N-acetylcysteine and sodium thiosulfate drug-eluting microparticles

Abstract

Methods for making and using hydrogels comprising therapeutic microparticle compositions are disclosed. Illustrative methods of the invention include methods of making a hydrogel composition, the method comprising combining at least 14% w / v of a non-ionic copolymer surfactant having the linear formula (C3H6OꞏC2H4O)x; microparticles comprising a polycaprolactone; and a therapeutic agent; such that the hydrogel composition is made. Embodiments of the invention further include compositions made by the methods disclosure herein as well as methods for using these compositions. For example, methods of the invention include disposing a composition in the ear of a subject being administered cisplatin, wherein amounts of the composition administered are sufficient to preserve hearing function in a mouse model of cisplatin ototoxicity.

Description

[0001] N-ACETYLCYSTEINE AND SODIUM THIOSULFATE DRUG-ELUTING MICROPARTICLES

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. Section 119(e) of copending and commonly-assigned U.S. Provisional Patent Application Serial No 63 / 685,419, filed on August 21, 2024 and entitled “N-ACETYLCYSTEINE AND SODIUM THIOSULFATE DRUG-ELUTING MICROPARTICLE HYDROGEL ’ which application is incorporated by reference herein.

[0004] STATEMENT REGARDING FEDERAL FUNDING

[0005] This invention was made with government support under K08DC019957 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] Embodiments of the disclosure concern at least the fields of medicine and material science.

[0008] BACKGROUND OF THE INVENTION

[0009] Injury to nerves from medications or physical manipulation is common. As there can be functional disability after injury that significantly affects patient quality of life, developing therapeutics to promote nerve healing or protect nerves (neuroprotection) is important. Two examples of the clinical importance of neuroprotection are noted below;

[0010] One medication source of nerve injury is cisplatin. Cisplatin is a chemotherapeutic medication known to cause ototoxicity. The resulting balance and hearing dysfunction cause significant disruptions to patients’ lives. Cisplatin has been shown to induce apoptosis in Schwann cells, providing evidence that Schwann cell injury is one of the mechanisms by which cisplatin induces ototoxicity. Additionally, Schwann cell damage has been shown to mimic a certain form of hearing loss known as hidden hearing loss. Schwann cells make up the myelin sheath that wraps around the axon of neurons to promote nerve conduction. When damage to neurons within the peripheral nervous system occurs, free radicals are created which adversely impact Schwann cell health by inducing oxidative stress.

[0011] As cisplatin therapies often require intermittent infusions over weeks to months, there is a need in the art for materials and methods that are able to locally deliver an otoprotective agent for a prolonged period of time so as to protect Schwann cells and reduce cisplatin-induced ototoxicity.

[0012] Another category of nerve injury that will benefit from expanded therapeutic options are stretch injuries from physical manipulation. Stretch injuries of nerves frequently occur during surgical procedures leading to dysfunction of important nerves such as the recurrent laryngeal nerve responsible for vocal fold movement and swallowing. Injury of the recurrent laryngeal nerve often occurs after thyroidectomy. Recovery takes weeks to months as the Schwann cells supporting nerves remyelinate the injured region.

[0013] There is a need in the art for materials and methods that are able to locally deliver one or more agent that promote Schwann cell rehabilitation.

[0014] SUMMARY OF THE INVENTION

[0015] Conventional hydrophilic medications are notoriously difficult to deliver for prolonged periods of time. The specific protocols and materials disclosed herein overcome such problems by providing microparticle compositions that are able to release therapeutic levels of a therapeutic agent for at least 1, 5, 7 or more days. The invention disclosed herein provides methods for making and using hydrogel compositions comprising polycaprolactone (PCL) and microparticles comprising a therapeutic agent such as N-acetylcysteine or sodium thiosulfate. In typical embodiments of the invention, the therapeutic agent release occurs over multiple days to multiple weeks. A model of Schwann cell injury was developed to demonstrate the protective effects of therapeutic compositions. Briefly, RSC96 cells, an immortalized rat Schwann cell line, were chosen as a Schwann cell model as they are conventionally used in studies of nerve injury. Prior studies have also investigated the use of antioxidants to blunt the effects of cisplatin ototoxicity. Building upon this knowledge, antioxidants such as N-acetylcysteine (NAC) were selected to counteract the influence of oxidative stressors seen during nerve injury and recovery. In addition, because a course of cisplatin therapy often requires intermittent infusions over weeks to months and nerve stretch injuries often recover over weeks to months, one strategy to reduce cisplatin-induced ototoxicity is to use a drug-eluting microparticle able to locally deliver an otoprotective agent for a prolonged period. In working embodiments of this invention discussed below, NAC was blended into poly lactic-co-glycolic acid (PLGA) microparticle composition. In embodiments of the invention, NAC was alternatively incorporated into polycaprolactone (PCL) microparticles using a double emulsion method. Both polymers were chosen for their widely accepted use as FDA approved biocompatible polymers, making them ideal vehicles to prolong the release of a therapeutic agent able to protect patients from cisplatin-induced inner ear injury. In both cisplatin and hydrogen peroxide nen e stretch injury models of nerve injury, NAC eluted from NAC-encapsulated microparticles was found to protect against the effects of hydrogen peroxide.

[0016] The invention disclosed herein has a number of embodiments. For example, embodiments of the invention include methods of making a otoprotective hydrogel composition optimized for clinical applications, the methods comprising combining at least 14% w / v of a non-ionic copolymer surfactant having the linear formula (CsHeO C2H4O)X; microparticles comprising a poly caprolactone; and a therapeutic agent; such that the otoprotective hydrogel composition is made. In such methods, the copolymer surfactant, the microparticles and the therapeutic agents are combined such that the hydrogel composition can be manually injected through a 27 gauge needle at room temperature and further forms a gel in less than 30 minutes upon exposure to human body temperature. Typically the methods comprise forming the composition so that no new bonds are created in the composition that alter the structures of the therapeutic agent as measured by Fourier transform infrared spectroscopy (FTIR). In certain embodiments, the methods comprise forming the composition to release > 100 pM of a therapeutic agent from the microparticle for at least one week 1 week. Embodiments of these methods can further comprise combining the hydrogel composition with live mammalian cells.

[0017] Embodiments of the invention also include otoprotective hydrogel compositions. Such compositions typically comprise a non-ionic copolymer surfactant having the linear formula (CvFEO C'iH-iOty: microparticles comprising a polycaprolactone; and a therapeutic agent. In typical embodiments of the invention, the composition comprises at least 14% w / v non-ionic copolymer surfactant; and / or less than 100 mg / mL microparticles comprising a poly caprolactone. In illustrative embodiments of the invention, the non-ionic copolymer surfactant comprises Pluronic*1F-127; the poly caprolactone comprises a C18E poly caprolactone; the therapeutic agent is disposed within the microparticles and comprises at least one of an antioxidant or an antibiotic or a steroid; the composition can be manually injected through a 27 gauge needle at room temperature; and / or the composition forms a gel in less than 30 minutes upon exposure to human body temperature. In certain compositions of the invention, the composition comprises 20% Pluronic Fl 27; the therapeutic agent comprises at least one of N-Acetylcysteine (NAC), Sodium Thiosulfate, steroid (e.g. dexamethasone), or antimicrobial (e.g. cilastatin); the composition further comprises a pharmaceutical excipient or an imaging agent; and / or the therapeutic agent comprises N- Acet l cysteine (NAC) and releases > 100 pM NAC from the microparticle for at least one week 1 week following disposing the composition in a tissue region of a subject. Typically, the composition does not comprise a chitosan.

[0018] Embodiments of the invention also include methods of delivering the otoprotective compositions disclosed herein to a subject, the method typically comprising disposing the hydrogel composition at a site where the hydrogel composition is in direct contact with a first tissue region of the subject (e.g. one comprising Schwann cells). In certain embodiments of these methods, the composition is delivered topically to the middle ear of the subject; the composition is delivered via injection transtympanically to the subject; the composition is delivered topically to stretched or injured nerves of the subject; and / or the composition is delivered to stretched or injured nen es of the subject by injection. In illustrative embodiments of these methods, the subject is selected to be a patient having been administered cisplatin or with concern for a nerve stretch injury. In certain embodiments of these methods, amounts of the composition administered are sufficient to reduce cellular injury in response to agents administered to the subject such as cisplatin or hydrogen peroxide. In certain working embodiments of these methods, amounts of the composition administered are sufficient to preserve hearing function in a mouse model of cisplatin ototoxicity. In illustrative embodiments of these methods, the composition is administered through a 27 gauge or larger gauge needle.

[0019] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

[0020] DRAWINGS

[0021] Figure 1. Determination of cisplatin LC50. A: RSC96 Cell viability at cisplatin concentrations from 1-500 pM as determined by CCK-8 assay. Curve fitted using a four-parameter logistic regression. The dose required to reduce absorbance by 50% was 3.76 pM and is indicated by a dashed line and LC50 label. B and C: 10X light micrographs of RSC96 cells exposed to 0 (Control, B) and the rounded LC50 value (Cisplatin, C).

[0022] Figure 2. NAC Dose Screening. RSC96 cells were co-dosed with the initial LC50 of 3.34 pM cisplatin and different concentrations of N-acetylcysteine (NAC) (0 pM, 1 pM, 10 pM, 100 pM, l OOOpM). Cells had improved viability’ when compared to cisplatin alone conditions at 100 pM and 1000 pM NAC.

[0023] Figure 3. Characterization of microparticles. A-F: 20x microparticle images of each condition. G: Microparticle diameter size distribution of six 20x images per microparticle.

[0024] Figure 4. Microparticle elution. NAC elution from microparticles as a function of the total amount eluted by the end of collection to account for differences in the total amount of mass from which NAC was eluted in each transwell.

[0025] Figure 5. Microparticle encapsulation. Total amount of NAC released based on the total mass of microparticle condition initially added to each transwell.

[0026] Figure 6. Bioactivity of eluted NAC assessment. All conditions were dosed with a rounded LC50 value of 4 pM of cisplatin. All replicate experiments were normalized to their respective controls. The control for eluted conditions was cells codosed with cisplatin and eluted solution from microparticles fabricated with no NAC. The control for resuspended conditions was cells codosed with cisplatin and media containing no NAC. A: NAC eluted from PLGA microparticles at 100 and 1000 pM exhibited similar increases in viability as resuspended NAC. B: NAC eluted from PCL microparticles at 100 pM exhibited similar increases in viability as resuspended NAC. C-F: Light micrographs (10X) of RSC96 cells dosed with cisplatin at 4 pM (C), cisplatin at 4 pM and resuspended NAC (D), cisplatin at 4 pM and eluted solution from PLGA microparticles at a concentration of 100 pM (E), and cisplatin at 4 pM and eluted solution from PCL microparticles at a concentration of 100 pM (F).

[0027] Figure 7. (A) details the 42 day saline and cisplatin injection timeline, and (B) is the supplemental and food and hydration regimen that CEMP and CNAC mice received during the duration of the study. Figure 8. The mass of NAC eluted from both our NAC and empty MPs in pg.

[0028] Figure 9. Mouse weights over the study period. This shows the weight lost in the cisplatin-treated CEMP and CNAC mice, versus the weight gain in the HCEMP group. Error bars (standard deviation) are shown for each group for each day weights were measured.

[0029] Figure 10. (A) Thresholds and (B) threshold shifts are displayed as dB HL, with green circles (HCEMP), red diamonds (CEMP), and blue squares (CNAC). (A) Solid symbols are pre-treatment. and hollow symbols are post-treatment.

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0032] Cisplatin is a chemotherapeutic medication known to cause ototoxicity .1'2The resulting balance and hearing dysfunction cause significant disruptions to patients’ lives.3 Cisplatin has been shown to induce apoptosis in Schwann cells, raising the possibility that Schwann cell injury is one of the mechanisms by which cisplatin induces ototoxicity7. 4,5 Additionally, Schwann cell damage has been shown to mimic a certain form of hearing loss known as hidden hearing loss.67 Schwann cells make up the myelin sheath that wraps around the axon of neurons to promote nerve conduction.8When damage to neurons within the peripheral nervous system occurs, free radicals are created which adversely impact Schwann cell health by inducing oxidative stress.4RSC96 cells, an immortalized rat Schwann cell line, were chosen as a Schwann cell model as they have been used in other studies with cisplatin. 10 Other studies have investigated antioxidants that blunt the effects of cisplatin ototoxicity.11,12Thus, an antioxidant like N-acetylcysteine (NAC) was hypothesized to counteract the influence of oxidative stressors.13 15A model of Schwann cell injury was then developed to investigate the protective effects of this antioxidant therapeutic.

[0033] Since a course of cisplatin therapy often requires intermittent infusions over weeks to months, one strategy to reduce cisplatin-induced ototoxicity is to design a drug-eluting microparticle able to locally deliver an otoprotective agent for a prolonged period. NAC, a hydrophilic antioxidant, was blended into poly lactic-co- glycolic acid (PLGA) microparticles. NAC was also incorporated into poly caprolactone (PCL) microparticles using a double emulsion method as previously described.15Both polymers were chosen for their widely accepted use as FDA approved biocompatible polymers, making them ideal vehicles to prolong the release of a therapeutic agent able to protect patients from cisplatin-induced inner ear injury.

[0034] Embodiments of the invention include a methodology for the creation of hydrogel compositions comprising polycaprolactone (PCL) and a therapeutic agent (e.g., antioxidants such as N-acetylcysteine or Sodium thiosulfate or the like, or steroids such as dexamethasone or the like, or antimicrobials such as cilastatin or the like) containing microparticles. Briefly, hydrophilic medications are notoriously difficult to deliver for prolonged periods of time and this protocol results in microparticles that are able to release therapeutic levels of each respective therapeutic (e.g., antioxidant) based on cell culture results. The release of a therapeutic agent currently occurs over days to weeks. This microparticle fabrication methodology7is generally adopted from: Murphy NP, Lampe KJ. Fabricating PLGA microparticles with high loads of the small molecule antioxidant N-acetylcysteine that rescue oligodendrocyte progenitor cells from oxidative stress. Biotechnol Bioeng. 2018 Jan;l 15(l):246-256. doi: 10.1002 / bit.26443. Epub 2017 Oct 6. PMID: 28872660.

[0035] Embodiments of the invention include methods of making a hydrogel composition, the methods comprising combining at least 14% w / v of a non-ionic copolymer surfactant having the linear formula (CsHeO C2H4O)x; microparticles comprising a polycaprolactone; and a therapeutic agent; such that the hydrogel composition is made. In such methods, the copolymer surfactant, the microparticles and the therapeutic agents are combined such that the hydrogel composition can be manually injected through a 27 gauge or larger needle at room temperature and further forms a gel in less than 30 minutes upon exposure to human body temperature. Typically, the methods comprise forming the composition so that no new bonds are created in the composition that alter the structures of the therapeutic agent as measured by FTIR. In certain embodiments, the methods comprise forming the composition to release > 100 pM of a therapeutic agent from the microparticle for at least one week 1 week. Typically this therapeutic agent release is observed / quantified using a transwell elution assay. Briefly, in one illustrative working embodiment of a transwell elution assay, amounts of drug eluted from the microparticle hydrogel were analyzed by disposing hydrogel microparticle compositions of the invention on permeable transwell inserts. The inserts were then placed into the wells of a tissue culture plate filled with water to a volume determined by the size of the tissue culture plate and transwell insert. This system is then kept in an incubator at 37 degrees Celsius. The water solution into which the drug was eluted is exchanged regularly over the course of elution (e.g., 1-2 months), with the amount of eluted agent then quantified from each sample. Elution is determined by plotting the amount of drug eluted over time.

[0036] In one illustrative method of the invention, the following procedure is followed. (1) Create a 2% polycaprolactone (PCL) in dichloromethane (DCM) solution. Let sit overnight in a fume hood capped.

[0037] (2) Vacuum fdter and prepare solutions of 1% polyvinyl alcohol (PVA) - 2 g in 200 mL DI at 50 degrees with Teflon coated magnets.

[0038] (3) Add agent of interest (NAC, STS, steroid, cilastatin) and sonicate the PCL / DCM solution. Solution should turn a milky white color after sonication.

[0039] (4) Add 15 mL of PCL / DCM solution dropwise to 75 mL 1% PVA with at least 2.5% agent of interest (NAC, STS, steroid, cilastatin) to exceed the solubility limit of the hydrophilic agent of interest while the homogenizer is running at 5,000 rpm for 15 minutes in 200mL beaker

[0040] (5) Add mixture to 225 mL 0.3% PVA (warmed in water bath) saturated with at least 5% agent of interest and stir at 700rpm w / Teflon coated magnet for 2 hrs at 40C.

[0041] (6) Perform three washes with deionized water with at least 2.5% agent of interest at a speed of least 4000 rpm in a centrifuge for 5 min, freeze, then lyophilize the frozen microparticle solution.

[0042] Embodiments of the invention also include hydrogel compositions made by the methods disclosed herein and comprising a non-ionic copolymer surfactant having the linear formula (CsHeO C^C x; microparticles comprising a polycaprolactone; and a therapeutic agent. In typical embodiments of the invention, the composition comprises at least 14% w / v non-ionic copolymer surfactant; and / or less than 100 mg / mL microparticles comprising a poly caprolactone. In illustrative embodiments of the invention, the non-ionic copolymer surfactant comprises Pluronic® F-127; the poly caprolactone comprises a C18E poly caprolactone; the therapeutic agent is disposed within the microparticles and comprises at least one of an antioxidant or a steroid; the composition can be manually injected through a 27 gauge needle at room temperature; and / or the composition forms a gel in less than 30 minutes upon exposure to human body temperature. In certain compositions of the invention, the composition comprises 20% Pluronic F127; the therapeutic agent comprises at least one of N- Acetylcysteine (NAC), Sodium Thiosulfate, steroid (e.g. dexamethasone) or an antimicrobial (e.g. cilastatin); the composition further comprises a pharmaceutical excipient or an imaging agent; and / or the therapeutic agent comprises N- Acetylcysteine (NAC) and releases > 100 pM NAC from the microparticle for at least one week 1 week following disposing the composition in a tissue region of a subject. Embodiments of these methods can further comprise combining the hydrogel composition with live mammalian cells. Typically, the composition does not comprise a chitosan.

[0043] Embodiments of the invention also include methods of delivering the composition disclosed herein to a subject, the method typically comprising disposing the hydrogel composition at a site where the hydrogel composition is in direct contact with a first tissue region of the subject. In certain embodiments of these methods, the composition is delivered topically to the middle ear of the subject; the composition is delivered via injection transtympanically to the subject; the composition is delivered topically to stretched or injured nerves of the subject; and / or the composition is delivered to stretched or injured nerves of the subject by injection. In certain embodiments of these methods, the subject is selected to be a patient having been administered cisplatin or undergone stretch injury of a nerve. In certain embodiments of these methods, amounts of the composition administered are sufficient to reduce cellular injury in response to agents administered to the subject such as cisplatin or hydrogen peroxide. In certain embodiments of these methods, amounts of the composition administered are sufficient to preserve hearing function in a mouse model of cisplatin ototoxicity; In illustrative embodiments of these methods, the composition is administered through a 27 gauge or larger gauge needle.

[0044] In certain embodiments of the invention, the compositions of the invention include additional constituents. In certain embodiments the compositions of the invention include one or more therapeutic agents such as an anti -infl ammatory agent, an agent that modulates coagulation, an antibiotic agent, a chemotherapeutic agent or the like. Compositions of the invention can be formulated for use as carriers or scaffolds of therapeutic agents such as drugs, cells, proteins, and other bioactive molecules. Certain embodiments of the compositions of the invention include, for example a pharmaceutical excipient such as one selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent. For compositions suitable for administration to humans, the term "excipient" is meant to include, but is not limited to, those ingredients described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) the contents of which are incorporated by reference herein.

[0045] As carriers, compositions of the invention can incorporate the agents and deliver them to a desired site in the body for the treatments of a variety of pathological conditions including cisplatin induced ototoxicity’ or nerve injury from physical manipulation. Certain illustrative materials and methods that can be adapted for use in such embodiments of the invention are found, for example in Hydrogels: Design, Synthesis and Application in Drug Delivery’ and Regenerative Medicine 1st Edition, Singh, Laverty and Donnelly Eds; and Hydrogels in Biology and Medicine (Polymer Science and Technology) UK ed. Edition by J. Michalek et al.

[0046] EXAMPLE 1 : N-ACETYLCYSTEINE MICROPARTICLES REDUCE CISPLATIN-INDUCED RSC96 SCHWANN CELL TOXICITY

[0047] Example 1 Summary

[0048] Objectives: Cisplatin is known to cause inner ear dysfunction. There is growing evidence that cisplatin-induced demyelination of spiral or Scarpa’s ganglion neurons may play an additional role in drug-induced ototoxicity alongside afferent neuron injury. As Schwann cells produce myelin, there may be an opportunity to reduce ototoxic inner ear damage by promoting Schwann cell viability. This work describes a cellular model of cisplatin-induced Schwann cell injury’ and investigates the ability' of the antioxidant N-acetylcysteine to promote Schwann cell viability. A local deliver^’ system of drug-eluting microparticles was then fabricated, characterized, and investigated for bioactivity.

[0049] Methods: RSC96 rat Schwann cells were dosed with varying concentrations of cisplatin to obtain a dose curve and identify the lethal concentration of 50% of the cells (LC50). In subsequent experiments, RSC96 cells were co-treated with cisplatin and both resuspended or eluted N-acefylcysteine. Cell viability was assessed with the CCK8 assay.

[0050] Results: The LC50 dose of cisplatin was determined to be 3.76 pM (p=2.2 x 10-16). When codosed with cisplatin and therapeutic concentration of resuspended or eluted N-acefylcysteine, Schwann cells had an increased viability compared to cells dosed with cisplatin alone.

[0051] Conclusion: RSC96 Schwann cell injury following cisplatin insult is characterized in this in vitro model. Cisplatin caused injury at physiologic concentrations and N-acetylcysteine improved cell viability’ and mitigated this injury. N-acefylcysteine was packaged into microparticles and eluted N-acefylcysteine retained its ability- to increase cell viability, thus demonstrating promise as a therapeutic to offset cisplatin-induced ototoxicity.

[0052] Materials and Methods

[0053] RSC96 Cell Culture

[0054] RSC96 rat Schwann cells were cultured in media consisting of Dulbecco’s modified Eagle’s medium, 1% penicillin-streptomycin and 10% fetal bovine serum. RSC96 cultures were maintained in an incubator kept at 37°C and 5% CO2.

[0055] CCK8 Viability Assay

[0056] The Cell Counting Kit 8 (CCK8) assay was used to assess cell viability. This kit was used to quantify cell viability by producing a formazan dye after reduction of a tetrazolium salt. LC50 Determination

[0057] RSC96 cells were plated at a density of 4000 cells / well in a 96-well plate and were allowed to grow for 24 hours. Following the 24-hour incubation period, the media was removed, after which each well received fresh media along with doses of cisplatin ranging from 0 pM-500 pM. Cell viability was determined after 48 hours using the CCK8 assay. The LC50 was determined using a four-parameter logistic regression.

[0058] NAC Screening with CCK8 Assay

[0059] Following the determination of the concentration of cisplatin resulting in 50% lethality of RSC96 cells (LC50), the protective capacity of NAC was assessed. RSC96 cells were plated at a density of 4000 cells / well in 96-well plates and were allowed to grow for 24 hours. Cells were then codosed with the cisplatin LC50 and increasing concentrations of NAC to obtain a total volume of 100 pL / well. After 48 hours, cell viability was determined. Six conditions were explored, each with 8 replicates: control, cisplatin alone, cisplatin with IpM NAC, cisplatin with lOpM NAC. cisplatin with lOOpM NAC, and cisplatin with lOOOpM NAC.

[0060] Microparticle Fabrication and Elution

[0061] A microparticle protocol from Murphy et al. was adapted to create PLGA and PCL microparticles as well as NAC-loaded PLGA and PCL microparticles. 15,16 Unloaded microparticles were fabricated as controls. Three separate batches were created by dissolving the following polymer formulations in dichloromethane (DCM): 2% 50:50 lactide: coglycolide poly(lactic-co-glycolic acid) (PLGA), 2% 75:25 lactide:coglycolide PLGA, or 2% poly caprolactone (PCL). Each polymer-DCM solution was then added dropwise to a 1% solution of poly (vinyl alcohol) (PVA) and homogenized at 3000 rpm for 15 minutes. The mixture was next poured into 0.3% PVA and stirred at 700 rpm for 2 hours at 40°C. The subsequent solution was then centrifuged and washed in double-distilled water three times before resuspending the microparticle solution and freezing it overnight at -80°C. Frozen microparticles were then lyophilized and stored at -20°C.

[0062] Microparticles fabricated with NAC were created by dissolving 2% 50:50 PLGA in DCM followed by 10 mg NAC in 100 pL water. The solution was then sonicated. This was then added dropwise to a 1% solution of PVA saturated with 2.5% NAC and homogenized at 3000 rpm for 15 minutes. The mixture was next poured into 0.3% PVA with 5% NAC and stirred at 700 rpm for 2 hours at 40°C. The mixture was then centrifuged and washed in double-distilled water with 2.5% NAC three times before freezing and lyophilization. Microparticles were similarly made with 75:25 PLGA and PCL.

[0063] Approximately 10 mg of each microparticle type was suspended in 150 pL double distilled water, a sample of which was placed on a glass slide for microparticle imaging using a Zeiss AxioPlan microscope with a 20x objective. Six images of each condition were obtained to ensure broad sampling. The imfindcircles function (Matlab Image Processing Toolbox) was used to identify and segment the microparticles, from which radii were determined. PCL images were converted to binary arrays using Fiji’s built-in "Make Binary” function and then ‘Till Holes” to prevent detection of within particle features otherwise identified as individual microparticles by the imfindcircles function. These within microparticle features were not detected in the PLGA microparticle images.

[0064] Microparticle Elution and RSC96 Dosing

[0065] Lyophilized microparticles were weighed directly into 12 mm 0.4 pm-pore polyester transwell inserts. The transwell inserts were filled with 0.6 mL of phosphate buffered saline (PBS) and placed into 1.5 mL of PBS within 12 well plates. Two replicates of each PLGA microparticle condition and four replicates of each PCL condition were used to characterize elution. Eluted solution was collected by exchanging the 1.5 mL of PBS below the transwell containing microparticles and placing this within cryotubes stored at -20°C. This was done daily for the first 5 days then twice weekly for the first month and weekly thereafter. The concentration of NAC in the eluted solution was determined via linear regression following incubation with Ellman's reagent, a compound that reacts with sulfhydryl groups to induce a color change that is then read on a plate reader at 412 nm.15

[0066] RSC96 cells were plated in 96 well plates at a density of 4000 cells / well for 24 hours and then co-dosed with cisplatin and resuspended NAC or eluted NAC solutions from 50:50 PLGA and PCL microparticles. As NAC was eluted into PBS, eluted solutions were diluted with media to the appropriate concentration for dosing. The CCK-8 assay was then used to determine cell viability.

[0067] Statistical Analyses

[0068] Preliminary statistical analyses were conducted using the R Studio statistical software platform (R Core Team, 2017). A bootstrap implementation of one- and two- way analyses of variance (ANOVA) were conducted using custom scripts written in the Igor Pro software environment. RSC96 viability under conditions of varying NAC concentration was analyzed using a single factor ANOVA with the Dunnef s post-hoc test. A two-way ANOVA was used to evaluate the effects of NAC source (i.e. resuspended or eluted) and concentration (0, 100, or 1000 pM; twofactor ANOVA), and Dunnef s post-hoc analysis was used to distinguish levels of NAC concentration. Figures were created with Igor Pro (WaveMetrics, 2022) and Adobe Illustrator (Adobe Inc, 2019) and Adobe Photoshop (Adobe Inc, 2019).

[0069] Results

[0070] LC50 Determination

[0071] The LC50 of two experiments after normalization was 3.76 pM (Figure 1). Therefore, all subsequent experiments were conducted using the rounded value of 4 pM cisplatin. NAC Screening

[0072] Cells co-dosed with 100 and 1000 pM NAC with the cisplatin LC50 resulted in an increase in CCK8 absorbance when compared to conditions dosed with cisplatin alone (Figure 2). Cell viability was improved at NAC concentrations of 100 pM (p < 2 x 10-6) and 1000 pM (p < 2 x 10'6) compared to cells dosed with cisplatin alone (resampled single factor ANOVA with Dunnet’s post hoc test). RSC96 viability associated with NAC concentrations less than 100 pM were similar to the control condition (4 pM cisplatin and 0 pM NAC).

[0073] Microparticle Characterization

[0074] The diameters of fabricated microparticles are represented in a histogram in Figure 3.

[0075] Microparticle Elution

[0076] To account for the different masses of microparticles weighed into each transwell at the beginning of elution, elution is expressed as a fraction of the total amount released by the end of collection versus time (Figure 4). By three days the PLGA microparticles had all released greater than 95% of the total amount eluted. The PCL microparticle elution was slightly more prolonged releasing 83.3% of what would be released after 3 days.

[0077] PCL microparticles with NAC incorporated released the largest amount of NAC relative to the initial mass added to their transwell, while 50:50 PLGA microparticles released the smallest amount of NAC relative to their initial mass (Figure 5).

[0078] NAC Eluted from Microparticles Showed An Increase in Cell Viability

[0079] To determine whether microparticle encapsulation altered the efficacy of NAC, RSC96 cells were dosed with cisplatin along with either resuspended NAC or NAC eluted from PLGA microparticles at concentrations of 100 pM and 1000 pM and from PCL microparticles at a concentration of 100 pM (Figure 6). Additional conditions were included that dosed cells with eluted solutions from empty microparticles to establish a control absorbance and ensure that a microparticle biproduct alone was not responsible for an increase in absorbance.

[0080] A protective effect was seen with NAC, whether from microparticles or resuspended. This was seen in both PLGA microparticles and PCL microparticles. Protection was seen with NAC eluted from PLGA microparticles at concentrations of 100 pM (p < 2 x IO’6) and 1000 pM (p < 2 x 10’6). NAC eluted from PCL microparticles increased viability at a concentration of 100 pM (p < 1 x 10'4). Additionally, there was a difference between cell viability' in conditions dosed with and without eluted solutions from microparticles. Cells were unable to be co-dosed with cisplatin and 1000 pM NAC from PCL microparticles due to the low concentration of eluted solution, which would not allow for diluting and dosing cells with 1000 pM without risk of significant media deprivation.

[0081] Discussion of Data

[0082] Development of a Schwann Cell Model of Cisplatin-Induced Injury

[0083] Given the deleterious effects of cisplatin on the inner ear, a cellular model can be a helpful first step in identifying therapeutic agents to mitigate this effect. In this RSC96 Schwann cell model the LC50 of cisplatin was 4 pM. Since standard chemotherapy treatments in humans attain a cisplatin concentration of 0.41 pM - 9.27 pM17, this experiment suggests that Schwann cells may undergo damage during chemotherapeutic treatment at concentrations attained therapeutically. The findings of the present study, therefore, may harbor high translational impact.

[0084] NAC as a Therapeutic Agent

[0085] The antioxidant NAC was explored for its ability to reduce Schwann cell damage. Two investigated concentrations of NAC were found to reduce cellular injury from cisplatin. NAC also did not demonstrate toxicity in this assay at concentrations approaching its solubility limit, a desirable property when considering that prolonged local delivery often requires high initial concentrations to attain sustained therapy. Furthermore, NAC eluted from both PLGA and PCL microparticles continued to demonstrate a protective effect at the concentrations identified during the screening assay. This is encouraging that the microparticle fabrication process did not affect the bioactivity of the eluted NAC, nor did microparticle degradation during NAC elution release any byproducts that negatively impacted NAC’s viability enhancement.

[0086] Designing a Method of Prolonging N-acetylcysteine Delivery with Microparticles

[0087] Microparticles synthesized with NAC eluted therapeutic concentrations (100 pM or greater) for 2-3 days from PLGA microparticles, and for up to 7 days from PCL microparticles. All conditions exhibited a large initial “burst release” with the most sustained release from PCL. Since variable amounts of microparticles were weighed into each condition’s transwell before measuring elution, the concentrations eluted were not directly comparable. The transwells containing PCL microparticles had a much lower microparticle mass loaded into the transwells, a result of lower overall yield in those conditions. When this is considered, it is likely that a larger mass of PCL, on the order of that weighed out for the PLGA conditions, would result in a therapeutic concentration of NAC for a period of time longer than 7 days.

[0088] The average diameter of PLGA microparticles was smaller than that of PCL. The PLGA microparticles were intentionally fabricated to be larger than those previously reported by our laboratory (3-4 pm versus 1 pm) in an effort to encapsulate more NAC and prolong elution, but this did not appear to significantly change the release profile.15

[0089] Since PCL microparticles had the longest sustained release, it is likely that their larger microparticle size allowed for more encapsulation of NAC and a longer duration of release as the microparticle degraded. The duration of therapeutic concentrations of NAC delivered may be increased in the future by increasing the mass of NAC microparticles added to each transwell. Larger masses of NAC microparticles eluted into smaller volumes may allow the concentration delivered to exceed 100 pM for longer periods of time. As the volume of the perilymph in the inner ear is 158.5 pL and we eluted into solutions of 1500 pL, the same weight of microparticles could attain a concentration of 100 pM for a longer period of time in a smaller more physiologic volume.18

[0090] In in vivo applications, PCL microparticles are suitable. PCL containing drug delivery systems demonstrate less of an inflammatory response than PLGA microparticles.19-20Additionally, PCL degrades at a slower rate in part because of its hydrophobic properties and higher molecular weight, making it more advantageous for a clinical application where chemotherapy treatment occurs over weeks to months.21,22The PCL microparticles also eluted a larger amount of NAC per weight.

[0091] Conclusion

[0092] Patients given cisplatin-based chemotherapy could benefit from the local administration of an agent that protects their inner ear from damage, ideally for a period of weeks to months. RSC96 Schwann cells were used as a model to investigate cisplatin-induced Schwann cell injury7. A promising therapeutic, N-acetylcysteine, was investigated both alone and when blended into several t pes of drug-eluting microparticles. The ability7of this therapeutic to demonstrate protection of RSC96 cells is encouraging that this agent may be an effective method of reducing cisplatin- induced Schwann cell injury7.

[0093] Example 1 References

[0094] 1. Callejo A, Sedo-Cabezon L, Domenech Juan I, Llorens J. Cisplatin-Induced Ototoxicity7: Effects, Mechanisms and Protection Strategies. Toxics. 2015;3(3):268- 293. doi:10.3390 / toxics3030268

[0095] 2. Karasawa T, Steyger PS. An integrated view of cisplatin-induced nephrotoxicity7and ototoxicity. Toxicol Lett. 2015;237(3):219-227. doi: 10. 1016 / j.toxlet.2015.06.012

[0096] 3. Rybak LP, Mukheijea D, Jajoo S, Ramkumar V. Cisplatin Ototoxicity and Protection:

[0097] Clinical and Experimental Studies. Tohoku J Exp Med. 2009;219(3): 177-186.

[0098] 4. Jirsova K, Mandys V, Gispen WH, Bar PR. Cisplatin-induced apoptosis in cultures of human Schwann cells. Neuroscience Letters. 2006;392(l):22-26. doi:10.1016 / j.neulet.2005.08.068

[0099] 5. Sugimoto T, Takeyama A, Fujita M, Ichikawa H, Takano-Yamamoto T. Peripheral neuroglial death induced by cisplatin administration in newborn rats. Neuroreport. 2001:12(1): 137-140. doi: 10. 1097 / 00001756-200101220-00035

[0100] 6. Wan G, Corfas G. Transient auditory nerve demyelination as a new mechanism for hidden hearing loss. Nat Commun. 2017;8(l): 14487. doi: 10.1038 / ncommsl4487

[0101] 7. Budak M, Grosh K, Sasmal A, Corfas G, Zochowski M, Booth V. Contrasting mechanisms for hidden hearing loss: Synaptopathy vs myelin defects. PLOS Computational Biology. 2021 ; 17(1 ):el 008499. doi: 10. 1371 / joumal.pcbi. 1008499

[0102] 8. Balakrishnan A, Belfiore L, Chu TH, et al. Insights Into the Role and Potential of Schwann Cells for Peripheral Nerve Repair From Studies of Development and Injury. Frontiers in Molecular Neuroscience. 202I;13. Accessed June 13. 2022. https: / / www.frontiersin.org / article / 10.3389 / fnmol.2020.608442

[0103] 9. Gui T, Wang Y, Zhang L, Wang W, Zhu H, Ding W. Kriippel-Like Factor 6 Rendered Rat Schwann Cell More Sensitive to Apoptosis via Upregulating FAS Expression. PLoS One. 2013;8(12):e82449. doi: 10.1371 / joumal.pone.0082449

[0104] 10. Brock PR, Maibach R, Childs M. et al. Sodium Thiosulfate for Protection from Cisplatin- Induced Hearing Loss. N Engl J Med. 2018;378(25):2376-2385. doi : 10. 1056 / NEJMoal 801109 11. Dickey DT, Wu YJ, Muldoon LL, Neuwelt EA. Protection against Cisplatin- Induced

[0105] Toxicities by N-Acetylcysteine and Sodium Thiosulfate as Assessed at the Molecular, Cellular, and in Vivo Levels. J Pharmacol Exp Ther. 2005;314(3): 1052-1058. doi:l 0.1124 / jpet. 105.087601

[0106] 12. Schwalfenberg GK. N- Acetyl cysteine: A Review of Clinical Usefulness (an Old Drug with New Tricks). J Nutr Metab. 2021;2021 :9949453. doi: 10. 1155 / 2021 / 9949453

[0107] 13. Tenorio MC dos S, Graciliano NG, Moura FA, de Oliveira ACM, Goulart MOF. NAcetylcysteine (NAC): Impacts on Human Health. Antioxidants (Basel). 2021;10(6):967. doi: 10.3390 / antioxl0060967

[0108] 14. Hong MK, Echanique KA. Hoffman LF, Kita AE. Designing a Prolonged Method of

[0109] Therapeutic Delivery to Support Rehabilitation From Ototoxic Damage in a Schwann Cell Model. Otol Neurotol. 2023;44(4):373-381. doi: 10.1097 / MAO.0000000000003839 15. Murphy NP, Lampe KJ. Fabricating PLGA microparticles with high loads of the small molecule antioxidant N- acetylcysteine that rescue oligodendrocyte progenitor cells from oxidative stress. Biotechnology and Bioengineering. 2018;l 15(l):246-256. doi: 10.1002 / bit.26443

[0110] 16. Urien S, Lokiec F. Population pharmacokinetics of total and unbound plasma cisplatin in adult patients. British Journal of Clinical Pharmacology. 2004;57(6):756- 763. doi: 10. 1111 / j. 1365-2125.2004.02082.x

[0111] 17. Buckingham RA, Valvassori GE. Inner Ear Fluid Volumes and the Resolving

[0112] Power of Magnetic Resonance Imaging: Can it Differentiate Endolymphatic Structures? Ann Otol Rhinol Laryngol. 2001 ; 1 10(2): 1 13-1 17. doi: 10. 1177 / 000348940111000204 18. Wong DY, Hollister SJ, Krebsbach PH, Nosrat C. Poly(epsilon-caprolactone) and poly (Llactic- co-glycolic acid) degradable polymer sponges attenuate astrocyte response and lesion growth in acute traumatic brain injury. Tissue Eng. 2007;13(10):2515-2523. doi:10.1089 / ten.2006.0440

[0113] 19. Ma S, Feng X, Liu F, Wang B, Zhang H, Niu X. The pro-inflammatory response of macrophages regulated by acid degradation products of poly(lactide-co-glycolide) nanoparticles. Engineering in Life Sciences. 2021;21(10):709-720. doi: 10.1002 / elsc.202100040

[0114] 20. Makadia HK, Siegel SJ. Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery’ Carrier. Polymers (Basel). 2011;3(3): 1377-1397. doi: 10.3390 / polym3031377

[0115] 21. Mohan N, Nair PD. Polyvinyl alcohol-poly (caprolactone) semi IPN scaffold with implication for cartilage tissue engineering. J Biomed Mater Res B Appl Biomater. 2008;84(2):584-594. doi:10. 1002 / jbm.b.30906

[0116] EXAMPLE 2: TRANSTYMPANIC INJECTION OF ANTIOXIDANT-ELUTING MICROPARTICLES FOR OTOPROTECTION FROM CISPLATIN TOXICITY IN A MOUSE MODEL

[0117] Example 2 Summary

[0118] Objective: Cisplatin is a chemotherapeutic agent with the undesirable side effect of ototoxicity. Transtympanic injections of antioxidant formulations may provide local otoprotection. We tested a novel antioxidant-eluting microparticle for its otoprotective capability from systemic cisplatin as measured by cochlear electrophysiology.

[0119] Study Design: Basic science.

[0120] Setting: Translational research laboratory’. Methods: Eighteen mice were assigned to 3 groups. All mice underwent baseline click-evoked auditory brainstem response (ABR) audiometry7and right ear microparticle injections before beginning 42-day intraperitoneal administration regimens of either saline (healthy control empty microparticle group - HCEMP) or cisplatin (cisplatin empty7microparticle group - CEMP and cisplatin N-acetylcysteine microparticle group - CNAC). These regimens consisted of three 4-day cycles of intraperitoneal saline or cisplatin administration followed by 10 rest days. HCEMP and CEMP received right-sided transtympanic empty microparticles and CNAC received transtympanic N-acetylcysteine eluting microparticles. On day 43, all mice underwent post-treatment ABR. ABR thresholds and threshold shifts were analyzed with mixed effects models and Tukey's post-hoc tests and were compared across pre- / post-treatment ears, treatment groups, and injected and non-injected ears.

[0121] Results: We found that threshold shifts in the ears that received a transtympanic injection of N-acetylcysteine and three cycles of intraperitoneal cisplatin were similar to the paired ears of mice that received no cisplatin. Mice that received a transtympanic injection without N-acetylcysteine and intraperitoneal cisplatin had increased thresholds compared to mice that received a transtympanic injection of N- acetylcysteine and cisplatin.

[0122] Conclusions: Transty mpanic N-acetylcysteine microparticle injections provided functional otoprotection in cisplatin-exposed mice.

[0123] Cisplatin is a commonly used chemotherapeutic that crosslinks DNA in cancer cells to cause cell cycle arrest, but it has many known adverse effects including irreversible ototoxicity .1 4Dose-dependent toxicity7occurs in both children and adults and is linked to genetic susceptibilities.4Its toxicity is likely a combination of oxidative stress, inflammation, apoptosis, and autophagy.4Cisplatin has been detected in the cochlea for months-to-years after therapy, emphasizing the need for a long- lasting therapeutic? There is no local therapeutic currently available to prevent cisplatin-mediated ototoxicity.6,7While systemic infusion of FDA-approved sodium thiosulfate (Pedmark) has been found to prevent ototoxicity in certain pediatric cancers, there are concerns that systemic therapies may limit the antitumor properties of cisplatin.6Additionally, approximately one-third of Pedmark treated patients still develop hearing deficits.8,9Other antioxidants, like N-acety lcysteine (NAC), may limit cisplatin’s ototoxic effects through suppression of the immune system. It can be hypothesized that NAC’s sulfhydryl groups protect hair cells and suppress inflammation through replenishing glutathione, scavenging free radicals, and inhibiting NF-KB inhibition.10 11Both intravenous and oral NAC are used to treat inflammatory conditions and toxic doses of acetaminophen, paracetamol, carbon monoxide, and x-ray contrast.10 12NAC’s versatility may allow it to be incorporated into an effective locally delivered otoprotective therapeutic, reducing concerns of interference with cisplatin’s anti-tumor effects.

[0124] Transtympanic injection (TTI) is a logical local drug delivery’ method, since other inner ear drug delivery methods, such as direct intracochlear drug administration and osmotic minipumps, are clinically difficult and require surgery.12 17TTIs can attain high perilymph concentrations of otoprotective agents through round or oval window diffusion.4TTIs routinely are performed in awake patients in the office by otolaryngologists, such as with steroids or gentamicin. Gausterer et al. demonstrated that a poloxamer hydrogel loaded with the antioxidant N-acetylcysteine deployed via TTI to the middle ear resulted in increased and sustained delivery of N-acetylcysteine to the perilymph.18Plasma concentrations of NAC remained unchanged, indicating that TTIs would not compromise the antineoplastic effects of cisplatin.18

[0125] Delivery of a therapeutic for a prolonged period is important given routine multi-cycle courses of cisplatin with cycles spaced weeks apart. We hypothesized that a combined NAC-eluting polycaprolactone (PCL) microparticle (MP) and thermosensitive hydrogel could be delivered via TTI to the middle ear in a mouse model. Furthermore, we hypothesized that this vehicle could prolong the delivery' of NAC to the inner ear via diffusion through the round window, thus protecting against cisplatin ototoxicity, as measured by click-evoked auditory' brainstem responses (ABR). Click-evoked ABRs have been used to measure the ototoxicity of cisplatin in many different animal models.13,19-21NAC was selected due to its promise in animal and clinical studies, and in our previous bioengineering and cell experiments where it was combined to make NAC PCL MPs.22-26PCL MPs are known for their hydrolytic breakdown, prolonged in vivo degradation times, and minimal inflammatory response.27We previously released NAC for nearly 2 weeks from PCL MPs at concentrations that promote cell viability after cisplatin exposure.22,23Poloxamer 407 (Pluronic) is a thermosensitive hydrogel that polymerizes at body temperature, allowing injection of the NAC PCL MP and hydrogel solution as a liquid. The injected solution can then polymerize over the round window when a patient is recumbent, limiting drug loss through the eustachian tube.27-29

[0126] Methods:

[0127] Microparticle Synthesis and Characterization:

[0128] We modified a protocol adapted from Murphy et al. to create NAC PCL MPs.30NAC (Sigma Aldrich, A7250 Saint Louis, MO) was added to reagents in multiple steps to enhance MP antioxidant loading. Batches of empty MPs (EMP; no NAC) were created as controls. We fully dissolved 2% PCL (Ashland, AS009, PolySciTech. West Lafayette, MI) in dichloromethane (DCM) overnight in a fume hood. The loaded PCL / DCM batches were then maximally saturated with NAC (50mg / mL) and pulse-sonicated for four cycles of 5 seconds on and 3 seconds off at a power setting of 5 on a Misonix Ultrasonic Liquid Processor (Misonix, XL-2000, Farmingdale, NY) before being added dropwise to 1% PVA / 2.5% NAC and mixed using a dispersing instrument (IKA, T18 Digital Ultra-Turrax, Wilmington, NC) at 3,000 rpm for 15 minutes. After homogenization, the mixture was poured into 0.3% PVA / 5% NAC and stirred at 700rpm for 2 hours at 40°C. The solution was then centrifuged at 4500 rpm for 5 minutes. Microparticles were subsequently centrifuged at 4500 rpm for 5 minutes. The supernatant was discarded and the pellet of microparticles was resuspended with 2.5% NAC in double distilled water (ddELO) three times. The final 1 mL of microparticle solution was stored at -80°C. The frozen MPs were then lyophilized and stored at -20°C. All EMP reagents and washes lacked NAC.

[0129] To confirm elution, MPs were suspended in 20% Pluronic (Sigma Aldrich, Poloxamer 407 P2443 Saint Louis, MO) in ddl EO at a concentration of lOmg / lOOpL. These solutions were added into transwells suspended in 600pL of ddELO in a 24- well plate (in triplicate). After 24 hours, eluted NAC was detected via incubation with Ellman’s reagent, which reacts with NAC’s sulfhydryl group to generate a colorimetric readout quantified by a plate reader.

[0130] Microparticle Sterilization:

[0131] We sterilized dry reagents in glass vials with UV-C light. Pluronic was spaced 24cm away from a biosafety cabinet’s 2542 UV-C germicidal bulb (Philips 30W TUV T8 Germicidal Fluorescent Tube, Amsterdam, Netherlands) for 1 hour. Lyophilized MPs were kept on cold packs and sterilized for 3 hours. Sterilized Pluronic and MPs were stored at room temperature and at -20°C, respectively.

[0132] Therapeutic Creation:

[0133] A 20% Pluronic solution was made by adding Pluronic to ddELO on ice and vortexing. The solution was stored at 4°C until use. On the day of transtympanic injection, MPs were added to the 20% Pluronic solution at a concentration of approximately lOOmg / mL. The solution was mixed by vortexing until uniformly suspended. Approximately 150-200pL of solution was loaded into a LOmL zero-dead space syringe and placed on ice until administration.

[0134] Animals: We adapted a clinically relevant cisplatin injury mouse model to study our TTI therapeutic in eighteen adult (9-10 week old) CBA / CaJ male and female mice (Jackson Laboratories, Bar Harbor, Maine, USA).31Younger mice were used to limit known age-related hearing loss, often seen by 16-18 months.32UCLA’s Animal Research Committee approved this protocol. Sex-balanced mice were assigned to one of three groups (n=6 per group). Same sex mice were housed together within each group (n=3 per cage) and provided food and water ad libitum. Nine week (Group 1) and ten week (Groups 2 and 3) mice began a 42-day intraperitoneal (IP) administration regimen of either saline (Group 1, control) or cisplatin (Groups 2 and 3). These regimens consisted of 3x 4-day cycles of IP saline or cisplatin separated by 10 rest days (Figure 7A). We injected 3.0 mg / kg of cisplatin (Fresenius Kabi, 100365 Lake Zurich, IL) daily, for each cycle. Weekly weights were recorded for Group 1 and daily for Groups 2 and 3. Cisplatin-injected mice received supplemental hydration and nutrition given cisplatin-associated weight loss (Figure 7B). Investigators and veterinary staff monitored all mice for health changes; which included body condition scores (BCS), grimace scale, and daily weights.33As mandated by our protocol, malnourished mice would be promptly euthanized if a BCS score was <2. No mice met this euthanasia criteria during our study. A size of 6 animals per group was selected due to the time required for bilateral hearing testing in each group in comparable time frames and the ability to administer three cycles of cisplatin, along with daily care with the necessary precautions required by our vivarium for ethical treatment and safe handling.

[0135] Auditory Brainstem Response Recording:

[0136] Cochlear function was assessed with ABR testing. Mice were anesthetized with isofl wane and placed in an anechoic booth. Warmed 50mL conical tubes of water were placed alongside each mouse to maintain core body temperature. Temperature was monitored with a rectal probe. Subdermal needle electrodes were placed at the vertex (non-inverting), each pinna (inverting), and at the tail base (ground). Acoustic stimuli were directed to each external auditory meatus via a tube coupled to a speaker (MF-1 speaker, Tucker-Davis Technologies (TDT); Alachua, FL, USA).

[0137] ABRs were recorded (RZ6 processor, BioSigRZ; TDT) in response to click stimuli delivered at alternating polarity and a rate of 21 / sec. Response windows of 10ms were amplified, filtered (0.3 to 3 kHz), and averaged (n=512) to produce response waveforms. Duplicate ABRs were recorded for each stimulus intensity level, starting at lOOdB and decreased in 5 dB steps. These traces were transferred to GraphPad Prism (Boston, MA, USA) and visually inspected. We examined replicate waveform traces for waves I-V, and thresholds were defined as the lowest stimulus magnitudes in which wave III was observed. ABRs were measured both at the start and at the completion of the 42-day injection protocol. Intensity levels of 70dB to 30dB were tested at the start of the protocol and intensity levels of lOOdB to 30dB were tested at the end of the protocol given the observed decline in hearing seen in control mice.

[0138] Transtympanic Therapeutic Injections:

[0139] Immediately after recording ABRs and under isoflurane anesthesia, mice underwent unilateral right ear TTI. A stereomicroscope was focused on the right ear. Mice were supinated and the external auditory canal skin was retracted to visualize the tympanic membrane (TM). A 26 gauge needle was used to perforate the TM pars tensa, and approximately 15pl of MP Pluronic therapeutic was administered into the middle ear space, sufficiently filling the middle ear in all mice. Mice remained supinated for 15 min to allow curing of the Pluronic before transferring to a recovery cage. IP regimens then began on the first day after ABR and TTI (see Fig. 7). Group 1 saline control mice received EMP TTI (HCEMP - healthy control empty microparticle group). Group 2 cisplatin mice also received EMP TTI (CEMP - cisplatin empty microparticle group), while Group 3 cisplatin mice were treated with NAC eluting microparticle TTI (CNAC - cisplatin NAC microparticle group). Ears were assessed under the stereomicroscope for persistent effusions and TM healing prior to post-treatment AB Rs.

[0140] Statistics:

[0141] Statistics were performed in GraphPad. Thresholds for all pre- / post-treatment ears were determined from averaged waveform traces. Pretreatment left ear thresholds were averaged and rounded to the nearest 5 dB. serving as our cohort's normal hearing level. This average, 55 dB, was used as our audiometric zero (0 dB HL) and all thresholds were normalized to this value. A mixed-effects analysis (a=0.05) was used to compare thresholds between saline and cisplatin treatment groups, pre- / post- IP cycles, and injected (right) and non-injected (left) ears. Analysis was followed with post-hoc Tukey’s multiple comparisons analyses. Pre- / post-treatment thresholds were used to display threshold shifts, which were compared with mixed-effects and post- hoc Tukey’s analyses. Adjusted means (Least Squares (LS) Means) were computed as part of post-hoc testing to compare unequal group sizes.

[0142] Results:

[0143] NAC Elution:

[0144] The elution of NAC from NAC MPs in Pluronic was evaluated using Ellman’s reagent to read NAC concentration from a standard curve. In Fig. 8, the mean quantity of NAC was 133.4 pg after 24 hours (n=3). No NAC was detected in the eluent from EMP in Pluronic, demonstrating the absence of confounding sources of fluorescence or contamination of our control therapeutic with NAC.

[0145] Systemic Cisplatin Toxicity:

[0146] Cisplatin treated mice lost 10-20% of their starting body weight over the 42 day protocol while mice not treated with cisplatin linearly gained roughly 20% in body weight. Mice weights are displayed as a percentage of initial pretreatment body weight (Figure 9). Cisplatin groups lost 10-20% of their body weight after each cisplatin cycle. During rest periods, some cisplatin-treated mice returned to baseline weights while others remained in the 10-15% deficit range. A drop of approximately 10% in weight was seen in the CEMP mice from days 24-25. There was one intraprotocol fatality in the CNAC group during an IP injection.

[0147] ABR Results:

[0148] We observed (1) otoprotection in ears treated with NAC injections, (2) systemic cisplatin induced ototoxicity in untreated ears, and (3) modest hearing loss secondary to our TTIs based on comparisons of pre- and post-treatment thresholds and threshold shifts (Figure 10). All groups had elevated post-treatment thresholds compared to their paired pre-treatment thresholds.

[0149] Post-treatment right CNAC ears had increased thresholds compared to their pre-treatment thresholds that while significant (least squares mean difference (LSMD)=17.66dB±4.070, p=0.0434), were less than the changes seen in their paired post-treatment control left ears (LSMD=-20.72dB±4.655, p=0.0375). Post-treatment right CNAC ears also had smaller thresholds compared to post-treatment CEMP right ears (LSMD=-20.67dB±4.919, p=0.0131). Smaller threshold shifts were also observed for CNAC right ears wften compared to untreated CNAC left ears (LSMD=- 13.75dB±5.454, P=0.0303), and CEMP right ears (LSMD=-16.25dB±5.550, p=0.0194). The less increased post-treatment thresholds seen in CNAC right ears after cisplatin therapy compared to paired pre-treatment thresholds was consistent with an otoprotective impact of eluted NAC. NAC treated ears also had post-treatment thresholds similar to FICEMP post-treatment right and left ears (LSMD=2.34dB±4.919, p>0.9999, LSMD=9.326dB±4.919, p=0.7508).

[0150] Ears that received cisplatin and no NAC had similarly increased posttreatment thresholds. Post-treatment left CNAC ears had thresholds similar to left CEMP ears (LSMD=-3.284dB±4.658, p=0.9998) and had thresholds significantly larger than the post-treatment HCEMP left ears (LSMD=30.05dB±4.658, p<0.0001). Thus, left ears illustrated the expected impact of cisplatin on unprotected cochleae. Comparisons of thresholds between post-treatment right and left ears in the HCEMP and CEMP group were similar (11.67dB±4.023, p=0.2761, & 3.333dB±4.023, p=0.9985 respectively), a finding which suggests that hearing loss seen is not secondary to the TTIs.

[0151] No persistent effusions were appreciated during post-treatment ear inspections. One post-treatment CNAC mouse’s ear was not completely visualized, likely secondary to clot formation, given the bleeding seen at the time of initial injection.

[0152] Discussion:

[0153] Otoprotective effects

[0154] The systemic effects of cisplatin are well established in humans and have been demonstrated to cause weight loss and hearing loss in mice. Most cisplatin treatments involve cyclical medication delivery, so replicating this in an animal model is more physiologic than single time-point cisplatin administration studies. Long-acting therapeutics that protect against the cyclical nature of cisplatin deliver}' are needed. Our study adapted the cisplatin ototoxicity protocol created by Fernandez et al. to show its usefulness as a model to study the otoprotective abilities of an NAC microparticle hydrogel therapeutic.31Mice treated with cisplatin developed significantly worse hearing loss compared to mice that did not receive cisplatin or that received the NAC microparticle hydrogel therapeutic. This suggests that intraperitoneally-administered cisplatin reached the inner ear and caused ototoxicity. Empty MP hydrogel TTIs did not show protection, suggesting that NAC is an effective otoprotectant against cisplatin and suggests that the PCL MP component and Pluronic hy drogel act only as vehicles for NAC. Furthermore, TTI was performed in all right ears without observed hearing loss attributable to the injection. This suggests that any conductive hearing loss from the TTI is not long-lasting. Post-treatment thresholds were elevated for all groups. This is likely secondary to loud noise exposure in our shared vivarium space. Vivariums commonly expose mice to moderate-level baseline noise, along with loud noises (>80dB), often related to human work activities (i.e., slamming doors, cage manipulations, etc.).34

[0155] Since multiple cycles of cisplatin are typically required, a prolonged-release therapeutic with a thermosensitive gel that targets release of microparticles is likely superior to NAC injection alone in suspension. Tn this model, an NAC microparticle hydrogel therapeutic provided otoprotection with NAC at a concentration of 100 mg / mL microparticles (10%). Lower concentrations of NAC have been less effective in animal and small clinical trials.25,35Yoo et al. observed no improvement in ABR thresholds compared to untreated ears in 11 patients receiving Cisplatin treated with 2% NAC TTIs.35Higher concentrations of NAC have been more effective. Riga et al. studied 20 patients and saw elevated thresholds in control ears compared to ears treated with 10% NAC injections.24Sarafraz et al. also used 10% NAC and compared thresholds at 8kHz in 120 patients treated with either NAC or dexamethasone.26They observed no significant threshold shift or incidence of tinnitus in the NAC treated group.26These findings suggest concentrations of NAC >10% could limit cisplatin mediated cochlear damage. Unlike our exploratory study, these trials administered NAC TTIs prior to every cisplatin cycle. Their NAC was not incorporated into a prolonged-release therapeutic and required frequent re-administration, unlike the microparticles explored in this study. While our data shows significant functional otoprotection, the exact quantity' of NAC that diffused across the round window is unknown. We further found that >10% concentrations of MPs were viscous and difficult to mix and inject.

[0156] Conclusions

[0157] An N-acetylcysteine antioxidant-releasing microparticle hydrogel therapeutic can be delivered locally through a single transtympanic injection and provide otoprotection after three cycles of cisplatin therapy in a mouse model. This is a promising first step in the development of a single administration prophylactic agent for otoprotection from platinum-based agents. Example 2 References

[0158] 1. Karasawa T, Sleyger PS. An integrated view of cisplatin-induced nephrotoxicity and ototoxicity. Toxicol Lett. 2015;237(3):219-227. doi:10.1016 / j.toxlet.2015.06.012

[0159] 2. Qi L, Luo Q, Zhang Y, Jia F, Zhao Y, Wang F. Advances in Toxicological Research of the Anticancer Drug Cisplatin. Chem Res Toxicol. 2019;32(8): 1469-1486. doi : 10. 1021 / acs . chemrestox.9b00204

[0160] 3. Dillard LK, Lopez-Perez L, Martinez RX, Fullerton AM, Chadha S, McMahon CM. Global burden of ototoxic hearing loss associated with platinumbased cancer treatment: A systematic review and meta-analysis. Cancer Epidemiol. 2022:79: 102203. doi : 10. 1016 / j . canep.2022. 102203

[0161] 4. Yu D, Gu J, Chen Y. Kang W, Wang X, Wu H. Current Strategies to Combat Cisplatin-Induced Ototoxicity. Front Pharmacol. 2020; 11:999. doi:10.3389 / fphar.2020.00999

[0162] 5. Breglio AM, Rusheen AE, Shide ED, et al. Cisplatin is retained in the cochlea indefinitely following chemotherapy. Nat Commun. 2017;8(l): 1654. doi: 10.1038 / s41467-017-01837-l

[0163] 6. Chattaraj A, Syed MP, Low CA, Owonikoko TK. Cisplatin-Induced Ototoxicity : A Concise Review of the Burden, Prevention, and Interception Strategies. JCO Oncol Pract. 2023;19(5):278-283. doi: 10.1200 / GP.22.00710

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[0165] 8. Brock PR, Maibach R, Childs M, et al. Sodium Thiosulfate for Protection from Cisplatin-Induced Hearing Loss. N Engl J Med. 2018;378(25):2376-2385. doi : 10. 1056 / NEJMoal 801109 9. Freyer DR, Chen L, Krailo MD, et al. Effects of sodium thiosulfate versus observation on development of cisplatin-induced hearing loss in children with cancer (ACCL0431): a multicentre, randomised, controlled, open-label, phase 3 trial. Lancet Oncol. 2017;18(l):63-74. doi: 10.1016 / S1470-2045(16)30625-8

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[0189] PUBLICATIONS

[0190] All publications mentioned herein (e.g., those listed above such as Hong et al., Tool Neurotol. 2023;44(4):373-381) are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification. The following references include descriptions of methods and materials in this field of technology.

[0191] CONCLUSION

[0192] This concludes the description of the illustrative embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

Claims

CLAIMS:

1. A method of making a hydrogel composition, the method comprising combining: at least 14% w / v of a non-ionic copolymer surfactant having the linear formula (CsHeO C2H4O)x; microparticles comprising a polycaprolactone; and a therapeutic agent; such that the hydrogel composition is made; wherein the copolymer surfactant, the microparticles and the therapeutic agents are combined such that the hydrogel composition can be manually injected through a 27 gauge needle at room temperature and further forms a gel in less than 30 minutes upon exposure to human body temperature.

2. The method of claim 1, wherein:The therapeutic agent comprises N-Acetylcysteine (NAC): and / or the method comprises forming the composition releases > 100 pM of the therapeutic agent from the microparticles over at least one week.

3. The method of claim 1, further comprising forming the composition so that no new bonds are created in the composition that alter the structures of the therapeutic agent as measured by Fourier transform infrared spectroscopy (FTIR).

4. The method of claim 2, further comprising combining the hydrogel composition with live mammalian cells.

5. A hydrogel composition comprising: a non-ionic copolymer surfactant having the linear formula (CsFLO CLFhOjx; microparticles comprising a polycaprolactone; and a therapeutic agent.

6. The composition of claim 5, wherein the composition comprises:at least 14% w / v non-ionic copolymer surfactant; and / or less than 100 mg / mL microparticles comprising a poly caprolactone.

7. The composition of claim 6, wherein: the non-ionic copolymer surfactant comprises Pluronic® F-127; the poly caprolactone comprises a C18E poly caprolactone; the therapeutic agent is disposed within the microparticles and comprises at least one of an antioxidant or a steroid; the composition can be manually injected through a 27 gauge needle at room temperature; and / or the composition forms a gel in less than 30 minutes upon exposure to human body temperature8. The composition of claim 7, wherein: the composition comprises 20% Pluronic F127; the therapeutic agent comprises at least one of an antioxidant, a steroid, or an antibiotic; the composition further comprises a pharmaceutical excipient or an imaging agent; and / or the therapeutic agent comprises N-Acetylcysteine (NAC) such that the composition releases > 100 pM NAC from the microparticle for at least one week following disposing the composition in a tissue region of a subject.

9. The composition of claim 5, wherein the composition does not comprise a chitosan.

10. A method of delivering the composition of claim 5 to a subject, the method comprising: disposing the hydrogel composition at a site where the hydrogel composition is in direct contact with a first tissue region of the subject.11 . The method of claim 10, wherein: the composition is delivered topically to the middle ear of the subject; the composition is delivered via injection transtympanically to the subject; the composition is delivered topically to stretched or injured nerves of the subject; the composition is delivered to stretched or injured nerves of the subject by injection.

12. The method of claim 11, wherein the subject is selected to be a patient having been administered cisplatin or other ototoxic agent or undergone nerve injury.

13. The method of claim 10, wherein amounts of the composition administered are sufficient to reduce cellular injury in response to agents administered to the subject such as cisplatin or hydrogen peroxide.

14. The method of claim 12, wherein amounts of the composition administered are sufficient to preserve hearing function in a mouse model of cisplatin ototoxicity.

15. The method of claim 10, wherein the composition is administered through an at least 27 gauge needle.

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