Patent Application
20250009942
This disclosure relates to biodegradable piezoelectric nanofibers, and in particular relates to piezoelectric polymer and amino acid materials and their use in medical applications.
Piezoelectric materials, a type of “smart” material that can convert mechanical force into electricity and vice versa, are the core of many medical devices including pressure sensors, actuators, and ultrasonic transducers. In many cases, these devices are implanted inside the body for various applications such as monitoring intra-organ pressures, generating ultrasound to facilitate drug-delivery, ablating diseased tissues, and/or stimulating tissue healing etc.
An appealing potential of piezoelectric devices is their ability to be self-powered by harvesting the body's own motion and thus avoiding the use of toxic batteries often required for medical implants. However, conventional piezoelectric ceramics (e.g., lead zirconate titanate (PZT)) or polymers (e.g., polyvinylidene fluoride (PVDF)) are not degradable and even contain toxic elements (e.g., lead in PZT). Thus, devices made from traditional piezoelectric materials may require an undesired removal surgery that can be invasive and can easily damage delicate tissues directly interfaced with the piezoelectric implants.
Recently, there have been reports on the potential for use of safe and biodegradable piezoelectric glycine, the simplest amino acid crystal, which possesses an extremely high piezoelectric constant, comparable to that of piezoelectric ceramics like PZT. Unfortunately, glycine crystals are brittle, difficult-to-handle, and quickly dissolve in aqueous solutions such as body fluid. Existing attempts to leverage the properties of glycine crystals use solvent-casting methods to integrate piezoelectric glycine crystals with soft polymeric matrices to effectively harvest its piezoelectricity. However, this method lacks adequate control over crystal growth direction, resulting in random orientations of crystal domains/dipole moments and leading to an overall low piezoelectric output from the film despite the high piezoelectric constant in each glycine crystal.
Other deficiencies and inferior characteristics of existing solvent cast implementations are discussed throughout the disclosure as well. Therefore, there is a need for a piezoelectric material utilizing glycine crystals in a flexible and easy-to-use form that can fully exploit the excellent piezoelectric effect of this organic material for medical device applications. There is a further need for a new approach to manufacturing such a material, including a method that can create oriented glycine crystals. There is a further need for such materials usable in transducers, as well as the transducers themselves, as well as methods of delivering therapeutics utilizing such materials through a blood-brain barrier in a subject.
One embodiment described herein is a composite material comprising: a biodegradable polymer; and a plurality of glycine crystals embedded in the biodegradable polymer. In one aspect, the biodegradable polymer comprises a plurality of biodegradable fibers. In another aspect, the plurality of biodegradable fibers are substantially parallel-aligned with each other as measured by scanning electron microscopy. In another aspect, each biodegradable fiber has a diameter of about 1 μm to about 5 μm. In another aspect, the biodegradable polymer has a number average molecular weight of about 10 kDa to about 200 kDa. In another aspect, the biodegradable polymer comprises one or more of poly (L-lactic acid) (PLLA), poly (D,L-lactide-co-glycolide) (PLGA), polycaprolactone (PCL), polyglycolic acid (PGA), polyhydroxybutyrate, silk, polyvinyl alcohol, chitosan, or combinations thereof. In another aspect, each glycine crystal has an α-form, a β-form, or a γ-form. In another aspect, each glycine crystal has a diameter of about 1 nm to about 5 μm. In another aspect, the plurality of glycine crystals are substantially parallel-aligned with each other as measured by X-ray scattering. In another aspect, the plurality of glycine crystals are uniformly distributed in the biodegradable polymer. In another aspect, the material comprises a weight ratio of about 0.25:1 to about 1:1, or in some cases, up to 2:1, of the plurality of glycine crystals to the biodegradable polymer (glycine:polymer). In another aspect, the glycine crystals that have the β-form substantially remain in the β-form for at least 30 days in vacuum. In another aspect, the composite material has an elastic modulus of about 10 MPa to about 2,000 MPa. In another aspect, the composite material has a piezoelectric output of greater than 600 kPa as measured by generated acoustic pressure.
Another embodiment described herein is a method of making a biodegradable piezoelectric composite material, the method comprising: electrospinning a mixture comprising a biodegradable polymer, a plurality of glycine crystals, and at least one solvent onto a collector drum having a speed of about 100 rpm to about 4,000 rpm to provide a piezoelectric composite material as described herein. In one aspect, electrospinning is performed at a voltage of about 10 kV to about 25 kV. In another aspect, electrospinning is performed at a flow rate of about 2 ml/h, at a humidity of about 30% to about 50%, or a combination thereof.
Another embodiment described herein is an ultrasonic transducer comprising: a first metal electrode; a second metal electrode; the composite material as described herein positioned between the first metal electrode and the second metal electrode; and an encapsulation layer covering the first metal electrode, the second metal electrode, and the composite material. In one aspect, the encapsulation layer comprises a biodegradable polymer. In another aspect, the first metal electrode, the second metal electrode, or both are electrically coupled to a wire. The ultrasonic transducer of clause 20, wherein the first metal electrode and the second metal electrode are electrically coupled to an ultrasonic generator through the wire.
Another embodiment described herein is method of delivering a therapeutic through a blood-brain barrier in a subject in need thereof, the method comprising: applying the ultrasonic transducer described herein to a craniotomy defect of the subject; transmitting an ultrasonic wave signal through the wire; and delivering a pulsed acoustic pressure to the craniotomy defect.
In one aspect, the method further comprises administering to the subject a therapeutic intravenously after transmitting the ultrasonic wave signal. In another aspect, the ultrasonic wave signal is driven at about 1 MHz to about 5 MHz.
Various embodiments and aspects of the inventions described herein are summarized by the following clauses:
Clause 1. A composite material comprising:
Clause 2. The composite material of clause 1, wherein the biodegradable polymer comprises a plurality of biodegradable fibers.
Clause 3. The composite material of clause 2, wherein the plurality of biodegradable fibers are substantially parallel-aligned with each other as measured by scanning electron microscopy.
Clause 4. The composite material of clause 2 or 3, wherein each biodegradable fiber has a diameter of about 1 μm to about 5 μm.
Clause 5. The composite material of any one of clauses 1-4, wherein the biodegradable polymer has a number average molecular weight of about 10 kDa to about 200 kDa.
Clause 6. The composite material of any one of clauses 1-5, wherein the biodegradable polymer comprises one or more of poly (
Clause 7. The composite material of any one of clauses 1-6, wherein each glycine crystal has an α-form, a β-form, or a γ-form.
Clause 8. The composite material of any one of clauses 1-7, wherein each glycine crystal has a diameter of about 1 nm to about 5 μm.
Clause 9. The composite material of any one of clauses 1-8, wherein the plurality of glycine crystals are substantially parallel-aligned with each other as measured by X-ray scattering.
Clause 10. The composite material of any one of clauses 1-9, wherein the plurality of glycine crystals are uniformly distributed in the biodegradable polymer.
Clause 11. The composite material of any one of clauses 1-10, wherein the material comprises a weight ratio of about 0.25:1 to about 1:1 of the plurality of glycine crystals to the biodegradable polymer (glycine:polymer).
Clause 12. The composite material of any one of clauses 7-11, wherein the glycine crystals that have the β-form substantially remain in the β-form for at least 30 days in vacuum.
Clause 13. The composite material of any one of clauses 1-12, wherein the material has an elastic modulus of about 10 MPa to about 2,000 MPa.
Clause 14. The composite material of any one of clauses 1-13, wherein the material has a piezoelectric output of greater than 600 kPa as measured by generated acoustic pressure.
Clause 15. A method of making a biodegradable piezoelectric composite material, the method comprising:
Clause 16. The method of clause 15, wherein electrospinning is performed at a voltage of about 10 kV to about 25 kV.
Clause 17. The method of clause 15 or 16, wherein electrospinning is performed at a flow rate of about 2 ml/h, at a humidity of about 30% to about 50%, or a combination thereof.
Clause 18. An ultrasonic transducer comprising:
Clause 19. The ultrasonic transducer of clause 18, wherein the encapsulation layer comprises a biodegradable polymer.
Clause 20. The ultrasonic transducer of clause 18 or 19, wherein the first metal electrode, the second metal electrode, or both are electrically coupled to a wire.
Clause 21. The ultrasonic transducer of clause 20, wherein the first metal electrode and the second metal electrode are electrically coupled to an ultrasonic generator through the wire.
Clause 22. A method of delivering a therapeutic through a blood-brain barrier in a subject in need thereof, the method comprising:
Clause 23. The method of clause 22, further comprising administering to the subject a therapeutic intravenously after transmitting the ultrasonic wave signal.
Clause 24. The method of clause 22 or 23, wherein the ultrasonic wave signal is driven at about 1 MHz to about 5 MHz.
Also provided is a method of making a biodegradable piezoelectric composite material. Such a method may include mixing a solution comprising biodegradable polymer, glycine crystal, and at least one solvent. The method further comprises electrospinning the solution and receiving the electrospun solution onto a collector drum having a speed of about 100 RPM to about 4,000 RPM. The resulting biodegradable polymer fibers are then substantially aligned with each other.
In some such embodiments, the method further includes growing or retrieving the glycine crystals for the solution, where the glycine crystals are needle-shaped. The method then includes grinding or otherwise crushing the glycine crystals to create glycine particles and incorporating the glycine particles into the solvent as the glycine crystal component.
In some embodiments, the needle-shaped glycine crystals are β-form crystals. In some embodiments, the glycine crystals are grown using a slow evaporation technique. In some embodiments, the glycine particles are ground using a homogenizer to a size of approximately 500 nm. In some embodiments, the glycine particles are themselves elongate crystals.
In some embodiments, the biodegradable polymer is polycaprolactone (PCL), poly (L-lactic acid) (PLLA), poly(D,L-lactide-co-glycolide) (PLGA), polyglycolic acid (PGA), polyhydroxybutyrate, silk, polyvinyl alcohol, chitosan, or combinations thereof.
Also provided are methods for making an ultrasonic transducer utilizing the piezoelectric composite material. In such an embodiment, the method further includes applying a first electrode to a first side of the biodegradable piezoelectric composite material, applying a second electrode to a second side of the biodegradable piezoelectric composite material opposite the first side, and encapsulating the composite material and the first and second electrodes with a biodegradable encapsulating layer.
In some such embodiments, the method further includes determining an idealized functional lifetime of the biodegradable piezoelectric composite material and selecting a thickness for the encapsulating layer based on the idealized functional lifetime.
In some embodiments of such a transducer, the idealized functional lifetime depends on a use case for the biodegradable piezoelectric composite material.
In some embodiments of a transducer, the encapsulating layer is formed from the biodegradable polymer.
Also provided is a method of therapy leveraging the transducer including implanting the ultrasonic transducer adjacent a craniotomy defect of a subject and transmitting an ultrasonic wave signal through a wire in electrical communication with the first or second electrode. The method may further include administering to the subject a therapeutic intravenously after transmitting the ultrasonic wave signal.
First,
As shown in
As shown in
As shown in
Representative optical images of a degrading glycine-PCL film on days 0, 2, 4, and 6 in DI water, 1×PBS, 10×PBS, and FBS at 37° C. (physiological temperature). Graphical representation of the % weight loss of the film is presented on the right.
The description of illustrative embodiments according to principles of the present invention is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description. In the description of embodiments of the invention disclosed herein, any reference to direction or orientation is merely intended for convenience of description and is not intended in any way to limit the scope of the present invention. Relative terms such as “lower,” “upper,” “horizontal,” “vertical,” “above,” “below,” “up,” “down,” “top” and “bottom” as well as derivative thereof (e.g., “horizontally,” “downwardly,” “upwardly,” etc.) should be construed to refer to the orientation as then described or as shown in the drawing under discussion. These relative terms are for convenience of description only and do not require that the apparatus be constructed or operated in a particular orientation unless explicitly indicated as such. Terms such as “attached,” “affixed,” “connected,” “coupled,” “interconnected,” and similar refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise. Moreover, the features and benefits of the invention are illustrated by reference to the exemplified embodiments. Accordingly, the invention expressly should not be limited to such exemplary embodiments illustrating some possible non-limiting combination of features that may exist alone or in other combinations of features; the scope of the invention being defined by the claims appended hereto.
This disclosure describes the best mode or modes of practicing the invention as presently contemplated. This description is not intended to be understood in a limiting sense, but provides an example of the invention presented solely for illustrative purposes by reference to the accompanying drawings to advise one of ordinary skill in the art of the advantages and construction of the invention. In the various views of the drawings, like reference characters designate like or similar parts.
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. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.
As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.
As used herein, the term “or” can be conjunctive or disjunctive.
As used herein, the term “substantially” means to a great or significant extent, but not completely.
As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to +10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “˜” means “about” or “approximately.”
All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to +10% of any value within the range or within 3 or more standard deviations, including the end points.
As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.
As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.
As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, therapeutic, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject's age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.
As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non-human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.
As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.
As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or therapeutic described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or therapeutic described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or therapeutic described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.
Described herein is a new material as well as a new strategy for material processing and device fabrication to (1) manufacture a biodegradable, flexible, easy-to-use, and highly piezoelectric nanofiber film of glycine crystals embedded inside a biodegradable polymer, such as a polycaprolactone (PCL) polymeric matrix (as shown in
Accordingly, and as discussed in more detail below,
As shown, the composite material 100 typically comprises a plurality of biodegradable fibers 140 formed from the polymer 120. As shown in
As can be seen in
As shown in those figures, in the embodiment shown, the glycine crystals 130 may be substantially uniformly distributed in the biodegradable polymer 120. As also shown in
As discussed below, the glycine crystals 130 referenced may be grown crystals or may be particles 190 of crushed glycine crystals. However, even when crushed, the particles 190 typically have a crystalline form, and are therefore generally referred to as glycine crystals 130 even once crushed.
In addition to the alignment of the biodegradable fibers 140, some embodiments provide the glycine crystals 130, or the crystals in particulate form 190, in alignment as well. As shown in
While ratios of the biodegradable polymer 120 to the glycine 130 may be varied in different embodiments, in the embodiment shown in
As can be seen in, e.g.,
The composite material 100 provided herein may have an elastic modulus of about 10 MPa to about 2,000 MPa.
As shown in
In preparing the biodegradable fibers 140 of the composite material 100 described herein, in some embodiments, electrospinning processes, such as that shown in
The electrospinning, shown in
Accordingly, as described in more detail below and as shown in
During dispensing of the mixture 150 from the needle 160, a voltage is applied by the power source 180 between the needle and collector 170. The voltage may be 14 kV, as noted elsewhere herein, and may typically be between 10 and 25 kV. In addition to the voltage applied by the power source 180 and the rotation speed of the collector drum 170, other variables may be controlled as well. For example, in some embodiments, the electrospinning may be performed at a flow rate of about 2 ml/h from the needle 160. The environment may be maintained at a humidity of about 30% to about 50%.
Especially, it is demonstrated that the glycine-PCL nanofiber device 100 produces a significant level of ultrasound to transiently open the BBB and deliver a drug model (dextran) deep into the brain tissue, exceeding the depth achieved by using a recently reported biodegradable PLLA ultrasonic transducer. By tailoring the encapsulation layer, the functional lifetime of the glycine ultrasound device can be controlled and optimized, which can be useful for different implant applications. Collectively, this highly piezoelectric, flexible, safe, and biodegradable glycine nanofiber film 100 could have a significant impact on medicine by offering many applications in different fields ranging from drug-delivery, tissue stimulation, cellular engineering to medical implant devices such as sensors, actuators, or transducers 2000.
It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the materials, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The materials and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the materials, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The ratios of the mass of any component of any of materials disclosed herein to the mass of any other component in the materials or to the total mass of the other components in the material are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.
Polycaprolactone (PCL, Polysciences MW 50000) solution, shown in
Hybrid electrospinning, as shown in
Samples were deposited on a standard SEM stub (Ted Pella) that was covered with carbon tape (Ted Pella) and then sputter-coated with Au/Pd (5 nm thickness) using a sputter coater (CCU-010, Safematic). The samples were then imaged using a Verios 460L SEM at 15 kV and 2500 magnifications. Data collection and analysis were carried out with xT microscope Control software. ImageJ software (NIH) was used for quantitative analysis of alignment and porosity.
A 3 μl aliquot of the glycine solution was deposited onto carbon-coated copper grids. Samples were adsorbed for 2 min. and rinsed with 100 μL of 0.5% uranyl acetate aqueous solution. Excessive uranyl acetate was blotted with filter paper and grids were air dried. Grids were visualized by using a FEI Tecnai Biotwin TEM (FEI, Eindhoven, Netherlands) operating at 80 KV.
FTIR spectra were acquired by Nicolet Magna 560 (Thermo Fisher Scientific) and attenuated total reflectance (ATR) ZnSe. The samples were cut 10 mm×10 mm and casted directly onto the ATR crystal. The measurements were performed and analyzed at several areas. Prior to each experiment, the diamond lens was cleaned using acetone, followed by deionized water. The FTIR spectra were background subtracted and baseline corrected with OMNIC software v.8.3.103.
XRD measurements of nanofiber films 100 were carried out at room temperature using a Bruker D2 Phaser instrument equipped with a high-speed linear detector and Cu-Kα radiation (λ=1.54184 Å). Diffractograms were recorded from 10° to 50° (2θ) with a scan speed of 0.2°/min. The patterns were compared with the ICDD powder diffraction file (PDF) database (PDF 00-032-1702 for α-glycine, 00-002-0171 for β-glycine, and 00-006-0230 for γ-glycine) for phase analysis.
2D WAXS frames were acquired at room temperature using an Oxford Xcalibur diffractometer equipped with a CCD detector and Cu-Kα radiation (λ=1.5418 Å) at 40 kV and 40 mA. Scattering data were processed by CrysAlisPro software.
The stress-strain relationships and tensile moduli of the films 100 were measured by Instron 1350 uniaxial tensile tester that was equipped with a 2525 Series Drop-through 100 N load cell and pneumatic grips. All the samples were prepared according to the ASTM D3822 standard. Data collection and analysis were carried out with Bluehill v.3 software.
The films 100 were cut 1 cm×1 cm and each piece was placed in a separate well of a 6-well plate and the well was filled with 5 mL of PBS (Gibco, pH 7.4) or DMEM (Gibco). The 6-well plates were placed on a hotplate (Fisherbrand) at 37° C. and imaged every 10 days.
Actuation Test (I.E., Film's Displacement Under Applied Electrical Field) was Performed By sandwiching the films between Al foil electrodes and half of the film/Al foil squares were encapsulated in polyimide tape (DuPont). The exposed Al foil electrode leads were then reinforced using copper tape (Ted Pella). The fabricated devices were firmly fixed on an Al beam in the actuation system using polyimide tape. Sinusoid waveforms were applied to the actuators from a function generator (BK Precision, 4054B) with varied voltage (20 V peak-to-peak) and frequency (1-4 Hz). The magnitude of the PLLA actuator's displacement was measured at the center of the exposed films using a laser displacement system (Keyence LK-G37). The laser was connected to a controller (Keyence, LKGD500), that sends the displacement data to a computer via USB. The data was then interpreted using LK Navigator software v. 1.6. Any drift in the measured signal was removed using a baseline function in Matlab 2018b.
Grinded glycine crystals 130, 190 were mounted on a Mo plate and placed between a grounded substrate and a high voltage (20 kV) needle on the top at 5 cm. Ionized air particles around the needle were accelerated and bombard towards the sample surface, causing electric field across the substrate. The surface potentials of glycine crystals 130 were measured by using a non-contact electrostatic voltmeter (ESVM-NC) at a relative humidity of 30%. Later, the samples (n=5 for each group) were positioned on an anti-static mat (Bertech, 1059-2X3BKT) to increase the accuracy of the measurement. The voltmeter was grounded and was held 5 mm from the sample surface.
A laser vibrometer (Polytec, PSV-I-500), mounted on an optical table and controlled with PSV 9.4 H-Acquisition software, was used for this experiment to measure the out-of-plane velocity of the films. All samples were held flat during the experiment using a pair of custom metal clamps designed to bolt into an optical table. The clamps were secured using metal bolts, which were tightened with a torque wrench (Neiko, 03727A) set to 1 Nm. A chirp waveform was generated by the vibrometer. The amplitude of the voltage waveform from the vibrometer was increased using a voltage amplifier (Piezo.com, EPA-104). The voltage output of the amplifier was then connected to a 50Ω splitter, with one output of the splitter connected to the device with a 50Ω BNC to alligator clip cable. The second output of the splitter connected to an RTM 30004 oscilloscope (Rhode and Schwarz) to ensure the voltage applied to all samples was at 37±1 Vpp. The vibrometer settings were as follows: the vibrometer scanned for vibration frequencies from 100-2000 Hz, applied a chirp waveform with frequencies of 100-2000 Hz, and had a resolution of 20 mm/s and a fast Fourier transform (FFT) size of 800.
A capsule hydrophone (HGL-0400, Onda Corp) and the transducers were mounted and immersed in a deionized (DI) water tank. DI water was selected as a testing medium because its acoustic impedance value is close to human tissue. The hydrophone was placed 2 cm away from the transducer and was aligned with the transducer to maximize the harvested signal. A continuous sinusoidal wave at 1 MHz with the amplitude of 0.1 Vrms from the function generator (BK Precision, 4054B) then amplified by an RF power amplifier (E&I RF Amplifier 1040L) was applied to the transducer. The acoustic pressure was recorded by an 8-channel oscilloscope (PicoScope 4000) as an output voltage. These voltage values were converted to acoustic pressure based on the calibrated sensitivity of the hydrophone.
Mouse adipose-derived stem cells (mADSC, iXCells Biotechnologies) were cultured in Dulbecco's Modified Eagle's Medium supplemented (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) at 37° C. and 5% CO2. At 80-90% confluence of passage 4, the cells were detached from the flasks using Trypsin/EDTA (Gibco) and seeded onto the electrospun films at a seeding density of 5×106 cells/mL. After 48 h, the cells were stained with a LIVE/DEAD™ cell imaging kit (Invitrogen). Live/dead images were taken using Leica SP8 confocal laser microscope (Leica Microsystems, Germany) with excitation/emission 488 nm/515 nm and excitation/emission 570 nm/602 nm.
mADSCs were purchased and cultured. The cells were incubated with the electrospun films for 22 h. Next, the medium was removed, and the cells were trypsinized and prepared for flow cytometry analysis on the BD LSRFortessa X-20 cytometer. Flow data were acquired and analyzed with BD FACSDive and FlowJo software. LIVE/DEAD™ viability/cytotoxicity kit (Invitrogen) was used for flow cytometry. The representative flow cytometry gating strategy used in this study is provided in
The electrospun mats 100 were cut into 5 mm×5 mm films. Encapsulating PLA films 2030 were obtained from compression molding using a Carver Press (3850-1011) at 200° C. and quenching using dry ice. Two 5 mm×5 mm squares attached to 1 mm wide wires were cut out of a 25 μm thick sheet of Molybdenum (ESPI Metals) using a pair of scissors. The electrodes 2010, 2020 were then hot embossed into PLA using the Carver Press at 110-130° C. The films 100 were sandwiched between the two electrodes 2010, 2020 that were embossed in PLA, and verification of resistance was done using a multimeter (Extech, MN35). All of the edges of the encapsulator were then sealed three times using a thermal bag sealer (ULINE, H-161) for 5 seconds.
All animal care and procedures were approved by the University of Connecticut Institutional Animal Care and Use Committee (IACUC). The experiment was conducted on 6 male CD-1 mice (Charles River) which were divided into three groups (n=2). The transducers were sterilized in 70% ethanol for 30 minutes and then exposed to UV light for another 30 minutes. A 5 mm diameter craniotomy defect was created on left side of the midline suture of the mouse anesthetized with ketamine (Covetrus)/xylazine (Covetrus). A drilling tool (Stoelting) was used to cut through the bone while keeping the dura intact. A 5 mm×5 mm single element ultrasound transducer 2000 was implanted on the craniotomy in the mouse's skull. The mouse was allowed to rest for 10 minutes after the defect was sutured. A bolus of 25 μL ultrasound contrast agent (VisualSonics, VS-11694) that contained microbubbles (2× 108 bubbles/mL) was injected into the tail vein of the mice approximately 5 minutes before the sonication. The transducer was operated at 1 MHz, burst rate at 10 Hz with a duty cycle of 20%, generating an acoustic pressure at 0.3 MPa in a series of two shots lasting 30 seconds each with a 30 second break between each shot. The animal received dextran (10 kDa, FITC-Lysine Fixable, Thermo Fisher) 5 minutes after the sonication process. Specifically, 100 μL of dextran was retro-orbitally injected on each eye of the mice (200 μL in total for an animal). After the injection, the mice were allowed to rest for an hour before being transcardially perfused.
Mice were anesthetized with an intraperitoneal injection of ketamine/xylazine. Following exposure of the heart by left anterolateral thoracotomy, the mouse was transcardially perfused (via the left ventricle) first with 20 mL of saline, to flush out the blood, and then with 10 mL of 4% paraformaldehyde (PFA). Each brain was removed carefully from the skull, and post-fixed in 4% PFA overnight at 4° C. The samples were cryoprotected in 15% sucrose and then in 30% sucrose solution until it sunk to the bottom of the container prior to freeze-embedding. The brains were placed in O.C.T. (optimal cutting temperature, Fisher HealthCare) compound and rapidly frozen to −80° C. Subsequently, 30 μm coronal cryosections were obtained using a Leica cryostat (maintained at −25° C.) on precleaned microscope slides (Epredia).
Sections were permeabilized with 0.5% Triton X-100 in PBS for 40 min, and nonspecific binding blocked by incubation with 1× Powerblock® (BioGenex) in UltraPure™ distilled water (Gibco) for 10 minutes. To identify blood vessels, the microvascular endothelium was stained using rat anti mouse CD31 (BD Pharmingen; 1:150 dilution), followed by incubation with goat anti-rat Alexa® 555 (Life Technologies; 1:300) as secondary antibody. The sections were washed in PBS prior to mounting in Mowiol®. Confocal images were acquired using a Leica SP8 confocal microscope.
The acoustic pressure generated by the devices was recorded by the hydrophone in the transmitting experiment. The device was encapsulated in 90 μm, 160 μm, and 260 μm thick PLA layers. They were immersed in PBS at 37° C. for degradation testing. The devices were removed, then, rinsed with deionized water and the acoustic pressure, under the input condition used for its initial calibration, was measured every 24 hours. This process was repeated with each device until failure. Subsequently, the samples were left in PBS for degradation analyses.
Glycine-PCL integrated nanofibers 140 include a PCL matrix 120 filled with aligned glycine crystals 130 (
Since β-glycine is the most piezoelectric phase of glycine, β-glycine needle-shaped crystals 130 were grown using slow evaporation technique, as shown in
Accordingly, while the embodiments shown and described are generally presented in terms of PCL as the biodegradable polymer used 120, a wide variety of such polymers are contemplated, including poly (
A method for making and using the biodegradable piezoelectric composite material 100 is outlined in
The method may then proceed with mixing a solution (5320) that includes a biodegradable polymer 120, separately retrieved (5330), the glycine crystal 130, either in crystal or particle 190 form, and at least one solvent. In some embodiments, it is noted that the glycine particles 190 themselves are elongate crystals 130. As discussed elsewhere herein, the orientation of such elongate crystals 130 may be aligned in the resulting fibers 140. A PCL solution 200 is shown in
As shown in
The glycine crystals 130 may be β-form crystals.
Images of stained glycine particles 190 and a stained PCL matrix 120 provide an insight into the distribution profile of glycine. As seen in
To determine the crystal phases, X-ray diffraction (XRD) was performed on glycine-PCL nanofibers 120 (as shown in
As described, glycine crystals 130 and solvent-casting polymeric film of these crystals are less flexible, rendering them incompatible for practical use in flexible, soft, and implanted devices. A comparison of the flexibility of films manufactured utilizing both techniques is shown in
Another advantage, in some scenarios, of the glycine-PCL nanofiber film 100 described herein is its ability to degrade completely when immersed in aqueous solutions, including buffers like phosphate buffered saline (PBS), and biofluids such as Dulbecco's modified eagle medium (DMEM). To illustrate various degradation stages of the nanofiber film,
In addition to the degradation study, contact angle measurements were used to characterize the surface wetting properties of PCL 120 and glycine-PCL films 100. It was found that glycine crystals 130 do not considerably modify the wetting properties of the PCL matrix 120, and the glycine-PCL film 100 is slightly hydrophobic, as shown in
Piezoelectricity in organic materials stems from alignment of molecular dipoles. This can be achieved through stretching (drawing) or application of a high external electrical field. Besides, piezoelectric property can be modulated by maximizing the macroscopic alignment of nanofibers over the entire electrospun mat 100. Following these strategies, hybrid electrospinning of glycine particles 130, 190 mixed with PCL solution at high voltage and high speed of the collector 170 in the electrospinning system, shown in
To gain insights into how alignment, both at the fiber 140 and crystal 130 levels and under applied electrical field 180, can enhance piezoelectric output, glycine-PCL films 100 were fabricated using the following three methods: solvent-casting 4500, shown in
Insight into the crystal alignment was obtained through a wide angle X-ray scattering (WAXS) analysis. As the crystals inside the film become more oriented, the WAXS pattern changes from full Debye rings indicating random crystal distribution to partial rings evidencing crystal alignment (
To evaluate the piezoelectric effect in the glycine-PCL nanofiber mat 100 for actuation performance (which is relevant to the ultrasound application presented later), an alternative electric field (1-4 Hz) was applied to the film 100 sandwiched between two metal electrodes 2010, 2020, which may be aluminum in a test scenario, while the displacement was measured by a laser displacement sensor (i.e., actuation test). The dynamic electrospun sample (4,000 rpm) exhibits an outstanding displacement of ˜12 μm, while the solvent-cast and static electrospun (0 rpm) samples with the same thickness exhibit no measurable displacement (
The strong piezoelectric response displayed by glycine-PCL nanofibers 140 in
To certify that the piezoelectricity of glycine-PCL nanofiber 140 arises from the glycine crystals 130 and not the PCL matrix 120, the piezoelectric effect of PCL and glycine-PCL fibers 140 were characterized using a laser scanning vibrometer to identify the vibration of the films 100 under applied voltages. Indeed, the glycine-PCL film 100 was clearly demonstrated, whereas bare PCL film 120 does not show any movement (
In addition to actuation test, it was demonstrated that the same glycine-PCL film 100 can be used to generate ultrasound waves. To do so, an ultrasonic transducer was fabricated by sandwiching the glycine-PCL nanofiber film 100 between two aluminum (Al) electrodes 2010, 2020 and then encapsulating it with a polyimide tape. During the ultrasound transmission testing, a capsule hydrophone was used to measure the acoustic pressure. Glycine-PCL device was driven by a function generator to produce a continuous ultrasonic wave at 1 MHz. As presented in
In order to compare the piezoelectric performance of glycine-PCL nanofibers 140 with state-of-the-art biodegradable piezoelectric materials, an ultrasound transmitting test was employed for electrospun PLLA and recently developed solvent-cast glycine-PVA films. The two way ANOVA results clearly demonstrate the superior performance of glycine-PCL nanofiber film compared to the others for actuation applications such as ultrasonic transducers (
The biocompatibility of glycine-PCL film 100 was examined through two different tests. First, cell imaging assay was used to visualize the effect of the film on the viability of mouse adipose-derived stem cells (mADSC). The mADSCs were seeded on the films at 37° C. for 48 h. Live cells (green stain, calcein AM, shown as outlines in the drawings) are distinguished by the presence of intracellular esterase activity, while dead cells (red stain, BOBO™-3 Iodide, shown as dark dots in the drawings) are detected by the presence of nucleic acids in damaged membranes. As shown in
Second, to quantify the number of viable cells in a large number of cells, and further certify the safety of glycine-PCL mat 100, flow cytometry assay was performed with the same mADSCs. All groups (n=5) exhibited a normal behavior, evidencing the non-cytotoxic nature of glycine-PCL film (
As an example of the usage of the glycine-PCL piezoelectric film in an implantable device,
The ultrasonic transducer 2000, made of the composite material 100 discussed herein, such as the piezoelectric glycine-PCL mat described, further comprises a first metal electrode 2010 adjacent the composite material 100 and a second metal electrode 2020 also adjacent the composite material 100 and opposite the composite material from the first metal electrode 2010. The metal electrodes 2010, 2020 may be formed from molybdenum (Mo). The electrode 2000 then further includes an encapsulation layer 2030 covering the first metal electrode 2010, the second metal electrode 2020, and the composite material 100. The encapsulating layer 2030 typically comprises a biodegradable polymer.
The electrodes 2010, 2020, corresponding wires 2040, 2050 coupled to the electrodes, and encapsulating layers 2030, which may be formed from polylactic acid (PLA) layers, can all safely degrade inside the body after a controllable time to avoid any invasive removal surgeries which are often required for conventional ultrasonic transducers (e.g., Sonocloud37) (
During use, the ultrasonic transducer 2000 described may be provided with the first metal electrode 2010 and the second metal electrode 2020 electrically coupled to an ultrasonic generator by way of the corresponding wires 2040, 2050.
The ultrasonic transducer 2000 described may then be used to deliver a therapeutic through a blood-brain barrier in a subject in need thereof. During use, the ultrasonic transducer 2000 is applied to a defect, such as a craniotomy defect, of the subject. The method then proceeds transmit an ultrasonic wave signal through the wire 2040, 2050 from the ultrasonic generator (not shown). The ultrasonic generator is then used to deliver a pulsed acoustic pressure to the craniotomy defect by way of the transducer 2000. In some embodiments, the ultrasonic wave signal is driven at about 1 MHz to about 5 MHz.
During treatment, the method may further include administering to the subject a therapeutic by way of an IV after transmitting the ultrasonic wave signal.
The perception of ultrasonic transducers for BBB disruption is schematically illustrated in
The brains were processed for immunofluorescence analysis to detect the leakage of dextran which penetrated the disrupted BBB. Two coronal brain regions, superficial and deep, which are 2 mm apart were imaged (
As seen in
Furthermore, in order to examine the penetration depth of the generated ultrasound into the brain tissue, a 2 mm region below the brain surface is imaged. As seen in
Finally, a functionality experiment was conducted to determine the operational lifetime of a transducer 2000 encapsulated with 90-260 μm thick layers of PLA 2030 and demonstrated the device has a lifetime of 10-25 days in PBS at 37° C. depending on the encapsulation thickness (
Accordingly, as further illustrated in
In doing so, a user may, in some embodiments, first determine an idealized functional lifetime (5380) of the biodegradable piezoelectric composite material 100. Such an idealized functional lifetime may depend on a use case for the biodegradable piezoelectric composite material 100. Accordingly, the functional lifetime may be based on a projected treatment process for a patient or subject (5390). Once an idealized functional lifetime is selected, the user may select a thickness (5400) for the encapsulating layer based on the idealized functional lifetime.
As noted above, following the encapsulation of the transducer 2000 in an encapsulating layer 2030, the ultrasonic transducer may be implanted (5400) adjacent a craniotomy defect 2410 of a subject as part of a method of therapy. Such a method may then proceed with transmitting (5410) an ultrasonic wave signal through one or more wire 2040, 2050 in electrical communication with the first or second electrode 2010, 2020. It is understood that although the method describes the actuation by way of an ultrasonic wave signal transmitted through a wire, other forms of actuation are contemplated as well.
In some embodiments, after transmitting the ultrasonic wave signal, the method may proceed with administering to the subject a therapeutic intravenously (5420).
Further, in some embodiments, prior to administering the therapeutic (at 5420), the subject may be injected with microbubbles (5415), which may increase the permeability of the BBB, as discussed above.
Disclosed is a unique biodegradable, soft and highly piezoelectric nanomaterial platform, made of glycine crystals 130 embedded inside a biodegradable matrix of PCL 120. While glycine crystals 130 are known to be safe and possess an attractive ultrahigh piezoelectric effect, there has not been any success to create the crystals in a flexible, easy-to-handle and stable form for useful device applications. Here, electrospinning processes were used to fabricate a soft, biodegradable, and stable glycine-embedded PCL nanofibers 140 and employ the nanofibers to create an exceptional biodegradable and powerful implantable ultrasound transducer 2000 for a significant application of blood-brain drug delivery. It was also demonstrated that the glycine-PCL nanofiber device 2000 exhibited an excellent actuation and ultrasonic performance, superior to those of the state-of-the-art biodegradable PLLA piezoelectric device that has been recently reported. Using only medical safe materials, the glycine-PCL nanofiber ultrasound device was created that is safe for cell culture and animal implantation. After a controllable and pre-defined lifetime, the glycine-PCL nanofiber device 2000 can self-degrade which is significant for implant application to avoid risky and invasive removal surgeries. Especially, the powerful piezoelectric performance of the nanofibers allowed the glycine-PCL ultrasonic devices, superficially implanted on the brain, to trigger the BBB opening even in deep brain regions. This ability could be important to the application of deep brain stimulation/modulation or the treatment of cancerous/diseased tissues deep inside the brain. Besides medical applications, the excellent piezoelectricity of this flexible, easy-to-use, and safe glycine platform could also enable the creation of “green” sensors, actuators, and transducers to replace the common lead-based or non-degradable piezoelectric devices which are used extensively in industry, and harmful to human and the environment. Overall, the disclosed glycine-PCL nanofiber platform offers a powerful and safe amino acid based piezoelectric nanomaterial which could bring about significant medical implant applications such as soft biodegradable sensors, actuators, or transducers.
While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention. Furthermore, the foregoing describes the invention in terms of embodiments foreseen by the inventor for which an enabling description was available, notwithstanding that insubstantial modifications of the invention, not presently foreseen, may nonetheless represent equivalents thereto.
This application is a continuation of International Patent Application No. PCT/US2023/016073, filed on Mar. 23, 2023, which takes priority from U.S. Provisional Patent Application No. 63/324,418, filed on Mar. 28, 2022, the entire contents of which are hereby incorporated by reference.
This invention was made with government support under grant number 1635103 awarded by the National Science Foundation. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63324418 | Mar 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/016073 | Mar 2023 | WO |
| Child | 18888959 | US |
