Hemostatic compositions and related methods

Abstract

A composition comprising a plurality of particles that can be prepared from chitosan salt and a method for preparing the same. The plurality of particles can have an average diameter in the range of about 100 μm to about 750 μm, for example, about 150 μm to about 500 μm, a steepness value in the range of about 30 to about 90, and an average aspect ratio of greater than 0.6. The plurality of particles can be, for example, substantially spherical. The preparation method includes electrospraying a chitosan salt solution through a needle.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to therapeutic materials and related methods of preparation and treatment. More particularly, the present invention comprises hemostatic materials for treating tissue. [Background technology]

[0002] Hemostatic agents are used to treat bleeding from internal and external injuries to prevent medical problems such as blood loss and infection. To treat internal injuries, such as gastrointestinal bleeding, hemostatic agents can be applied via appropriate medical devices. However, delivering such agents to target sites within the body presents challenges. Summary of the Invention [Means for solving the problem]

[0003] The present invention includes therapeutic materials and methods for their preparation and treatment. For example, the present invention includes compositions containing a plurality of particles that can be prepared from a polysaccharide salt, such as a chitosan salt. Thus, for example, the plurality of particles can contain chitosan. The chitosan of the particles can include, or be in the form of, for example, chitosan with a crosslinker (e.g., a crosslinker for chitosan) and / or a chitosan salt. According to some embodiments, the plurality of particles includes chitosan and a crosslinker, and the plurality of particles can be prepared from a chitosan salt. The particles may have an average diameter (average CE diameter) in the range of about 100 μm to about 1 mm, e.g., about 100 μm to about 750 μm, about 150 μm to about 500 μm, about 250 μm to about 900 μm, about 250 μm to about 500 μm, about 400 μm to about 600 μm, about 550 μm to about 750 μm, or about 300 μm to about 450 μm. The particles may have a steepness value in the range of about 30 to about 90, e.g., about 50 to about 80, about 70 to about 90, or about 60 to about 80. Additionally or alternatively, the particles may have an average aspect ratio greater than 0.6, greater than 0.7, or greater than 0.8. For example, the particles may be approximately spherical. The average maximum distance of the plurality of particles may be, for example, within a range of about 500 μm to about 700 μm.

[0004] According to some embodiments of the present invention, the plurality of particles can have an average diameter in the range of about 150 μm to about 500 μm, an average aspect ratio greater than 0.6, and optionally a steepness value in the range of about 30 to about 90. For example, the plurality of particles can have an average diameter in the range of about 150 μm to about 500 μm, an average aspect ratio greater than 0.7, and optionally a steepness value in the range of about 50 to about 80. Further, for example, the plurality of particles can have an average diameter in the range of about 150 μm to about 500 μm, an average aspect ratio greater than 0.6, and optionally a steepness value in the range of about 60 to about 80. In at least one example, the plurality of particles has an average diameter in the range of about 300 μm to about 450 μm, an average aspect ratio greater than 0.6, and optionally a steepness value in the range of about 30 to about 90. Further, for example, the plurality of particles can have an average diameter in the range of about 300 μm to about 450 μm, an average aspect ratio of greater than 0.7, and optionally a steepness value in the range of about 30 to about 90. In at least one example, the plurality of particles can have an average diameter in the range of about 300 μm to about 450 μm, an average aspect ratio of greater than 0.7, and optionally a steepness value in the range of about 50 to about 80. Further, for example, the plurality of particles can have an average diameter in the range of about 300 μm to about 450 μm, an average aspect ratio of greater than 0.8, and optionally a steepness value in the range of about 30 to about 90.

[0005] The bulk density of the plurality of particles can be in the range of about 0.05 g / mL to about 0.7 g / mL, e.g., about 0.1 g / mL to about 0.7 g / mL, about 0.2 g / mL to about 0.7 g / mL, about 0.05 g / mL to about 0.4 g / mL, about 0.3 g / mL to about 0.5 g / mL, or about 0.1 g / mL to about 0.2 g / mL. For example, the plurality of particles can have an average diameter in the range of about 150 μm to about 500 μm, an average aspect ratio of greater than 0.6, optionally a steepness value in the range of about 30 to about 90, and a bulk density in the range of about 0.1 g / mL to about 0.7 g / mL. Further, for example, the plurality of particles can have an average diameter in the range of about 150 μm to about 500 μm, an average aspect ratio of greater than 0.7, optionally a steepness value in the range of about 30 to about 90, and a bulk density in the range of about 0.3 g / mL to about 0.5 g / mL. The plurality of particles can have a porosity of greater than 80% as measured by mercury intrusion porosimetry, e.g., a porosity in the range of about 85% to about 95%. In some examples herein, the plurality of particles have an average diameter in the range of about 150 μm to about 500 μm, an average aspect ratio of greater than 0.6, and optionally a steepness value in the range of about 30 to about 90, a bulk density in the range of about 0.1 g / mL to about 0.7 g / mL, and a porosity of greater than 80% as measured by mercury intrusion porosimetry. For example, the particles may have an average diameter in the range of about 150 μm to about 500 μm, an average aspect ratio of greater than 0.7, and optionally a steepness value in the range of about 30 to about 90, a bulk density in the range of about 0.1 g / mL to about 0.7 g / mL, and a porosity in the range of about 85% to about 95% as measured by mercury intrusion porosimetry.

[0006] In some examples, chitosan salts that can be used to prepare particles include chitosan acetate, chitosan lactate, chitosan succinate, chitosan glutamate, chitosan glycolate, or chitosan citrate. The compositions herein can include a plurality of particles comprising chitosan and a crosslinker, and optionally, the crosslinker comprises a tripolyphosphate, such as sodium tripolyphosphate. Additionally, the compositions can further include a fluid, such as a liquid. Additionally or alternatively, the compositions can be in the form of a powder.

[0007] Also disclosed herein is the use of a composition as a hemostatic agent, which may be the composition described above and / or elsewhere herein. The present invention also includes a method for preparing the above-described compositions and / or compositions described elsewhere herein, the method comprising electrospraying a chitosan salt solution through a needle. Electrospraying the chitosan salt solution through a needle can produce a plurality of particles containing chitosan. For example, electrospraying the chitosan salt solution through a needle into an aqueous solution containing a crosslinker can produce a plurality of particles containing chitosan and a crosslinker. The chitosan salt solution used to prepare the particles can have a viscosity at 25°C within the range of about 1,500 mPa·s (1,500 cPs) to about 8,000 mPa·s (8,000 cPs), e.g., about 1,500 mPa·s (1,500 cPs) to about 3,000 mPa·s (3,000 cPs), and can additionally or alternatively contain about 0.5 wt% to about 10 wt% chitosan, based on the volume of the solution. Electrospraying can include applying a voltage of about 9 kV to about 25 kV or about 10 kV to about 30 kV to a needle. The needle can be, for example, a 21-gauge needle, a 22-gauge needle, or a 23-gauge needle. The method can include electrospraying a chitosan salt solution into an aqueous solution including a crosslinker, such as sodium tripolyphosphate; lyophilizing the plurality of particles, where lyophilization can be performed for a time period ranging from 30 minutes to 48 hours, e.g., from 2 hours to 12 hours or from 4 hours to 6 hours; and rinsing the plurality of particles with a liquid after electrospraying and / or before lyophilization. In some examples, the liquid can include water, alcohol, or a mixture thereof.

[0008] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various exemplary embodiments and, together with the detailed description, serve to explain the principles of embodiments of the invention. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is an exemplary schematic diagram illustrating an electrospray system according to some embodiments of the present invention. [Figure 2] 10A-10C are images showing chitosan particles subjected to different drying treatments, as described in Example 2. [Figure 3] 10A-10C are images showing chitosan particles subjected to different drying treatments, as described in Example 2. [Figure 4] 10A-10C are images showing chitosan particles subjected to different drying treatments, as described in Example 2. [Figure 5] 1 is an image showing chitosan particles according to some embodiments of the present invention, as described in Example 3. [Figure 6] 1 is an image showing chitosan particles according to some embodiments of the present invention, as described in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0010] Certain aspects of the invention are described in more detail below. In the event of a conflict with terms and / or definitions incorporated by reference, the terms and definitions provided herein shall control. As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, composition, article, or device that includes a list of elements does not include only those elements, but may include other elements not expressly listed or inherent in such process, method, composition, article, or device. The term "exemplary" is used in the sense of "example" rather than "ideal."

[0011] As used herein, the singular forms "a," "an," and "the" include plural references unless the context dictates otherwise. The terms "approximately" and "about" mean nearly the same as a referenced number or value. As used herein, the terms "approximately" and "about" should be understood to encompass the specified amount or value ±5%.

[0012] Embodiments of the present invention include materials and related methods for preparing hemostatic agents or compositions thereof. The hemostatic agents herein may comprise one or more compounds, including one or more biomaterials derived from biocompatible and / or biological materials.

[0013] The hemostatic agents herein may include particles, such as particles having a controlled size and / or shape. The particles may be prepared by a customized (modified) electrospraying process using parameters selected to produce particles with desired properties. Electrospraying generally refers to a technique for generating droplets from a fine liquid aerosol via electrostatic charging. In the modified electrospraying process of the present invention, an electric field is used to force a liquid (e.g., a chitosan salt solution) through a needle, thereby breaking up the liquid stream and forming individual droplets. Thus, for example, embodiments of the present invention do not generate aerosols like traditional electrospraying processes. Once particles are formed by electrospraying, they may be dried for immediate or future use. The parameters and / or drying method used for electrospraying may produce particles with a controlled size and / or shape compared to particles prepared by other methods. For example, the hemostatic agents herein may include approximately spherical particles with a relatively narrow size distribution.

[0014] The compositions herein may include natural materials, such as natural polymers, that have the ability to act as hemostatic agents to reduce, stop, and / or prevent tissue bleeding. Some particulate materials can act as good mechanical barriers and coagulants, but the size and shape of their particles can adversely affect the delivery of such compounds and compositions thereof. For example, compounds with relatively large particle sizes and / or fragmented particle shapes can clog medical devices, making it difficult to deliver the hemostatic agent to the target site.

[0015] Without being bound by theory, it is believed that selection of desired parameters for electrospraying and the modified electrospraying process of the present invention, and / or further processing of the particles after electrospraying, can produce particles with desired properties, e.g., particle size, bulk density, and / or shape, to facilitate delivery to the target site for effective hemostatic treatment.

[0016] The compositions herein may include one or more materials that act as hemostatic agents. The hemostatic agent may be natural or derived from natural materials. In at least one example, the hemostatic agent may be charged, such as cationic. For example, the hemostatic agent may include one or more polysaccharides, such as chitosan and / or derivatives thereof, including, but not limited to, thiolated chitosan, PEGylated chitosan, catechol-modified chitosan, and carboxymethyl chitosan, starch (e.g., potato starch or other vegetable starch), cellulose, alginate, or combinations thereof. In some examples herein, the hemostatic agent includes chitosan or a derivative thereof, optionally in combination with one or more other hemostatic agents.

[0017] Chitosan is a linear polysaccharide formed from glucosamine units derived from chitin, a structural component of the exoskeleton of crustaceans:

[0018] [ka] Chitosan is typically prepared by deacetylating chitin with an alkaline reagent such as sodium hydroxide to produce a water-soluble material. Chitosan is antimicrobial and cationic, and has natural bioadhesive properties that allow it to bind to negatively charged surfaces, such as mucous membranes, and concentrated blood components, such as red blood cells, to promote coagulation. The hemostatic compositions herein may include chitosan. For example, the compositions may include chitosan with a crosslinker (e.g., particles of a composition containing crosslinked chitosan) and / or chitosan in the form of a biocompatible salt. Exemplary crosslinkers for chitosan include tripolyphosphates, such as sodium tripolyphosphate (TPP). Without being bound by theory, it is believed that crosslinking of chitosan and tripolyphosphate through ionic interactions involves the positively charged amine groups (e.g., protonated amine groups) of chitosan interacting with the negatively charged phosphate groups of the tripolyphosphate to form a crosslinked structure. Exemplary chitosan salts of the compositions herein and / or that may be prepared and used in the electrospraying processes herein include, but are not limited to, chitosan acetate, chitosan lactate, chitosan succinate, chitosan glutamate, chitosan glycolate, and chitosan citrate.

[0019] Particles of the hemostatic agents herein can be prepared to have a desired particle size to facilitate administration to an internal bleeding site, such as via a catheter or other suitable medical device for endoscopic delivery. As described below, the preparation methods herein can provide particles with a relatively narrow size distribution to facilitate controlled flow of the particles through a suitable fluid medium (e.g., a liquid such as water, saline solution, other aqueous solutions, or a gas such as air, nitrogen, or oxygen).

[0020] Particle properties, including size parameters, can be characterized by morphological analysis, for example, by imaging, such as using optical microscopy. Statistical analysis of multiple particles can be performed by static automated imaging using an optical microscope. For example, a Malvern Morphologi 4 instrument can be used to analyze various particle parameters using static automated imaging with optical microscopy. Such particle properties that can be determined by optical microscopy include circle equivalent (CE) diameter, length, width, perimeter, area, maximum distance, sphere equivalent (SE) volume, and aspect ratio. Properties of multiple particles, including average values and particle distribution data, can be calculated from individual particle values (e.g., average CE diameter, average maximum distance, average aspect ratio, etc.).

[0021] As used herein and for purposes of the present invention only, the terms "average particle diameter" and "average diameter" refer to the average CE diameter of a plurality of particles, where the CE diameter is defined as the diameter of a circle having the same area as the particle. According to some examples herein, the particles of the hemostatic agent may have an average particle diameter within a range of about 100 μm to about 1 mm, e.g., about 100 μm to about 750 μm, about 150 μm to about 500 μm, about 250 μm to about 900 μm, about 200 μm to about 450 μm, about 250 μm to about 500 μm, about 250 μm to about 450 μm, about 250 μm to about 400 μm, about 300 μm to about 500 μm, about 300 μm to about 450 μm, about 550 μm to about 750 μm, or about 350 μm to about 450 μm. In some examples herein, the average particle diameter can be about 250 μm±60 μm, about 300 μm±60 μm, about 350 μm±60 μm, or about 450 μm±60 μm. Furthermore, for example, the particles herein can have a maximum distance within a range of about 100 μm to about 1.2 mm, e.g., about 150 μm to about 1 mm, about 200 μm to about 550 μm, about 275 μm to about 950 μm, about 250 μm to about 500 μm, about 275 μm to about 550 μm, about 300 μm to about 700 μm, about 350 μm to about 550 μm, about 500 μm to about 700 μm, or about 400 μm to about 650 μm. The term "maximum distance" of a particle as used herein refers to the maximum distance among the set of all linear distances between any two points on the periphery of the particle. According to some embodiments of the present invention, the average maximum distance of a plurality of particles can be in the range of about 400 μm to about 800 μm, e.g., about 450 μm to about 700 μm, about 500 μm to about 650 μm. To avoid confusion, each individual particle of the plurality of particles has a CE diameter and a maximum distance, and references to "average particle diameter" or "average diameter" of a plurality of particles refer to the average of the CE diameters of the particles, and references to "average maximum distance" of a plurality of particles refer to the average of the multiple maximum distances of the plurality of particles.

[0022] In at least one example, the particles have an average diameter (average CE diameter) in the range of about 400 μm to about 600 μm, and the average maximum distance of the particles is in the range of about 500 μm to about 700 μm. In at least one example, the particles have an average diameter (average CE diameter) in the range of about 450 μm to about 550 μm, and the average maximum distance of the particles is in the range of about 500 μm to about 650 μm.

[0023] d 50 The value is the particle CE diameter below which 50% of the particles have a smaller CE diameter (corresponding to the median particle diameter of the particle size distribution). 10 The value is the particle CE diameter below which 10% of the particles have a smaller CE diameter, d 30 The value is the particle CE diameter below which 30% of the particles have a smaller CE diameter, d 70 The value is the particle CE diameter below which 70% of the particles have a smaller CE diameter, and d 90 The value is the particle CE diameter below which 90% of the particles have a smaller CE diameter. According to some examples herein, the plurality of particles may have a d in the range of about 450 μm to about 850 μm, e.g., about 500 μm to about 750 μm, or about 600 μm to about 700 μm. 50 value, and / or d in the range of about 300 μm to about 600 μm, for example, about 400 μm to about 550 μm, or about 450 μm to about 500 μm. 10 value, and / or d in the range of about 500 μm to about 1 mm, for example, about 650 μm to about 950 μm, or about 800 μm to about 900 μm. 90 It has a value.

[0024] Information about the particle size range is given by the ratio d 30 / d 70 This ratio can be obtained from the steepness of the particle size distribution, which is defined as d × 100. 30 and d 70The steepness value can be calculated from the value of the mean particle size. A higher steepness value indicates less deviation from the average particle size. The particles herein can have a relatively high steepness value indicative of a relatively narrow size distribution, for example, a steepness value in the range of about 30 to about 90, e.g., about 50 to about 80, about 60 to about 90, about 70 to about 90, about 80 to about 90, about 75 to about 85, or about 60 to about 80.

[0025] The aspect ratio (ρ) of a particle provides information about its shape. The aspect ratio of two dimensions is defined as the length along the particle's width divided by its length and can be determined based on the particle's major and minor axes. The major axis is the axis passing through the particle's center of mass in an orientation corresponding to the shape's minimum rotational energy. The minor axis is the axis passing through the particle's center of mass at a right angle to the major axis. To determine the length of a particle, lines from all points on the particle's periphery are projected onto the major axis (the projected lines are parallel to each other and perpendicular to the major axis). The longest distance between the points where two of these line projections intersect the major axis is the length. Similarly, to determine the width of a particle, lines from all points on the particle's periphery are projected onto the minor axis (the projected lines are parallel to each other and perpendicular to the minor axis). The widest distance between the points where two of these projected lines intersect the minor axis is the width. An aspect ratio approaching 1 indicates a more uniformly rounded particle; any cross section of a sphere has an aspect ratio of 1. The aspect ratio of a particle can be determined by imaging (e.g., optical microscopy) and software capable of determining the minor axis, major axis, width, and length of each particle. The average aspect ratio of multiple particles can be determined by averaging the aspect ratios of the particles, such as by static automated imaging.

[0026] The particles herein can have a rounded, spherical shape that facilitates flow through a fluid medium within a delivery device. In some examples, the particles can have an average aspect ratio greater than 0.6, greater than 0.7, or greater than 0.8, e.g., in the range of about 0.6 to 1.0, about 0.7 to 1.0, or about 0.8 to 1.0. For example, the average aspect ratio can be in the range of about 0.7 to about 0.98, about 0.8 to about 0.95, or about 0.85 to about 0.95. For purposes of the present invention, the term "substantially spherical" refers to a rounded, three-dimensional shape having the general shape of a sphere. For example, any cross-sectional dimension passing through the center point of a substantially spherical particle varies by less than 10% compared to any other cross-sectional dimension passing through the center point of the particle. In other words, the longest cross-sectional dimension of a substantially spherical particle varies by less than 10% compared to the shortest cross-sectional dimension.

[0027] In at least one example, the particles have an average diameter (average CE diameter) in the range of about 400 μm to about 600 μm, an average maximum distance in the range of about 500 μm to about 700 μm, and / or an average aspect ratio in the range of about 0.7 to about 0.9. In at least one example, the particles have an average diameter (average CE diameter) in the range of about 450 μm to about 550 μm, an average maximum distance in the range of about 500 μm to about 650 μm, and / or an average aspect ratio in the range of about 0.7 to about 0.9. In these examples, the particles can be approximately spherical.

[0028] In at least one example, the plurality of particles have an average diameter (average CE diameter) in the range of about 400 μm to about 600 μm, an average aspect ratio in the range of about 0.7 to about 0.9, and / or a steepness value in the range of about 60 to about 90. In at least one example, the plurality of particles have an average diameter (average CE diameter) in the range of about 400 μm to about 600 μm, an average aspect ratio in the range of about 0.7 to about 0.9, and / or a steepness value in the range of about 70 to about 80. In at least one example, the plurality of particles have an average diameter (average CE diameter) in the range of about 450 μm to about 550 μm, an average aspect ratio in the range of about 0.7 to about 0.9, and / or a steepness value in the range of about 70 to about 80. In these examples, the particles can be approximately spherical.

[0029] The particles herein may also have a suitable bulk density to facilitate delivery to the target site. Lower bulk densities generally result in fluffier particles, which may be desirable for effective delivery. However, too low a bulk density may result in static conditions that hinder delivery. Therefore, to facilitate delivery to the target site, the particles herein may have a relatively low bulk density that is not too low to cause static conditions. In some examples, the bulk density of the particles is less than 0.7 g / L, e.g., from about 0.05 g / mL to about 0.7 g / L, from about 0.08 g / mL to about 0.126 g / mL, from about 0.1 g / mL to about 0.7 g / L, from about 0.2 g / mL to about 0.7 g / mL, from about 0.05 g / mL to about 0.6 g / mL, from about 0.2 g / mL to about 0.5 g / mL, or about 0.05 g / mL. The bulk density can be in the range of about 0.1 to about 0.5 g / mL, about 0.3 g / mL to about 0.6 g / mL, about 0.3 g / mL to about 0.5 g / mL, about 0.05 g / mL to about 0.4 g / mL, about 0.05 g / mL to about 0.3 g / mL, about 0.05 g / mL to about 0.2 g / mL, about 0.1 g / mL to about 0.2 g / mL, or about 0.2 g / mL to about 0.3 g / mL. Bulk density can be measured by filling a known volume with particles and weighing the powder (without compressing the material) to calculate the bulk density in g / mL.

[0030] Exemplary methods for preparing particles herein are described below with reference to Figure 1. Methods for preparing particles herein may include preparing a solution, electrospraying the solution to form particles, washing the resulting particles, and / or drying the particles. It is understood that one or more of these steps may be omitted. For example, the method may include preparing a solution, electrospraying the solution to form particles, drying the resulting particles (e.g., optionally washing the particles), or electrospraying the solution to form particles and washing and / or drying the resulting particles.

[0031] Preparing the solution can include combining a hemostatic agent, e.g., chitosan, with a suitable acid, e.g., one or more organic acids, e.g., acetic acid, lactic acid, succinic acid, glutamic acid, glycolic acid, citric acid, or a combination thereof. In at least one example, preparing the solution includes combining chitosan with citric acid and / or acetic acid. In some examples, the solution can include up to 10% chitosan by weight based on the total weight or volume of the solution, e.g., about 0.5% to about 10.0% chitosan by weight or about 0.5% to about 3.0% chitosan by weight based on the total weight or volume of the solution. For example, the solution may contain about 0.5% to about 7.5%, about 1.0% to about 6.0%, about 2.5% to about 5.5%, about 3.0% to about 6.5%, about 5.5% to about 9.5%, about 4.5% to about 7.0%, about 0.5% to about 2.5%, about 1.0% to about 2.0%, about 1.0% to about 3.0%, about 2.0% to about 3.0%, or about 2.5% to about 4.5% by weight of chitosan, based on the total weight or volume of the solution. In some examples, the solution may contain up to about 5% by weight of organic acid, e.g., about 0.5% to about 5.0% by weight, or about 1.0% to about 2.5% by weight, based on the total weight of chitosan. For example, the solution can include about 0.5% to about 7.0%, about 1.0% to about 5.0%, about 0.5% to about 3.0%, or about 1.0% to about 2.0% by weight of an organic acid (e.g., citric acid and / or acetic acid) based on the total weight of chitosan. The organic acid can be present as an aqueous solution, e.g., a citric acid solution, an acetic acid solution, etc. In at least one example, chitosan is combined with a solution including citric acid, acetic acid, and water (e.g., citric acid added to an aqueous acetic acid solution).The chitosan / organic acid solution (or other chitosan salt solution according to the present disclosure) may have a viscosity at 25° C. of less than 10,000 cPs, e.g., less than 8,000 cPs, less than 5,000 cPs, less than 3,500 cPs, less than 3,200 cPs, less than 3,000 cPs, less than 2,800 cPs, less than 2,500 cPs, less than 2,200 cPs, less than 2,000 cPs, less than 1,800 cPs, or less than 1,500 cPs, e.g., from about 1,000 cPs to about 10,000 cPs, from about 1,500 cPs to about 8,000 cPs, from about 1,500 cPs to about 7,500 cPs. cPs, about 2,000 cPs to about 5,000 cPs, about 3,000 cPs to about 7,000 cPs, about 1,000 cPs to about 4,000 cPs, about 2,500 cPs to about 5,500 cPs, about 1,000 cPs to about 3,200 cPs, about 1,200 cPs to about 1,500 cPs, about 1,200 cPs to about 2,000 cPs, about 1,400 cPs to about 1,800 cPs, about 1,500 cPs to about 3,000 cPs, about 1,500 cPs to about 2,500 cPs, or about 2,000 cPs to about 2,500 cPs. The viscosity can be measured with a viscometer. For example, viscosity at 25°C can be measured with a viscometer using a circulating water temperature of 25°C, a speed of 50 RPM (revolutions per minute), and testing a 12 mL sample for a total test time of 8 minutes, with the viscosity reported as the average of the viscosity measured in the last 3 minutes of the total test time. An exemplary device for measuring viscosity is a Brookfield viscometer DV2T Extra. Higher viscosities can be expected to increase the time to prepare particles. Depending on the viscosity, at least in some cases, the higher the viscosity, the more difficult it can be to achieve a consistent, uniform flow during electrospraying. Furthermore, in some cases, the viscosity can decrease over time, for example, facilitating electrospraying.

[0032] The chitosan / organic acid solution (or other chitosan salt solution according to the present disclosure) can then be passed through an electrospraying system. FIG. 1 illustrates an exemplary electrospraying system, in which a syringe 130 containing the solution is connected to a pump 120 (e.g., optionally part of an automatic injection system). The pump 120 can be, for example, a mechanical pump or a peristaltic pump. When the pump 120 is activated, the solution is pumped from the syringe 130, for example, via a connecting supply tube, to an electrospraying device, whereupon the solution is ejected through a needle 140 of the device, which acts as a nozzle. 1 illustrates one syringe 130 and one electrospray needle 140, the system may include two or more syringes and / or two or more electrospray needles to increase the amount of droplets and the volume of particles produced (e.g., each syringe connected to a corresponding needle, two or more syringes connected to one needle, one syringe connected to two or more needles in parallel or in series, etc.). The amount of droplets may also be increased by replacing the syringes with a pressure pot and using a pressurized system to pump the solution into the electrospraying device (e.g., using a mechanical or peristaltic pump).

[0033] Because the needle 140 is electrically charged, the solution passes through an electric field toward the collection unit 150 as it exits the needle 140. The electric field is generated by the applied voltage from the power supply 110 and the distance from the collection unit 150 to the needle 140. Electrode 190a connects the power supply 110 to the needle 140, and electrode 190b connects the power supply 110 to the collection unit 150. The solution may also be electrically charged to have a positive charge, for example, due to the positive charge of a hemostatic agent in the solution. As the solution flows through the needle 140, which functions as a nozzle, the solution breaks up into individual charged droplets. According to some examples herein, the amount of voltage applied can cause unstable droplets to form. For example, the charged droplets may exhibit a generally cone-shaped region 160, somewhat resembling a Taylor cone, but they do not form the stable Taylor cone characteristic of conventional electrospraying processes. Thus, for example, the electric field may guide unstable droplets toward collection unit 150 and break the surface tension of the liquid above needle 140. At the tip of region 160, the droplets (e.g., unstable droplets) form a fine jet spray. This jet spray also becomes unstable and breaks up into a mist of fine droplets represented as plume 170. The droplets repel each other and move toward the ground medium provided by collection unit 150. The particles thus generated are received by collection unit 150.

[0034] As described above, the parameters of the modified electrospraying process herein can be selected to produce particles of a desired size and shape. To change the particle size distribution, for example, the applied voltage can be increased to accelerate the flow of solution (droplet velocity of the solution) through the tip of the needle 140. This affects the stability of the droplets (e.g., due to the lack of a stable Taylor cone) and, as a result, changes the size of the droplets. A droplet of solution can be generated at the tip of the needle 140 and fall when surface tension can no longer hold the droplet in place. The electric field at least partially determines the size of the droplets as they leave the needle 140 to form particles that are collected in the collection unit 150. Exemplary voltages according to the present invention can be in the range of about 10 kV to about 30 kV, or about 9 kV to about 25 kV. The voltage can be selected to be below a threshold expected to cause damage to the hemostatic agent. For example, in the case of chitosan, the polysaccharide structure is expected to break down at temperatures above about 50°C. Therefore, to avoid damaging the chitosan in solution, a voltage of less than 25 kV may be used.

[0035] Other aspects of the electrospraying system herein can be selected to produce particles with desired characteristics, such as size and / or shape. For example, the size of the electrospraying needle, the distance from the tip of the needle (i.e., nozzle) to the grounded medium (collection unit), etc. In some examples, the needle can have a gauge size within the range of 16-23 or 16-22, e.g., 16, 18, 20, 21, 22, or 23. Different gauge sizes can vary droplet size. For example, larger gauge sizes can produce larger droplets and therefore larger particles, while smaller gauge sizes can produce smaller droplets and therefore smaller particles. Furthermore, for example, the distance from the tip of the electrospraying needle to the collection unit can be within the range of about 5 cm to about 15 cm.

[0036] According to some embodiments of the present invention, a voltage in the range of about 10 kV to about 30 kV and a 23 mm needle gauge, or a voltage in the range of about 15 kV to about 23 kV and a 22 mm needle gauge, can be used to produce particles having an average diameter in the range of about 200 μm to about 800 μm.

[0037] The grounding medium may include a liquid suitable for receiving the particles. For example, in the case of a water-soluble natural polymer such as chitosan, an aqueous solution may be used. Volatile solvents such as alcohol are less suitable because the water-soluble polymer is less likely to remain distributed throughout the medium and may aggregate and clump together. In some examples, the collection unit 150 may include an aqueous solution containing an ionic crosslinker, e.g., a reversible ionic crosslinker. Exemplary crosslinkers (also referred to herein as crosslinkers) suitable for the methods herein include, but are not limited to, tripolyphosphates (TPP), such as sodium tripolyphosphate, ethanol, and sodium hydroxide. In some examples, the crosslinker, e.g., TPP, may be present in the solution in an amount ranging from about 1% to about 15% by weight, e.g., from about 3% to about 15% by weight, e.g., from about 5% to about 10% by weight, or from about 5% to about 7% by weight, based on the total weight or volume of the solution. Without being bound by theory, it is believed that the crosslinker (crosslinking agent) forms spherical complexes around the particle droplets as they enter the aqueous solution of the collection unit 150. For example, the complexes, e.g., TPP complexes, may help prevent the particles from agglomerating or otherwise sticking together. Furthermore, the crosslinker (crosslinking agent) may crosslink with the functional groups (e.g., amine groups) of the chitosan. Thus, for example, TPP as a crosslinker (crosslinking agent) may crosslink with the amine groups (e.g., protonated amine groups) of the chitosan to form chitosan-TPP crosslinked particles. Therefore, the crosslinker (crosslinking agent) may aid in particle formation while avoiding agglomeration, so that the particles maintain their porous structure, their round shape, and a relatively uniform size.

[0038] Once electrospraying is completed or a desired amount of particles have been collected in the collection unit 150, the particles can be separated from the solution via any suitable technique, such as filtration, a funnel, etc. The particles can optionally be rinsed or washed to remove residual crosslinker solution and excess crosslinker complexes around the particles. For example, the particles can be rinsed or washed with water (e.g., deionized water), an alcohol such as ethanol or isopropyl alcohol, or a mixture thereof. The particles can be rinsed / washed once or any number of times sufficient to remove residual crosslinker. In some examples, the particles are not rinsed or washed. According to some embodiments of the present disclosure, the rinsed / washed particles can be placed on a sieve and spread into a thin layer to separate the particles from one another. The particles can optionally be further rinsed / washed with water (e.g., deionized water), an alcohol (e.g., ethanol, isopropyl alcohol, or other alcohols), or a mixture thereof.

[0039] The methods herein include, for example, drying the particles to further maintain the desired size and particle shape. Because the aqueous solutions and ground media used in the modified electrospraying processes herein are not as volatile as some other liquids, such as alcohol, the rinsed / washed particles may contain residual water. This moisture can cause the particles to agglomerate, resulting in the formation of larger, irregularly shaped particles that are more difficult to deliver to the target site in a medical procedure.

[0040] Therefore, the particles may be subjected to at least one drying process. Various drying processes may affect the shape, size, and / or porosity of the particles. In the methods herein, the drying process may include freezing, exposure to reduced pressure (e.g., vacuum pressure), mild heating, or a combination thereof. According to some embodiments of the present invention, the particles are freeze-dried. For example, the particles are cooled until frozen and then placed in a vacuum chamber to sublimate the solid ice crystals. Sublimation may avoid substantial agglomeration observed when the particles contain liquid water. See Example 2 below. As noted above, excess water present on the particles may result in relatively large, agglomerated, irregularly shaped particles. Sublimation of water present in the particles may reduce particle agglomeration and the formation of irregularly shaped particles.

[0041] In some examples, particles can be frozen to or at a temperature within the range of about -5°C to about -30°C, e.g., about -10°C to about -20°C, and the temperature can be gradually reduced from room temperature (about 22°C) to the target temperature over a period of minutes to hours. According to some examples herein, particles can be dried using a dynamic freeze-drying system, e.g., using a rotating drum apparatus. For example, particles can be frozen and maintained at a freezing temperature (e.g., about -55°C) while rotating an inner drum. The outer drum can also be circulated at an elevated temperature (-20°C) to promote uniform drying of the particles as they rotate around the dryer. Dynamic freeze-drying systems can operate at reduced pressure.

[0042] At the time of freezing and / or as part of the freezing process, the particles may be placed in a vacuum chamber for sublimation. For example, the vacuum chamber may be maintained at a pressure less than 150 Pa (1.13 mmHg), e.g., a pressure in the range of about 5 Pa to about 150 Pa (0.038 to 1.13 mmHg), or about 50 Pa to about 150 Pa (0.38 to 1.13 mmHg), e.g., about 5 Pa, about 10 Pa, about 50 Pa, about 75 Pa, or about 100 Pa. According to some embodiments of the present invention, the pressure in the vacuum chamber may be reduced from atmospheric pressure for a period of several minutes to several hours, e.g., for a period of about 10 minutes to about 4 hours, about 30 minutes to about 3 hours, or about 1 hour to about 2 hours. The duration in the vacuum chamber may be in the range of 2 to 3 hours to 2 to 3 days. For example, the particles can be placed in a vacuum chamber for a duration ranging from about 30 minutes to about 4 days, e.g., from about 1 hour to about 48 hours, from about 1 hour to about 24 hours, from about 1.5 hours to about 12 hours, from about 1 hour to about 4 hours, from about 2 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 12 hours, from about 12 hours to about 18 hours, from about 12 hours to about 24 hours, or from about 24 hours to about 48 hours. Optionally, the vacuum chamber can be gently heated.

[0043] The particles produced according to the present invention can be porous. For example, the particles can have a porosity greater than 80%, e.g., between about 85% and about 95%, or between about 87% and about 97%. Porosity can be measured by mercury intrusion porosimetry at a pressure of about 413.6 MPa (60,000 psia). An exemplary mercury intrusion porosimeter suitable for measuring the porosity of the particles herein is the Micromeritics® AutoPore V, which is capable of measuring pore diameters from 3 nm to 1100 μm. Various factors of the manufacturing process can be adjusted to achieve the desired porosity value, including the chitosan concentration in the chitosan salt solution, the TPP concentration in the collection bath, and the amount of freezing time. For example, lower concentrations of chitosan and TPP are expected to result in lower crosslink density and more porous particles. Furthermore, for example, longer freezing times are expected to promote the retention of particle structure without agglomeration or collapse of pore structure during the drying process. Different temperature ramping techniques are feasible to create different pore structures in the resulting particles.

[0044] The modified electrospraying and drying process described above can be used to prepare approximately spherical particles of a desired size, but the particles can optionally undergo one or more additional processing steps. In some instances, the dried particles can be milled or ground to further reduce particle size. The particles can be milled or ground by any suitable method or device, such as a pestle or grinder.

[0045] (Example) The following examples are intended to illustrate the invention but are not limiting in nature, and it is understood that the invention includes other aspects and embodiments consistent with the above description and the following examples.

[0046] Example 1 Chitosan acetate was prepared by mixing 2% solids chitosan (by weight based on the volume of the solution) and 1% citric acid (by weight based on the weight of chitosan) in a 1% acetic acid / water solution (by weight based on the volume of the solution). The mixed chitosan was then passed through a 22-gauge needle and an electric field generated by an applied voltage of 20-25 kV using a modified electrospraying process described herein. The particles were then dropped into a collection bath composed of water and 7% sodium tripolyphosphate (TPP). Once a selected volume of chitosan was electrosprayed, the particles were gently removed from the collection bath and washed multiple times with deionized water to remove excess solution (salt and uncrosslinked TPP). The cleaned particles were placed on a single layer of sieve and cleaned again with deionized water via a squirt bottle, followed by isopropyl alcohol. The water in the particles was replaced with alcohol to remove salt crystals. The sieve was then placed in a freezer (e.g., at a temperature ranging from -80°C to -20°C). Once frozen, the particles were placed in a vacuum chamber to slowly remove water by sublimation. The resulting chitosan-TPP crosslinked particles had an average diameter of 300-500 μm, a bulk density of approximately 0.3-0.7 g / mL, and an average aspect ratio of greater than 0.6.

[0047] Example 2 Three different batches of chitosan particles prepared according to the modified electrospraying method of Example 1 were dried according to different methods to investigate the effect on particle size and shape.

[0048] The first batch of chitosan particles was air-dried in the open air. The particles had a CE diameter ranging from approximately 100 μm to approximately 900 μm. As shown in Figure 2, the dried particles appeared as ellipsoidal fragments. The size bar in Figure 2 is 500 μm.

[0049] The second batch of chitosan particles was placed in a vacuum chamber and dried for approximately 96 hours. The particles had an average diameter ranging from approximately 200 μm to approximately 400 μm and a bulk density of approximately 0.7 g / mL. As shown in Figure 3, the particles were more spherical, but still had some agglomeration. The size bar in Figure 3 is 500 μm.

[0050] The third batch of chitosan particles was frozen at a temperature of approximately -20°C and then placed in a vacuum chamber and evacuated to a pressure of 0.7 mmHg for 2 to 5 hours. The temperature was then maintained at 35°C for 2 hours, drying the particles via sublimation. The particles had an average diameter ranging from approximately 200 μm to approximately 425 μm and a bulk density ranging from approximately 0.2 g / mL to approximately 0.3 g / mL. As shown in Figure 4, the particles were more spherical and separated compared to the particles shown in Figure 3. The size bar in Figure 4 is 1000 μm.

[0051] Example 3 Chitosan particles were prepared according to the modified electrospraying method of Example 1. In this example, the particles were dried using a dynamic freeze-drying system, which maintained the particles frozen at a freezing temperature of approximately -55°C while rotating the inner drum. The outer drum also circulated at an elevated temperature of approximately -20°C, rotating the particles around the dryer to ensure uniform drying. The resulting material was highly porous and had a more uniform and homogenous population of particles with a more uniform particle morphology and density. The particles are shown in Figure 5 (optical microscopy) and Figure 6 (scanning electron microscopy). The size bar in Figure 5 is 1000 μm, and the size bar in Figure 6 is 200 μm.

[0052] Aspects of the invention include the following numbered exemplary embodiments: 1. A composition comprising a plurality of particles of chitosan, such as chitosan with a crosslinker and / or a chitosan salt, wherein the plurality of particles have an average diameter in the range of about 150 μm to about 500 μm, e.g., about 300 μm to about 450 μm, and optionally, the plurality of particles have a steepness value in the range of about 30 to about 90, e.g., about 50 to about 80, or about 60 to about 80, and an average aspect ratio of greater than 0.6, greater than 0.7, or greater than 0.8, and the plurality of particles are approximately spherical in shape.

[0053] 2. The composition of embodiment 1, wherein the bulk density of the plurality of particles is in the range of about 0.2 g / mL to about 0.7 g / mL, for example, about 0.3 g / mL to about 0.5 g / mL. 3. The composition of embodiment 1 or 2, wherein the chitosan salt is chitosan acetate, chitosan lactate, chitosan succinate, chitosan glutamate, chitosan glycolate, or chitosan citrate.

[0054] 4. The composition of any one of embodiments 1 to 3, wherein the composition further comprises a liquid. 5. A method for preparing a composition according to any one of embodiments 1 to 4, comprising electrospraying a chitosan salt solution through a needle.

[0055] 6. The method of embodiment 5, wherein the chitosan salt solution has a viscosity in the range of about 1,500 cPs to about 3,000 cPs. 7. The method of embodiment 5 or 6, wherein the chitosan salt solution comprises about 0.5% to about 10% by weight of chitosan.

[0056] 8. The method of any one of embodiments 5-7, wherein electrospraying comprises applying a voltage of about 9 kV to about 25 kV to the needle. 9. The method of any one of embodiments 5-8, wherein the needle is a 21 gauge needle or a 22 gauge needle.

[0057] 10. The method of any one of embodiments 5 to 9, wherein the chitosan salt solution is electrosprayed into an aqueous solution containing a cross-linking agent such as sodium tripolyphosphate. 11. The method of any one of embodiments 5-10, wherein the method further comprises lyophilizing the plurality of particles.

[0058] 12. The method of embodiment 11, wherein the freeze-drying is carried out for 30 minutes to 48 hours, such as 2 hours to 12 hours, or 4 hours to 6 hours. 13. The method of any one of embodiments 5-12, wherein the method further comprises rinsing the plurality of particles with a liquid after electrospraying and / or before lyophilization.

[0059] 14. The method of embodiment 13, wherein the liquid comprises water, alcohol, or a mixture thereof. 15. Use of the composition according to any one of embodiments 1 to 4 as a hemostatic agent.

[0060] 16. A method of preparing a composition, the method comprising: electrospraying a chitosan salt solution through a needle to produce a plurality of particles of chitosan, such as chitosan and / or chitosan salt with a crosslinker; and freeze-drying the plurality of particles, wherein the plurality of particles have an average diameter in the range of about 150 μm to about 500 μm and an average aspect ratio of greater than 0.6, and the plurality of particles are substantially spherical in shape.

[0061] 17. The method of embodiment 16, wherein the chitosan salt comprises chitosan acetate, chitosan lactate, chitosan succinate, chitosan glutamate, chitosan glycolate, or chitosan citrate.

[0062] 18. The method of embodiment 16, wherein the average diameter of the plurality of particles is in the range of about 300 μm to about 450 μm. 19. The method of embodiment 16, wherein the plurality of particles have an average aspect ratio of greater than 0.8.

[0063] 20. The method of embodiment 16, wherein the plurality of particles has a steepness value in the range of about 50 to about 80. 21. The method of embodiment 16, wherein the chitosan salt solution is electrosprayed into an aqueous solution containing a cross-linking agent.

[0064] 22. The method of embodiment 21, wherein the cross-linking agent comprises sodium tripolyphosphate. 23. A method of preparing a composition, the method comprising: electrospraying a chitosan salt solution through a needle to produce a plurality of particles of chitosan, such as chitosan and / or chitosan salt comprising a crosslinker; and freeze-drying the plurality of particles, wherein the plurality of particles have an average diameter in the range of about 300 μm to about 500 μm, an average aspect ratio of greater than 0.6 (the plurality of particles are substantially spherical in shape), and a bulk density in the range of about 0.2 g / mL to about 0.7 g / mL.

[0065] 24. The method of embodiment 23, wherein the chitosan salt solution comprises about 0.5% to about 10% by weight of chitosan. 25. The method of embodiment 23, wherein the chitosan salt solution has a viscosity in the range of about 1,500 cPs to about 3,000 cPs.

[0066] 26. The method of embodiment 23, wherein electrospraying comprises applying a voltage of about 9 kV to about 25 kV to the needle. 27. The method of embodiment 23, wherein the needle is a 21 gauge needle or a 22 gauge needle.

[0067] 28. The method of embodiment 23, wherein the freeze-drying is carried out for a time period ranging from 30 minutes to 48 hours. 29. The method of embodiment 23, wherein the method further comprises rinsing the plurality of particles with a liquid after electrospraying and before freeze-drying.

[0068] 30. The method of embodiment 29, wherein the liquid comprises water, alcohol, or a mixture thereof. 31. A composition comprising a plurality of particles of chitosan, such as chitosan and / or chitosan salts comprising a crosslinker, wherein the plurality of particles have an average diameter in the range of about 250 μm to about 500 μm, an average aspect ratio of greater than 0.6 (the plurality of particles are substantially spherical in shape), and an average bulk density in the range of about 0.2 g / mL to about 0.7 g / mL.

[0069] 32. The composition of embodiment 31, wherein the chitosan salt comprises chitosan acetate, chitosan lactate, chitosan succinate, chitosan glutamate, chitosan glycolate, or chitosan citrate.

[0070] 33. The composition of embodiment 31, wherein the composition is in the form of a powder. 34. The composition of embodiment 31, wherein the plurality of particles have a steepness value in the range of about 50 to about 80.

[0071] 35. The composition of embodiment 31, wherein the bulk density is within the range of about 0.3 m / mL to about 0.5 g / mL. Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the examples disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. A composition comprising a plurality of particles comprising chitosan crosslinked solely by a crosslinking agent, wherein the plurality of particles are prepared from a chitosan salt, the crosslinking agent is a tripolyphosphate salt, and the plurality of particles are an average diameter in the range of 100 μm to 750 μm, and Average aspect ratio greater than 0.6 A composition comprising:

2. 10. The composition of claim 1, wherein the average diameter of the plurality of particles is in the range of 150 μm to 500 μm.

3. 3. The composition of claim 1, wherein the average diameter of the plurality of particles is in the range of 400 μm to 600 μm.

4. The composition of any one of claims 1 to 3, wherein the plurality of particles have an average aspect ratio greater than 0.7, or greater than 0.

8.

5. The composition of any one of claims 1 to 4, wherein the plurality of particles have a steepness value in the range of 30 to 90, or in the range of 70 to 90.

6. The composition according to any one of claims 1 to 5, wherein the average maximum distance between the plurality of particles is in the range of 500 µm to 700 µm.

7. The composition of any one of claims 1 to 6, wherein the plurality of particles are spherical.

8. 8. The composition of claim 1, wherein the bulk density of the plurality of particles is in the range of 0.1 g / mL to 0.7 g / mL, 0.2 g / mL to 0.7 g / mL, 0.3 g / mL to 0.5 g / mL, or 0.1 g / mL to 0.2 g / mL.

9. 9. The composition of any one of claims 1 to 8, wherein the plurality of particles have a porosity greater than 80%, or in the range of 85% to 95%, as measured by mercury intrusion porosimetry.

10. 10. A method for preparing the composition of any one of claims 1 to 9, comprising electrospraying a chitosan salt solution through a needle into an aqueous solution containing the crosslinker.

11. 11. The method of claim 10, wherein the chitosan salt solution has a viscosity in the range of 1,500 cPs (1,500 mPa s) to 8,000 cPs (8,000 mPa s) at 25°C, or in the range of 1,500 cPs (1,500 mPa s) to 3,000 cPs (3,000 mPa s) at 25°C.

12. 12. The method of claim 10 or 11, wherein the chitosan salt solution comprises 0.5% to 10% chitosan by weight based on the volume of the solution.

13. The method of any one of claims 10 to 12, wherein the method further comprises freeze-drying the plurality of particles.

14. 14. The method of claim 10, further comprising rinsing the plurality of particles with a liquid after electrospraying and / or before freeze-drying, wherein the liquid comprises water, an alcohol, or a mixture thereof.

15. The composition according to any one of claims 1 to 9, which is used as a hemostatic agent.

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