Injectable polymer scaffolds

Abstract

Provided herein are injectable polymer scaffolds that provide a niche for the evaluation of health status and systemic disease without the need for surgical implantation and biopsy of scaffolds. In some embodiments, the implant serves as a synthetic metastatic niche that can capture the changing immune responses observed during disease progression and recapitulate immune and phenotypes found in the natural metastatic niche ( e.g., lung).

Description

[0001] UM-44857.601

[0002] INJECTABLE POLYMER SCAEFOLDS

[0003] The present application claims priority to United States Provisional Application Serial Number 63 / 776,569, filed March 24, 2025, the disclosure of which is herein incorporated by reference in its entirety.

[0004] STATEMENT REGARDING FEDERAL FUNDING

[0005] This invention was made with government support under CA272940 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] Provided herein are injectable polymer scaffolds that provide a niche for the evaluation of health status and systemic disease without the need for surgical implantation and biopsy of scaffolds.

[0008] BACKGROUND

[0009] Breast cancer has the highest rates of incidence and mortality in women worldwide. Specifically, triple-negative breast cancer (TNBC), characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, presents a particularly poor prognosis due to its highly metastatic nature and elevated risk of distant recurrence. Despite significant advances in characterizing primary tumors through imaging, histological analysis, and sequencing for mutation identification, these methods primary focus on the primary tumor and often fail to adequately address metastatic disease. Extensive efforts to identify biomarkers for cancer staging and personalized treatment at the primary tumor site have yet to provide conclusive results regarding metastatic potential.

[0010] Current methods to detect metastatic progression involve imaging techniques such as computed tomography, which often rely on late-stage self-reporting by patients and only detect the disease when the tumor has reached a significant size and has already caused organ dysfunction. Alternatively, in vitro molecular diagnostics for assessing metastatic potential involve analyzing biological samples from the patient and conducting laboratory tests to identify specific biomarkers, including gene signatures. While this method offers more information compared to traditional imaging and pathology methods, it faces significant limitations. LiquidUM-44857.601

[0011] biopsies, such as blood samples, capture circulating tumor cells that may not provide sufficient information about the specific characteristics and behavior of metastatic tumors, as these cells lack tissue context and are often low in abundance. Conversely, tissue biopsies carry the risk of impairing vital organs and may not be feasible or accessible for all patients. The difficulty in capturing a limited number of circulating tumor cells from liquid biopsies and analyzing non-metastatic tumor cells has hindered accurate diagnosis. Furthermore, adaptive changes occurring at the metastatic site contribute to the failure of conventional treatments, including chemotherapy and immunotherapy. Better systems are needed.

[0012] SUMMARY

[0013] Provided herein are injectable polymer scaffolds that provide a niche for the evaluation of health status and systemic disease without the need for surgical implantation and biopsy of scaffolds. In some embodiments, the implant serves as a synthetic metastatic niche that can capture the changing immune responses observed during disease progression and recapitulate immune and phenotypes found in the natural metastatic niche (e g., lung).

[0014] To address the limitations of late detection that plague existing technologies, provided herein are technologies for early assessment of disease progression. Cancer cell dissemination to specific organs is influenced by receptive microenvironments characterized by supportive cells such as fibroblasts and immune cells, which secrete matrix proteins, cytokines, and chemokines to facilitate cancer cell migration, invasion, proliferation, and angiogenesis.

[0015] Previously, our research has demonstrated that implantable biomaterial scaffolds recapitulate characteristics of the natural metastatic niche (see e.g., US20140072510 and US20190008971, herein incorporated by reference in their entireties). The scaffold’s porous structure supports cellular infiltration and vascular growth, while the foreign body response attracts immune cells to its surface. Immunosuppressive immune cells are recruited to the scaffold, reflecting the systemic changes in the immune response during cancer progression and effectively transforming the scaffold into a metastatic niche.

[0016] The compositions and method herein provide an advanced diagnostic tool that assesses the microenvironment conditions at metastatic sites without invasive surgical implantation. The injectable polymer scaffolds, for example, mirror the size and geometry of the implantableUM-44857.601

[0017] scaffold pores; hence, forming a porous structure post-bulk injection. Experiments conducted during the development of embodiments of the technology demonstrated that these injectable scaffolds recruit immune cells reflective of disease progression. The injectable scaffolds allow for the recruitment of tumor cells prior to metastasis, providing an early indication of disease advancement. Fine needle aspiration was performed to collect immune cells from the scaffold for downstream analysis. Examination of the gene expression profiles from these fine needle aspirates demonstrated the capability to trace disease progression, underscoring the diagnostic use of the injectable scaffold. This approach offers minimally invasive compositions and methods to monitor disease state, guiding early therapeutic interventions and improving patient outcomes.

[0018] In some embodiments, provided herein are injectable implants comprising a polymer configured to form a scaffold when injected in vivo into a subject, wherein said scaffold is configured to recruit cells or other biological materials (e.g., circulating metastatic cells, immune cells, etc.). In some embodiments, the injectable implants are contained a gel or a gel precursor material. In some embodiments, the polymer comprises poly(e-caprolactone) (PCL), poly(lactic acid) (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLG or PLGA), or combinations thereof. In some embodiments, the implant comprises a plurality of unconnected cubes, disks, spheres, fibers, or pyramids. In some embodiments, the plurality of cubes, disks, spheres, fibers, or pyramids have a maximum dimension of less than 300 pm (e.g., less than 250 pm, 200 pm, 150 pm, 100 pm, 50 pm, of 20 pm).

[0019] Further provided herein are methods comprising, injecting a polymer into a subject (e.g., wherein the polymer is configured to recruit circulating metastatic cells). In some embodiments, the polymer comprises poly(s-caprolactone) (PCL), poly(lactic acid) (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLG or PLGA), or combinations thereof. In some embodiments, the injecting comprises injecting said polymer into a gel or gel precursor contained in a subcutaneous region of said subject. In some embodiments, a gelling agent is administered to the subject to form a gel from the gel precursor materials. In some embodiments, the method further comprises removing a sample from said polymer. In some embodiments, the method further comprises analyzing said sample. In some embodiments, theUM-44857.601

[0020] analyzing comprises detecting one or more biomarkers (e.g., biomarkers indicative of metastasis).

[0021] Further provided herein are methods of manufacturing an injectable scaffold, comprising one or more or each of the steps of a) forming a polymer mold on template, b) forming a sacrificial film on the mold, c) removing the sacrificial film, d) coating the removed sacrificial film with a scaffold polymer, and e) dissolving sacrificial film with a solvent to generate a released scaffold polymer. In some embodiments, the polymer mold comprises polydimethylsiloxane (PDMS). In some embodiments, the template comprises a silicon wafter template. In some embodiments, the template comprises a plurality of wells having a cube, disk, fiber, or pyramid shape. In some embodiments, the generated injectable scaffold has a cube, disk, sphere, fiber, or pyramid shape. In some embodiments, the plurality of wells have a maximum dimension of less than 300 pm (e.g., less than 250 pm, 200 pm, 150 pm, 100 pm, 50 pm, of 20 pm). In some embodiments, the sacrificial film comprises polyvinyl alcohol. In some embodiments, an emulsion process is used to make scaffold polymers having certain shapes (e.g., discs and spheres). In some embodiments, the scaffold polymer comprises poly(s-caprolactone) (PCL), poly(lactic acid) (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLG or PLGA), or combinations thereof. In some embodiments, the solvent comprises water. In some embodiments, the dissolving step further comprises filtering to collect individual injectable scaffolds. Further provided herein are injectable polymer scaffolds made by any such method.

[0022] Further provided herein are methods of manufacturing an injectable scaffold, comprising milling a porous polymer scaffold to an injectable particle size. In some embodiments, the porous polymer scaffold comprises poly(£-caprolactone) (PCL), poly(lactic acid) (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLG or PLGA), or combinations thereof. In some embodiments, the milling comprises mechanical milling using a blade mill. In some embodiments, the porous polymer scaffold has a pore size of from about 63 pm to about 425 pm (e.g., from about 63 pm to about 250 pm, or from about 250 pm to about 425 pm). In some embodiments, the milled injectable scaffold is suspended in a gel or gel precursor prior to injection. Further provided herein are injectable polymer scaffolds made by any such method.UM-44857.601

[0023] Further provided herein are methods of manufacturing an injectable scaffold, comprising mixing a polymer with a particulate porogen at an elevated temperature, and leaching the porogen with a solvent to generate a porous scaffold. In some embodiments, the porogen comprises salt crystals. In some embodiments, the salt crystals have a size of from about 104 pm to about 250 pm. In some embodiments, the polymer comprises poly(s-caprolactone) (PCL), poly(lactic acid) (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLG or PLGA), or combinations thereof. In some embodiments, the mixing is performed at a ratio of from about 20: 1 to about 40: 1 porogen to polymer by weight (e.g., about 30:1). In some embodiments, the mixing is performed at a temperature of from about 80°C to about 100°C (e.g., about 90°C). In some embodiments, the leaching solvent comprises water. In some embodiments, the generated injectable scaffold is suspended in a gel or gel precursor prior to injection. Further provided herein are injectable polymer scaffolds made by any such method.

[0024] Further provided herein are uses of an injectable implant or injectable polymer scaffold as described above or elsewhere herein. In some embodiments, the use comprises collection of a biological sample (e g., to isolate and / or analyze one or more biomarkers associated with a disease or condition or health status of a subject).

[0025] Definitions

[0026] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0027] As noted herein, the disclosed embodiments have been presented for illustrative purposes only and are not limiting. Other embodiments are possible and are covered by the disclosure, which will be apparent from the teachings contained herein. Thus, the breadth and scope of the disclosure should not be limited by any of the described embodiments but should be defined only in accordance with claims supported by the present disclosure and their equivalents. Moreover, embodiments of the subject disclosure may include a process, method, system, composition, device, apparatus, or kit which may further include any and all elements from any otherUM-44857.601

[0028] disclosed processes, methods, systems, compositions, devises, apparatuses, or kits including any and all elements corresponding to controlled structures of polymer scaffolds. In other words, elements from one or another disclosed embodiment may be interchangeable with elements from other disclosed embodiments. Moreover, some further embodiments may be realized by combining one and / or another feature disclosed herein with processes, methods, systems, compositions, devices, apparatuses, kits and one or more features thereof, disclosed in materials incorporated by reference. In addition, one or more features or elements of disclosed embodiments may be removed and still result in patentable subject matter (and thus, resulting in yet more embodiments of the subject disclosure).

[0029] As used herein, “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise.

[0030] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed.

[0031] It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for anyUM-44857.601

[0032] combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0033] As used herein, “disease,” “pathologic condition,” and “condition” are used interchangeably, unless indicated otherwise herein, to describe a deviation from the condition regarded as normal or average for members of a species or group (e g. humans), and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species or group. Such diseases and conditions include, but are not limited to, cancer, autoimmune disease, cell and organ transplant rejection, inflammatory conditions, metabolic conditions, and pregnancy complications such as pre-eclampsia.

[0034] As used herein, “subject” refers to any animal (e.g. mammal, reptiles, avians, amphibians), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject, unless indicated otherwise.

[0035] As used herein, “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Sample may includer cells, cell lysates or purified forms of the enzymes, peptides, and / or polypeptides described herein (e.g., a purified protein sample). The term sample may also include purified samples, such as purified protein samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention.

[0036] As used herein, “protein” is used synonymously with “peptide,” “polypeptide,” or “peptide fragment.”

[0037] As used herein, “cancer” means a disease or condition involving unregulated and abnormal cell growth. Non-limiting exemplary cancers herein include multiple myeloma, leukemia, pancreatic cancer, breast cancer, colorectal cancer, cachexia, melanoma, cervical cancer, ovarian cancer, lymphoma, gastrointestinal, lung cancer, prostate cancer, renal cellUM-44857.601

[0038] carcinoma, metastatic kidney cancer, solid tumors, non-small cell lung carcinoma, nonHodgkin's lymphoma, bladder cancer, oral cancer, myeloproliferative neoplasm, B-cell lymphoproliferative disease, and plasma cell leukemia.

[0039] As used herein, “comprise” (or variations thereof), “contain” (or variations thereof), “have” (or variations thereof), or “include” (or variations thereof), are not intended to be limiting, are inclusive or open-ended and do not exclude additional, unrecited additives, components, integers, elements, or method steps. For example, a process, method, system, composition, kit, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, system, composition, device, apparatus, or kit.

[0040] As used herein, “biomarker” is any measurable characteristic that indicates the presence or absence of disease or the biological response to a stimulus, typically an exposure or intervention than can be used clinically for detecting or measuring the disease in terms of a subject’s susceptibility and risk, diagnosis, monitoring, prognostication, predictive determinations, pharmacodynamic and treatment response, and / or safety.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a fabrication schematic of exemplary injectable scaffolds.

[0042] FIG. 2 shows SEM images of individual injectable scaffolds.

[0043] FIG. 3 shows injection of polymer scaffold in mice.

[0044] FIG. 4 shows H&E staining of injectable scaffolds explanted after 14 days in vivo.

[0045] FIG. 5 shows quantification of innate immune cells recruited at the injectable scaffold in a 4T1 murine model.

[0046] FIG. 6 shows injectable scaffold immune flow compared to implantable scaffold.

[0047] FIG. 7 shows ultrasound image of the injectable scaffold after 14 days post-injection.

[0048] FIG. 8 shows total live cell and RNA isolated from fine-needle aspiration at different time-points post-tumor inoculation.

[0049] FIG.9 shows characterization of the immune cell populations at the injectable scaffold.UM-44857.601

[0050] FIG. 10 shows gene analysis of the injectable scaffold and its ability to track disease progression. FIG. 11 shows a comparison of biomarker levels in CD45+ cells between implantable and injectable scaffolds and lung.

[0051] FIG. 12 shows a comparison of biomarker levels in CD45+ cells at different time points comparing healthy and tumor samples.

[0052] FIG. 13 shows characterization of injectable cube scaffolds at varying fibrin gel concentrations and injection volumes.

[0053] FIG. 14 shows fabrication and characterization of injectable sphere scaffolds produced by a solvent-emulsion process.

[0054] FIG. 15 shows fabrication and characterization of injectable disc scaffolds produced by a solvent-emulsion process.

[0055] FIG. 16 shows cellular recruitment at injectable scaffolds of different geometries seven days post-injection in a mouse model.

[0056] FIG. 17 shows live cell counts on injectable scaffolds of different geometries across varying polymer concentrations.

[0057] FIG. 18 shows the effect of scaffold geometry on scaffold number per unit mass and live cell counts following normalization for scaffold number.

[0058] FIG. 19 shows the influence of polymer coating thickness on cube scaffold morphology and live cell counts.

[0059] FIG. 20 shows histological analysis of injectable cube scaffolds in healthy and tumor-bearing mice at days 7, 14, and 21 post-injection.

[0060] FIG. 21 shows immunofluorescent analysis of vascularization of injectable cube scaffolds at days 7, 14, and 21 post-injection.

[0061] FIG. 22 shows flow cytometry analysis of innate immune cell populations and tumor cell capture at injectable cube scaffolds in tumor-bearing and tumor-free mice.UM-44857.601

[0062] FIG. 23 shows a comparison of immune cell populations at injectable and implantable PCL scaffolds at different time points in a tumor-bearing mouse model.

[0063] FIG. 24 shows fine-needle aspiration of injectable scaffolds for minimally invasive collection of cells and RNA to monitor disease progression.

[0064] FIG. 25 shows random forest prediction probabilities for injectable scaffolds implanted at different time points prior to explant in a tumor-bearing mouse model.

[0065] FIG. 26 shows a fabrication schematic and characterization of milled injectable PCL scaffolds. FIG. 27 shows a fabrication schematic and scanning electron micrograph of injectable PCL scaffolds produced by a salt-coating and leaching process.

[0066] FIG. 28 shows subcutaneous scaffold formation and cellular recruitment of coated-salt injectable scaffolds following injection in a mouse model.

[0067] FIG. 29 shows flow cytometry characterization of CD45+ immune cell populations recruited by coated-salt injectable scaffolds at one week post-implantation.

[0068] DETAILED DESCRIPTION

[0069] In some embodiments, injectable scaffolds are synthesized using a top-down approach. Initially, a master template (e.g., silicon master template) is created (e.g., using optical lithography). In some embodiments, the master template comprises an array of wells (e.g., with 100 pm edge length and a depth of 100 pm). The size and shape of the template may be selected to control the size shape of the generated injectable scaffolds. In some embodiments, the shapes are cubes, disks, spheres, fibers, pyramids, or the like. In some embodiments, the maximum dimension (e.g., length, width, height, diameter) of the selected shape is 300 pm (e.g., 250 pm, 200 pm, 150 pm, 100 pm, 50 pm, 20 pm).

[0070] Next, a polymer (e.g., polydimethylsiloxane (PDMS)) replica of the silicon template is generated. This polymer (e.g., PDMS) mold serves as a template for producing sacrificial polymer (e.g., polyvinyl alcohol (PVA)) films on its surface through, for example, solvent evaporation (e.g., at 20°C for 12 hours). Subsequently, the wells in the film (e.g., PVA film) are filled with a polymer mixture, such as polycaprolactone (PCL). The polymer-coated films (e.g.,UM-44857.601

[0071] PVA films) are dissolved (e.g., in deionized water), and the injectable scaffolds are recovered through sequential filtering.

[0072] In some embodiments, injectable scaffolds are synthesized using a milling approach. A bulk porous polymer scaffold (e g., comprising poly caprolactone (PCL)) is first fabricated with a selected pore size. In some embodiments, the pore size of the bulk scaffold is from about 63 pm to about 425 pm (e.g., from about 63 pm to about 250 pm, or from about 250 pm to about 425 pm). The bulk scaffold is then mechanically milled (e.g., using a benchtop blade mill) to reduce the scaffold to injectable particle dimensions. The milling process yields particles of a size suitable for passage through a syringe needle (e.g., 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, 30-gauge needle). The resulting milled scaffold particles may be suspended in a gel or gel precursor material (e.g., fibrin gel) prior to injection to ensure localization at the injection site following subcutaneous administration.

[0073] In some embodiments, injectable scaffolds are synthesized using a salt-coating and leaching approach. A particulate porogen (e.g., salt crystals having a size of from about 104 pm to about 250 pm) is mixed with a polymer (e.g., polycaprolactone (PCL)) at an elevated temperature (e.g., at about 90°C) for a sufficient time to allow coating of the porogen particles with the polymer (e.g., for about one hour). In some embodiments, the porogen and polymer are mixed at a ratio of from about 20: 1 to about 40: 1 by weight (e.g., about 30:1). Following mixing, the porogen is removed by leaching (e.g., by submerging the mixture in water), yielding a porous polymer scaffold. The size and shape of the resulting scaffold pores are determined by the size and shape of the porogen particles. The resulting scaffolds may be suspended in a gel or gel precursor material (e.g., fibrin clottable protein (e.g., 5 mg / mL) with a gelling agent (e.g., thrombin (e.g., 1 U / mL))) and injected subcutaneously (e.g., using a 21-gauge needle), whereupon gel formation localizes the scaffold at the injection site.

[0074] In some embodiments, the polymer mixture used to form the injectable scaffolds comprises one or more backbone monomers selected from the group consisting of polycaprolactone (PCL), poly(lactic acid) (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLG or PLGA), poly(vinyl alcohol) (PVA), polyethylene glycol) (PEG), poly(ethylene oxide), poly(ethylene oxide)-co-poly(propylene oxide) block copolymers (poloxamers, meroxapols), poloxamines, polyanhydrides, polyorthoesters, poly(hydroxy acids),UM-44857.601

[0075] polydioxanones, polycarbonates, polyaminocarbonates, poly(vinyl pyrrolidone), poly(ethyl oxazoline), carboxymethyl cellulose, hydroxyalkylated celluloses such as hydroxyethyl cellulose and methylhydroxypropyl cellulose, and natural polymers such as nucleic acids, polypeptides, polysaccharides or carbohydrates such as polysucrose, hyaluronic acid, dextran and similar derivatives thereof, heparan sulfate, chondroitin sulfate, heparin, or alginate, and proteins including without limitation gelatin, collagen, albumin, or ovalbumin, or copolymers, or blends thereof.

[0076] In some embodiments, the polymer mixture comprises co-polymers. These co-polymers may have varying molar ratio. Suitable co-polymer ratio of modified particles may be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0. In another embodiment, the co-polymer may be periodical, statistical, linear, branched (including star, brush, or comb co-polymers) co-polymers.

[0077] In some embodiments, injectable scaffolds are administered by incorporating the polymer into a gel (e.g., fibrin gel) to ensure the localization of the polymer after injection. Gels may include natural and / or synthetic components and many include components such as collagen, Matrigel, hyaluronic acid, alginate, chitosan, gelatin methacryloyl or other acrylated proteins, polyethylene glycol, poloxamers, polyvinyl alcohol, hydroxypropyl methylcellulose, selfassembling proteins or polymers, or carboxymethyl cellulose.

[0078] In some embodiments, the injectable scaffolds are resuspended in a gel precursor material (e.g., fibrinogen (2.5 mg / mL)) and injected into a subcutaneous space of the subject (e.g., using a 25 gauge-needle). Subsequently, at the same injection site, a gelling agent (e.g., thrombin (1 U / mL)) is added to allow the formation of the gel. In some embodiments, to harvest cells or other biomaterials from the injectable scaffolds in vivo, fine-needle aspiration is performed as a non-invasive biopsy technique. Since the injectable scaffolds are not physically connected, cells or other biomaterials recruited to the polymer scaffold are collected by inserting a needle into the injection site and applying negative pressure on the plunger of the syringe to suction the cells or other materials. The fine-needle aspirates are then evaluated (e.g., used to track immune infiltrates overtime in the same subject) and for downstream analysis (e.g., flow cytometry and sequencing).UM-44857.601 EXAMPLES

[0079] Example 1

[0080] Injectable Scaffold Fabrication

[0081] Injectable scaffolds were fabricated using a “top-down” process (FIG. 1).

[0082] Polydimethylsiloxane (PDMS) mold was formed from a silicon wafer template. On top of the PDMS mold, 6% w / v polyvinyl alcohol (PVA) solution was applied to create sacrificial films. These films were gently removed to be coated with 10% w / v polycaprolactone (PCL) paste. Once evaporated, coated-films were wiped with dichloromethane (DCM) to rid of excess PCL. These coated-films were dissolved in deionized water and filtered to collect the individual injectable scaffolds. Injectable scaffolds can also be formed by creating spheres or disks at a size that can be passed through a syringe.

[0083] FIG. 1 shows fabrication and characterization of injectable scaffolds, including a schematic of the multi-step, top-down fabrication process used to create open-top cube scaffolds. FIG. 2 shows scanning electron micrograph of the injectable PCL scaffold. SEM image shows the open-top structure of the cube scaffolds. Scanning electron micrograph (SEM) of the injectable PCL scaffold, highlighting its open-top structure. Injectable PCL scaffolds were embedded in fibrin gel at varying concentrations: 4 mg / mL, 8 mg / mL, and 12 mg / mL. The 12 mg / mL polymer concentration yielded a pore distribution comparable to that of an implantable scaffold. FIG. 3 shows an image of an injectable scaffold 4 days post-injection in a mouse, showing localization in the subcutaneous space. Quantification of cells collected from scaffolds 4 days post-injection was conducted at different volumes. Cell recruitment increased with injection volume (50 pL and 100 pL, n = 2; 250 pL and 500 pL, n = 3). Statistical significance was assessed at *p<0.05, **p<0.01 between the compared groups, determined by one-way ANOVA.

[0084] Histological and immunofluorescent analysis of the injectable scaffold in a tumor-bearing model was conducted. H&E staining of tumor sections at days 7, 14, and 21, showed progressive cell integration within the scaffold. Higher-magnification images of H&E-stained sections highlighted cell accumulation from the outer edge toward the scaffold center over time. GSL-1 staining demonstrated vascularization of the injectable scaffolds. Vascular structures are presentUM-44857.601

[0085] at day 7, increase at the scaffold periphery by day 14, and extend into the scaffold center by day 21. tdTOM-4Tl staining confirming the presence of tumor cells within the scaffold. A tumorbearing mouse, at day 14, showed a 4T1 tumor cell within the scaffold.

[0086] Characterization of immune cell populations was conducted at the injectable scaffold. Innate immune cells within the injectable scaffolds were quantified using flow cytometry in 4T1 tumor-bearing mice and healthy controls at specified time points. Adaptive immune cells were also quantified in 4T1 tumor-bearing mice at days 14 and 21 post-tumor inoculation. Statistical significance was determined using a series of t-tests.

[0087] Gene analysis of the injectable scaffold can track disease progression. FIG. 10A shows a diagram illustrating the 4T1 breast cancer model setup. Scaffolds were injected at the time of tumor-inoculation. The whole scaffold was explanted after euthanizing each mouse at specified time points. FIG. 10B shows a heatmap of gene expression across samples from tumor-bearing (TB) and tumor-free (TF) mice at days 7, 14, and 21. Gene expression in the tumor-bearing mice was normalized to the injectable scaffolds from healthy mice. FIG. 10D shows a boxplot of random forest prediction scores for gene expression in injectable scaffolds from tumor-bearing and healthy mice. FIGS. 10E-N show fold-change of the 10 signature genes at the three collection time points, highlighting gene expression changes over time. Statistical significance is indicated as *p<0.05, **p<0.01 between the compared groups, determined by two-way ANOVA.

[0088] Example 2 - Characterization and Biological Performance of Injectable Scaffolds Injectable polymer scaffolds of varying geometry were fabricated, characterized, and evaluated for their ability to recruit immune and tumor cells in vivo. Studies examined the effects of scaffold geometry, polymer concentration, and coating thickness on cellular recruitment, and assessed the utility of fine-needle aspiration for minimally invasive disease monitoring.

[0089] Fabrication and Characterization of Injectable Scaffolds of Varying Geometry Injectable cube scaffolds were embedded in fibrin gel at concentrations of 2.5 mg / mL, 5 mg / mL, and 10 mg / mL and characterized by photomicrography (4x magnification) to assess scaffold distribution within the gel matrix (FIG. 13A-C). Corresponding well images confirmed uniform distribution of scaffolds throughout a 500 pL fibrin volume at each concentration (FIG.

[0090] 13D-F). Scaffold localization in the subcutaneous space was confirmed by optical imaging fourUM-44857.601

[0091] days post-injection in a mouse model (FIG. 13G). Quantification of cells collected from scaffolds at four days post-injection demonstrated that cell recruitment increased with injection volume, across volumes of 50 pL, 100 pL, 250 pL, and 500 pL (FIG. 13H; 50 pL and 250 pL, n = 3; 100 pL, n = 4; 500 pL, n = 5).

[0092] Sphere-shaped injectable scaffolds were fabricated using a solvent-emulsion process that generated both large and small polycaprolactone (PCL) microspheres (FIG. 14A). SEM imaging confirmed the morphology of large (FIG. 14B) and small (FIG. 14C) microspheres.

[0093] Quantification of microsphere diameters demonstrated that large microspheres had an average diameter of approximately 105 pm and small microspheres had an average diameter of approximately 48 pm (FIG. 14D).

[0094] Disc-shaped injectable scaffolds were fabricated using an analogous solvent-emulsion process (FIG. 15 A). Photomicrography (20* magnification) and SEM imaging confirmed the discdike morphology of the resulting scaffolds (FIG. 15B-C).

[0095] Effect of Scaffold Geometry on Cellular Recruitment

[0096] The influence of scaffold geometry on cellular recruitment was evaluated by injecting cube, large sphere, and disc scaffolds subcutaneously in a mouse model. Optical imaging confirmed localization of each scaffold type in the subcutaneous space seven days post-injection (FIG. 16A-C). Quantification of total live cells collected at seven days post-injection demonstrated that cube scaffolds collected more live cells than large sphere or disc scaffolds when injected at a concentration of 5 mg / mL (FIG. 16D).

[0097] Live cell counts were further evaluated across cube, large sphere, and disc scaffolds at polymer concentrations of 5, 10, and 20 mg / mL. No statistically significant differences in live cell counts were observed among the different concentrations for any scaffold geometry (FIG.

[0098] 17).

[0099] To account for geometry-dependent differences in scaffold number per unit mass, the number of scaffolds per unit mass was quantified for cubes, large spheres, small spheres, and discs (FIG. 18A). Scaffold injection concentrations were then adjusted to deliver equivalent scaffold numbers across geometries, using 5 mg / mL cubes as the reference condition; discs were injected at 11.1 mg / mL, large spheres at 44.5 mg / mL, and small spheres at 4.4 mg / mL. AfterUM-44857.601

[0100] normalization for scaffold number, no statistically significant differences in live cell counts were observed between scaffold geometries (FIG. 18B).

[0101] Effect of Polymer Coating Thickness on Scaffold Morphology and Cellular Recruitment The influence of polymer coating thickness on cube scaffold morphology and cellular recruitment was examined by comparing scaffolds coated with one versus five layers of PCL polymer paste. SEM imaging demonstrated that five-layer-coated scaffolds exhibited increased coating thickness relative to single-layer-coated scaffolds, with attendant reduction in accessible open-top surface area (FIG. 19A-B). Live cell counts revealed that single-layer-coated scaffolds supported significantly greater cellular recruitment compared to five-layer-coated scaffolds (FIG. 19C), consistent with the interpretation that greater accessible surface area enhances cellular infiltration and recruitment.

[0102] Histological and Immunofluorescent Analysis of Injectable Scaffolds In Vivo Histological analysis of injectable cube scaffolds was performed using hematoxylin and eosin (H&E) staining of scaffold center sections at days 7, 14, and 21 post-injection in both healthy and tumor-bearing mice. In healthy mice, progressive cellular infiltration from the scaffold periphery toward the center was observed over the three-week period (FIG. 20A-C). A similar pattern of cellular infiltration was observed in tumor-bearing mice (FIG. 20D-F), demonstrating a similar cellular infiltration pattern from the edge to the center.

[0103] Vascularization of injectable cube scaffolds was assessed by GSL-1 immunostaining. Vascular structures were detectable at day 7, increased at the scaffold periphery by day 14, and extended into the scaffold center by day 21 (FIG. 21A-C), demonstrating vascularization of the injectable scaffolds.

[0104] Immune Cell Recruitment and Tumor Cell Capture at Injectable Scaffolds

[0105] Innate immune cell populations at injectable cube scaffolds were characterized by flow cytometry at days 7, 14, and 21 post-tumor inoculation in tumor-bearing (TB) and tumor-free (TF) mice (FIG. 22). Dendritic cells (CD1 lc+) were slightly elevated in TF scaffolds, particularly at later time points (FIG. 22A). Macrophages (F4 / 80+) were enriched at early time points in both TF and TB scaffolds but declined by day 21 (FIG. 22B). Monocytes (Ly6C+)UM-44857.601

[0106] showed similar frequencies between groups, with a modest trend toward higher levels in TB scaffolds (FIG. 22C). Neutrophils (Ly6G+) were significantly enriched in TB scaffolds at days 14 and 21, reflecting disease-associated neutrophil accumulation also observed in the lung metastatic niche (FIG. 22D). The presence of tumor cells within the scaffold was confirmed by RT-qPCR detection of tdTomato expression in day 14 scaffolds from tumor-bearing mice (FIG.

[0107] 22E).

[0108] Comparison of Injectable and Implantable Scaffold Immune Profiles

[0109] The immune cell composition at injectable scaffolds was compared to that of surgically implanted PCL scaffolds at matched time points in tumor-bearing mice (FIG. 23). Despite differences in delivery method, subcutaneous injection versus surgical implantation, both scaffold formats recruited broadly similar innate immune cell populations, including macrophages, monocytes, dendritic cells, and neutrophils. These findings demonstrated that the injectable scaffold recapitulated the immunological microenvironment established by the implantable scaffold, supporting the conclusion that the injectable format can serve as a functional, minimally invasive alternative to surgically implanted scaffolds for immune monitoring and disease surveillance.

[0110] Robustness of Disease Tracking to Scaffold Injection Timing

[0111] The sensitivity of scaffold-based disease tracking to the timing of scaffold injection relative to scaffold explant was evaluated by comparing random forest prediction probabilities for scaffolds injected 3, 5, 7, and 21 days prior to explant in tumor-bearing mice (FIG. 25). No statistically significant differences in random forest prediction probability were observed between any of the earlier injection time points and the 21 -day condition. These results demonstrated that the gene expression signature captured at the injectable scaffold was not materially influenced by the duration of scaffold residence prior to collection, indicating that the diagnostic signal was robust to variability in injection-to-collection timing.

[0112] Fine-Needle Aspiration for Minimally Invasive Disease Monitoring

[0113] Fine-needle aspiration was employed as a minimally invasive biopsy technique to collect cells from injectable scaffolds in vivo without scaffold explantation. The scaffold injection site was visualized by optical imaging at seven days post-injection (FIG. 24 A), and scaffoldUM-44857.601

[0114] localization in the subcutaneous space was confirmed by ultrasound imaging at fourteen days post-injection (FIG. 24B). Total live cells and total RNA collected via fine-needle aspiration were quantified in TB and TF mice across time points (each condition, n = 10), demonstrating consistent recovery of viable cells and sufficient RNA for downstream analysis (FIG. 24C-D). Random forest classification of gene expression profiles from fine-needle aspirates yielded prediction scores that distinguished tumor-bearing from tumor-free mice across all time points examined (FIG. 24E; TF-Day 7, n = 5; TF-Day 14, n = 7; TF-Day 21, n = 6; TB-Day 7, n = 9; TB-Day 14, n = 6; TB-Day 21, n = 5).

[0115] Example 3 - Additional Methods of Injectable Scaffold Fabrication

[0116] Additional methods were developed to manufacture injectable polymer scaffolds of tunable pore size and geometry. Two approaches were employed: (1) mechanical milling of bulk porous scaffolds and (2) a salt-coating and leaching process. Both methods yielded injectable scaffolds capable of subcutaneous delivery and immune cell recruitment in vivo.

[0117] Milled Injectable Scaffolds

[0118] Microporous polycaprolactone (PCL) scaffolds with tunable pore sizes were fabricated and subsequently reduced to injectable dimensions using abenchtop blade mill (FIG. 26A). Scaffolds were milled until a powdered form was achieved (FIG. 26B). Scanning electron microscopy (SEM) confirmed the structural characteristics of the resulting milled scaffold particles across two pore size ranges: 250-425 pm (FIG. 26C) and 63-250 pm (FIG. 26D). To assess biological performance, milled scaffolds were suspended in a fibrin / thrombin gel and injected subcutaneously in vivo. Cell recruitment from the injected scaffolds was quantified and demonstrated successful recruitment of cells at both pore size ranges (FIG. 26E).

[0119] Coated-Salt Injectable Scaffolds

[0120] A salt-coating and leaching process was employed as an alternative fabrication approach (FIG. 27A). Salt crystals sized between 104 and 250 pm were mixed with PCL polymer at a 30: 1 ratio at 90°C for one hour. Following mixing, the salt was leached by submerging the material in water, yielding a porous PCL scaffold. SEM imaging of the resulting scaffolds confirmed the formation of porous PCL structures from this salt coating-leaching process (FIG. 27B; 114x magnification, Zeiss Crossbeam).UM-44857.601

[0121] The coated-salt scaffolds were evaluated for injectability and scaffold formation in situ. Scaffolds were suspended in 5 mg / mL fibrin clottable protein with 1 U / mL thrombin and injected subcutaneously via a 21 -gauge needle. Gel formation occurred subcutaneously following injection (FIG. 28 A). Cell recruitment was assessed at one week post-injection at two scaffold concentrations (5 mg / mL and 10 mg / mL), and both concentrations recruited equivalent numbers of cells (FIG. 28B).

[0122] The immune cell populations recruited by the coated-salt injectable scaffolds were further characterized by flow cytometry at one week post-implantation (FIG. 29). Both the 5 mg / mL and 10 mg / mL scaffold injection concentrations recruited similar numbers and compositions of CD45+ immune cells (FIG. 29A-B). The number of CD45+ cells per scaffold was comparable across concentrations (FIG. 29C). Further immunophenotyping revealed recruitment of CD1 lb+ myeloid cells (FIG. 29D-E), F4 / 80+ macrophages (FIG. 29F), and CD1 lc+ F4 / 80-low dendritic cells (FIG. 29G) among the CD45+ population, demonstrating that the coated-salt scaffolds effectively established an immune microenvironment consistent with that observed for other injectable scaffold formats.

Claims

UM-44857.601CLAIMS1. An injectable implant comprising a polymer configured to form a scaffold when injected in vivo into a subject, wherein said scaffold is configured to recruit circulating cells.

2. The injectable implant of claim 1, wherein said polymer comprises poly(e-caprolactone) (PCL).

3. The injectable implant of claim 1, wherein the implant comprises a plurality of unconnected cubes, disks, spheres, fibers, or pyramids.

4. The injectable implant of claim 3, wherein plurality of cubes, disks, spheres, fibers, or pyramids have a maximum dimension of less than 300 pm.

5. A method comprising, injecting a polymer into a subject, wherein the polymer is configured to recruit circulating metastatic cells.

6. The method of claim 5, wherein said polymer comprises poly(s-caprolactone) (PCL).

7. The method of claim 5, wherein said injecting comprises injecting said polymer into a gel or gel precursor contained in a subcutaneous region of said subject.

8. The method of claim 7, wherein a gelling agent is administered to the subject to form a gel.UM-44857.6019. The method of claim 5, further comprising removing a sample from said polymer.

10. The method of claim 9, further comprising analyzing said sample.

11. The method of claim 10, wherein said analyzing comprising detecting one or more biomarkers indicative of metastasis.

12. A method of manufacturing an injectable scaffold, comprising: a) forming a polymer mold on template, b) forming a sacrificial film on the mold, c) removing the sacrificial film, d) coating the removed sacrificial film with a scaffold polymer, and e) dissolving sacrificial film with a solvent to generate a released scaffold polymer; or forming said injectable scaffold using an emulsion process.

13. The method of claim 12, wherein the polymer mold comprises polydimethylsiloxane (PDMS).

14. The method of claim 12, wherein the template comprises a silicon wafter template.

15. The method of claim 12, wherein the mold comprises a plurality of wells having a maximum dimension of less than 300 pm.

16. The method of claim 12, wherein the sacrificial film comprises polyvinyl alcohol.UM-44857.60117. A method of manufacturing an injectable scaffold, comprising milling a porous polymer scaffold to an injectable particle size.

18. A method of manufacturing an injectable scaffold, comprising: a) mixing a polymer with a particulate porogen; and b) leaching the porogen with a solvent to generate a porous injectable scaffold.

19. The method of claim 18, wherein the particulate porogen comprises salt crystals.

20. The method of claim 19, wherein the salt crystals have a size of from about 104 pm to about 250 pm.

21. The method of any of claims 12-20, wherein the generated injectable scaffold has a cube, disk, sphere, fiber, or pyramid shape.

22. The method of any of claims 12-21, wherein the scaffold polymer comprises polycaprolactone (PCL).

23. The method of claim 12, wherein the solvent comprises water.

24. The method of claim 12, wherein the dissolving step further comprises filtering to collect individual injectable scaffolds.

25. An injectable polymer scaffold made by the method of any of claims 12-24.UM-44857.60126. Use of an injectable implant or injectable polymer scaffold of claims 1-4 or 25.

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