method of Aspect 16, wherein the method comprises electrospinning.

Aspect 18. The method of Aspect 17, wherein the electrospinning comprises conjugated electrospinning.

Aspect 19. The method of Aspect 17, wherein the electrospinning comprises electrospinning from a solution of PHBHX and a plurality of solvents.

Aspect 20. The method of Aspect 19, wherein the plurality of solvents comprises at least one of chloroform and acetic acid.