WO2022099093A1 - Échafaudages pour amélioration des neutrophiles et utilisations associées - Google Patents

Échafaudages pour amélioration des neutrophiles et utilisations associées Download PDF

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WO2022099093A1
WO2022099093A1 PCT/US2021/058359 US2021058359W WO2022099093A1 WO 2022099093 A1 WO2022099093 A1 WO 2022099093A1 US 2021058359 W US2021058359 W US 2021058359W WO 2022099093 A1 WO2022099093 A1 WO 2022099093A1
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subject
innate
interleukin
scaffold
level
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PCT/US2021/058359
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WO2022099093A8 (fr
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Nisarg J. SHAH
Matthew D. KERR
David J. Mooney
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President And Fellows Of Harvard College
The Regents Of The University Of California
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Publication of WO2022099093A1 publication Critical patent/WO2022099093A1/fr
Publication of WO2022099093A8 publication Critical patent/WO2022099093A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Neutrophil deficiency contributes to opportunistic infections and could impair tissue regeneration in affected individuals 4-7 .
  • pre-conditioning myelosuppressive regimens can contribute to a marked transient post-therapy impairment of neutrophils and render recipients susceptible to immune deficiency-associated complications for up to several weeks 6, 8-11 .
  • Post-HSCT neutrophil regeneration follows successful bone marrow engraftment of transplanted hematopoietic cells 12, 13 , facilitated by granulocyte colony stimulating factor (G- CSF)-mediated granulopoiesis of hematopoietic cells 14-16 .
  • G- CSF granulocyte colony stimulating factor
  • Neutropenia is typically treated as an emergency and, in a subset of patients, the risk of neutropenia may be prophylactically addressed with post-HSCT subcutaneous injection of recombinant human G-CSF (filgrastim) to facilitate recovery 6, 14, 17, 18 .
  • Daily injections are used as G-CSF has a half-life of a 3 – 4 hours, which can be extended by conjugating G-CSF with polyethylene glycol (PEGylation) 19, 20.
  • PEGylation polyethylene glycol
  • immune responses against PEG have been demonstrated to enhance clearance of PEG-G-CSF in an antibody-dependent manner 21 . As multiple cycles of PEG-G-CSF treatment are common, long-term treatment could be rendered ineffective.
  • compositions and methods for delivering an active agent to a subject provide a means to treat and/or prevent diseases associated with a deficiency in levels of functional innate leukocytes, e.g., neutrophils.
  • the present invention provides a method of delivering an active agent to a subject in need thereof, wherein the subject has a deficiency in levels of functional innate leukocytes, e.g., neutrophils, the method comprising: administering to the subject a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby delivering the active agent to the subject.
  • a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby delivering the active agent to the subject.
  • the present invention provides a method of modulating the activity of an innate leukocyte, e.g., neutrophils, restoring levels of functional innate leukocytes, reconstituting the levels of functional innate leukocytes and/or enhancing the regeneration of functional innate leukocytes in a subject in need thereof, wherein the subject has a deficiency in levels of functional innate leukocytes, the method comprising: administering to the subject a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby modulating the activity of an innate leukocyte, reconstituting the levels of innate leukocyte and/or enhancing the regeneration of an innate leukocyte in the subject.
  • an innate leukocyte e.g., neutrophils
  • restoring levels of functional innate leukocytes restoring levels of functional innate leukocytes
  • the active agent (i) modulates the activity of an innate leukocyte; (ii) restores levels of functional innate leukocytes, e.g., neutrophils; (iii) reconstitutes levels of functional innate leukocytes; (iv) enhances the regeneration of functional innate leukocytes in the subject; (v) enhances immunity of the subject in need thereof; and/or (vi) treats or prevents a disorder in the subject, wherein the disorder is caused by or associated with a deficiency in functional innate leukocytes.
  • the present invention provides a method for detecting immune recovery in a subject having a deficiency in levels of functional innate leukocytes, e.g., neutrophils, comprising: administering to the subject a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof, assessing the level of degradation of the scaffold composition, wherein degradation of the scaffold composition is indicative of immune recovery in the subject.
  • the scaffold composition further comprises an active agent.
  • the method provides for controlled, sustained, and/or delayed release of the active agent upon administration to the subject.
  • the method provides for sustained release of the active agent upon administration to the subject. In some embodiments, the method provides for controlled release of the active agent upon administration to the subject. In some embodiments, the method provides for delayed release of the active agent upon administration to the subject. In some embodiments, the release of the active agent is delayed at least about 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more days after administration.
  • degradation of the scaffold composition is indicative of (i) modulation of the activity of an innate leukocyte; restoration of levels of functional innate leukocytes, e.g., neutrophils; reconstitution of levels of functional innate leukocytes; regeneration of functional innate leukocytes in the subject; enhanced immunity of the subject; and/or treatment or prevention of a disorder in the subject, wherein the disorder is caused by or associated with a deficiency in functional innate leukocytes.
  • the scaffold composition degrades upon exposure to functional innate leukocytes, e.g., neutrophils.
  • the innate leukocyte (i) is selected from the group consisting of a natural killer cell, a mast cell, an eosinophil, a basophil, a macrophage, a dendritic cell, a gamma-delta ( ⁇ ) T cell, and a neutrophil; and/or (ii) is not a dendritic cell.
  • the innate leukocyte comprises a neutrophil.
  • the active agent comprises an amino acid, a peptide, a protein, a nucleic acid, an oligonucleotide, a small molecule, or a combination thereof.
  • the active agent comprises a protein, optionally, wherein the protein is an isolated protein and/or a recombinant protein. In some embodiments, the active agent comprises a cytokine, a chemokine, and/or a growth factor. In some embodiments, the active agent comprises a cytokine, optionally, a pro-inflammatory cytokine and/or an anti- inflammatory cytokine.
  • the active agent is selected from the group consisting of a granulocyte colony stimulating factor (G-CSF), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a bone morphogenetic protein 2 (BMP2), a stromal cell-derived factor (SDF), a platelet activating factor (PAF), a tumor necrosis factor ⁇ (TNF ⁇ ), an interleukin-1 (IL-1), an interleukin-1 ⁇ (IL-1 ⁇ ), an interleukin-1 ⁇ (IL-1 ⁇ ), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin- 6 (IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin-10 (IL-10), an interleukin-11 (IL-11), an interleukin-12 (IL-12), an
  • the active agent comprises a G- CSF. In some embodiments, the active agent comprises a polyethylene glycol conjugated granulocyte colony-stimulating factor (PEG-G-CSF). In some embodiments, the active agent active agent is G-CSF. In various embodiments of the above aspects or any other aspect of the invention described herein, the method further comprises administering an additional active agent. In some embodiments, the additional active agent is encapsulated in the scaffold. In some embodiments, the additional active agent is administered systemically. In various embodiments of the above aspects or any other aspect of the invention described herein, the subject is or has undergone therapy that depletes or reduces the innate leukocyte in the subject.
  • PEG-G-CSF polyethylene glycol conjugated granulocyte colony-stimulating factor
  • the therapy is a chemotherapy or a radiotherapy for the treatment of a cancer.
  • the composition further comprises a detectable agent.
  • the detectable agent is attached to the active agent.
  • the detectable agent is for detecting the scaffold and/or active agent in vivo.
  • the detectable agent is for assessing the level of degradation of the scaffold composition.
  • the detectable agent is selected from the group consisting of a dye, a fluorophore, a fluorescent compound, a luminescent compound, a chemiluminescent compound, a radioisotope, a paramagnetic metal ion, a nanoparticle, and a quantum dot, or a combination thereof.
  • the detectable agent comprises a dye.
  • the detectable agent is detectable by eye, fluorescence imaging, magnetic resonance imaging (MRI), surface-enhanced Raman scattering (SERS), multi-mode imaging, ultrasound imaging (USI), photoacoustic imaging (PAI), and/or optical coherence tomography (OCT).
  • the method further comprises assessing the level of degradation of the scaffold.
  • assessing the level of degradation of the scaffold comprises detecting the level of the detectable agent and comparing to a detectable agent reference level, optionally, wherein the detectable agent reference level is the level of the detectable agent upon administration of the scaffold composition or at an earlier time point after administration of the scaffold composition.
  • the method further comprises obtaining a measurement of (i) the level of the detectable agent and/or active agent in the scaffold prior to administration of the scaffold to the subject; (ii) the level of functional innate leukocytes, e.g., neutrophils in the subject prior to administration of the scaffold to the subject; (iii) the level of the innate leukocytes in the subject prior to administration of the scaffold to the subject; (iv) the level of the detectable agent and/or active agent in the scaffold after administration of the scaffold to the subject; (v) the level of functional innate leukocytes after administration of the scaffold to the subject; (vi) the level of the innate leukocytes in the subject prior to administration of the scaffold to the subject; (vii) the level of functional innate leukocytes in the subject before the subject receives a therapy that depletes or reduces the level of the innate leukocyte; and/or (viii) the level of functional innate leukocyte
  • the level of functional innate leukocytes is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% or more as compared to a reference level.
  • the level of functional innate leukocytes, e.g., neutrophils is increased by at least about 1-fold, about 2-fold, or about 3-fold or more as compared to a reference level.
  • the reference level is a standardized level.
  • the reference level is the level of functional innate leukocytes, e.g., neutrophils prior to administration of the scaffold composition or at an earlier time point after administration of the scaffold composition.
  • the scaffold composition does not comprise a differentiation factor that induces the differentiation of a hematopoietic stem cell into a T cell progenitor cell.
  • the scaffold composition degrades upon exposure to an enzyme produced by the innate leukocyte.
  • the scaffold composition degrades by neutrophil-mediated oxidation.
  • the degradation of the scaffold composition results in and/or enhances the release of the active agent.
  • the degradation of the scaffold composition results in and/or enhances the release of the detectable agent.
  • the scaffold is macroporous or wherein the scaffold comprises pores having a diameter of about 50 microns to about 150 microns, about 80 microns to about 180 microns, or about 40 microns to about 90 microns.
  • the subject is a human.
  • the composition is administered to the subject via injection, optionally, intravenously, intramuscularly, or subcutaneously.
  • the subject has a disorder selected from the group consisting of primary neutropenia, acute neutropenia, severe chronic neutropenia (SCN), severe congenital neutropenia (Kostmann's syndrome), severe infantile genetic agranulocytosis, benign neutropenia, cyclic neutropenia, chronic idiopathic neutropenia, secondary neutropenia, syndrome associated neutropenia, and immune-mediated neutropenia, chronic granulomatous disease, Chediak-Higashi syndrome, Griscelli Syndrome-II, Hermansky-Pudlak Syndrome type 2, leukocyte adhesion deficiency, specific granule deficiency, x-linked neutropenia, myeloperoxidase deficiency, and hyper-IgE syndrome.
  • SCN severe chronic neutropenia
  • Kostmann's syndrome severe congenital neutropenia
  • severe infantile genetic agranulocytosis benign neutropenia
  • the subject has a neutropenia, optionally, wherein the neutropenia is caused by or associated with radiation, alcoholism, drugs, allergic disorders, aplastic anemia, autoimmune disease, T- ⁇ lymphoproliferative disease (T- ⁇ LPD), myelodysplasia, myelofibrosis, dysgammaglobulinemia, paroxysmal nocturnal hemoglobinuria, cancer, vitamin B12 deficiency, folate deficiency, viral infection, bacterial infection, spleen disorder, hemodialysis, or transplantation, toxins, bone marrow failure, Schwachman-Diamond syndrome, cartilage-hair hypoplasia, dyskeratosis congenita, glycogen storage disease type IB, splenomegaly of any cause, and intrinsic defects in myeloid cells or their precursors.
  • T- ⁇ lymphoproliferative disease T- ⁇ lymphoproliferative disease (T- ⁇ LPD)
  • myelodysplasia myelo
  • the subject has or is receiving a hematopoietic stem cell transplantation (HSCT).
  • HSCT hematopoietic stem cell transplantation
  • the administration of the composition is prior to, concurrently with, or subsequent to the HSCT.
  • the method reconstitutes the innate leukocyte within 7 days, 14 days, 21 days, or 28 days.
  • the reconstitution of the innate leukocyte results in the level of innate leukocyte to be at least about 75%, 80%, 85%, 90%, 95% or 99% of a reference level.
  • the reference level is (i) a standardized level; (ii) the level of the innate leukocyte of the subject before depletion of the level of the innate leukocyte; and/or (iii) the level of the innate leukocyte of the subject before the subject receives a therapy the depletes or reduces the level of the innate leukocyte.
  • the method results in (i) an increase in the number of the innate leukocytes in the subject; (ii) an increase in the number of functional innate leukocytes, e.g., neutrophils in the subject; (iii) a reduction in the incidence, severity, and/or duration of an opportunistic infection in the subject; (iv) a reduction in the incidence, severity, and/or duration of a symptom of neutropenia in the subject; (v) a reduction in the incidence, severity, and/or duration of a symptom of an immunodeficiency-associated complication in the subject; (vi) an increase in the level of tissue regeneration in the subject; (vii) an increase in the therapeutic efficacy of a therapy that depletes or reduces the innate leukocyte in the subject; (viii) an increase in the degradation of the scaffold in the subject; and/or (ix) an increase in the release of a detectable agent, an increase in the level of tissue regeneration in the subject; (vii) an increase in the
  • the subject has or is receiving systemic G-CSF and/or PEG-G-CSF.
  • the administration of the composition is prior to, concurrently with, or subsequent to the systemic G-CSF and/or PEG-G-CSF.
  • the method further comprising administering to the subject systemic G-CSF and/or PEG-G-CSF.
  • the subject has or is at risk of developing anti-PEG antibody (APA)-mediated rapid clearance.
  • the subject has pre-existing or induced anti-PEG antibody (APA)-mediated rapid clearance.
  • the subject has failed or is at risk of failing to respond to systemic G-CSF and/or PEG-G-CSF.
  • the present invention provides a method of treating a subject for cancer in need thereof, wherein the subject is undergoing chemotherapy or radiotherapy, comprising administering to a subject suffering from depleted or reduced innate leukocytes, e.g., functional neutrophils, wherein the composition comprises a biodegradable scaffold and a cytokine that enhances the generation of the innate leukocyte; assessing the levels of the innate leukocyte in the subject; administering chemotherapy or radiotherapy to the subject wherein the levels of innate leukocyte have attained a reference level following administration of the composition.
  • innate leukocytes e.g., functional neutrophils
  • the innate leukocytes are neutrophils.
  • the cytokine is G-CSF.
  • the subject is suffering from depleted or reduced innate leukocytes as a result of previous exposure to chemotherapy or radiotherapy.
  • the reference level is a standardized level, optionally, (i) wherein the reference level is the level of the innate leukocyte of the subject before depletion of the level of the innate leukocyte; and or (ii) the reference level is the level of the innate leukocyte of the subject before the subject receives a therapy the depletes or reduces the level of the innate leukocyte.
  • the present invention provides an injectable composition, comprising a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof, and at least one of (i) an active agent that increases the number of and/or modulates the activity of an innate leukocyte; and/or (ii) a detectable agent that allows for assessment of degradation of the scaffold composition and/or assessment of the levels of functional innate leukocytes, e.g., neutrophils, in vivo.
  • the injectable composition comprises both the active agent and the detectable agent.
  • the innate leukocytes are neutrophils.
  • the active agent is G-CSF.
  • the present invention provides a method for detecting immune recovery in a subject having a deficiency in levels of functional innate leukocytes, e.g., neutrophils, comprising administering the injectable composition of any one of the above aspects or any other aspect of the invention described herein and assessing the degradation of the scaffold composition and/or levels of functional innate leukocytes, e.g., neutrophils, in vivo.
  • functional innate leukocytes e.g., neutrophils
  • the present invention provides a method of modulating the activity of an innate leukocyte, restoring levels of functional innate leukocytes, e.g., neutrophils, reconstituting the levels of functional innate leukocytes and/or enhancing the regeneration of functional innate leukocytes in a subject in need thereof, wherein the subject has a deficiency in levels of functional innate leukocytes, the method comprising administering to the subject the composition of any one of the above aspects or any other aspect of the invention described herein.
  • FIGS.1A-1E depict macroporous HA cryogel synthesis and fabrication.
  • FIG.1A shows tetrazine and norbornene functionalization of HA.
  • FIG.1B shows cross linking of tetrazine functionalized hyaluronic acid (HA) and norborene functionalized HA.
  • FIG.1C depicts sub-zero cross-linking process to create macroporous HA cryogels.
  • FIG.1D shows a representative SEM image depicting HA cryogels made from 0.8% and 7% degree of substitution (DOS) tetrazine (Tz) functionalized HA (Tz-HA).
  • Top row scale bar 500 ⁇ m.
  • Bottom row scale bar 100 ⁇ m.
  • FIGS.2A-2B show that pore size distribution is unaffected after lyophilization.
  • FIG. 2A shows representative confocal microscopy images of cy5 functionalized HA cryogels after thawing, after lyophilization, and after lyophilization and rehydration.
  • FIGS.3A-3E shows that cross-linking density influences HA degradation.
  • FIG.3A is a schematic showing cy5-conjugated tetrazine functionalization of norbornene functionalized HA and subsequent cross-linked structure after cross-linking with tetrazine functionalized HA.
  • FIG.3B shows a photograph of cy5-conjugated HA-cryogel.
  • FIG.3C shows degradation scores of HA cryogels synthesized with 0.8% and 7% degree of substitution (DOS) tetrazine functionalized hyaluronic acid.
  • FIG.3D shows representative IVIS fluorescence images of gel degradation in mice.
  • FIG.3E shows tracking of HA cryogel degradation by quantification of total radiant efficiency normalized to initial day 3 timepoint.
  • FIGS.4A-4G depict in vivo deployment and gel degradation in models of immunodeficiency.
  • IVIS in vivo imaging system
  • FIG.4F shows hematoxylin and eosin (H&E) stain of explanted HA cryogels from immune competent mice, T-cell deficient mice, B-cell deficient mice, and macrophage deficient mice at days 1, 5, and 10.
  • FIG.4G depicts an analysis of H&E stains to quantify cell density on outer 50 ⁇ m of HA cryogel and interior of HA cryogel. Data represents measurements from 2-4 H&E stains for no treatment (immune competent), B-cell deficient, T-cell deficient, and macrophage deficient mice for HA cryogels excised 1 day, 5 days, and 10 days after implant.5 areas are sampled on outer edge and interior of each H&E sample.
  • FIGS.5A-5D shows innate immune cell infiltration into HA cryogels.
  • FIG.5A depicts representative flow cytometry plots of F4-80 and CD11b expression in HA cryogels 1 day after administration.
  • FIG.5B shows representative flow cytometry plots of F4-80 and CD11b expression in HA cryogels 10 days after administration.
  • FIGS.5C-5D depicts FACs quantification of total number of CD45 + CD11b + myeloid cells (FIG.5C) and FIG.5D CD45 + CD11b + F4-80 + macrophages (FIG.5D) infiltrating HA cryogels in immune competent mice, T-cell depleted mice, B-cell depleted mice, and macrophage depleted mice.
  • FIGS.6A-6F show that degradation kinetics of HA cryogels is impaired during transient immunodeficiency following bone marrow transplant (BMT).
  • FIG.6A shows the schedule of administration of lethal total body irradiation followed by BMT and simultaneous injection of Cy5-HA cryogel.
  • B6 mice were irradiated with 10Gy (1 dose) 48 hours before transplant with 2 x 10 5 lineage-depleted syngeneic bone marrow cells.
  • FIG.6B shows epresentative IVIS fluorescence images of gel degradation in both immune competent mice and mice that received a BMT.
  • FIG.6C depicts tracking gel degradation by quantification of total radiant efficiency normalized to initial day 3 timepoint.
  • FIG.6D depicts peripheral blood CD45 + CD11b + myeloid cells reconstitution after BMT with respect to time. Radiation administered at day -2 for 10Gy radiation group and HA cryogels injected on day 0.
  • FIGS. 6E-6F show cell infiltration of CD45 + CD11b + myeloid cells (FIG.6E) and CD45 + CD11b + F4-80 + macrophage cells (FIG.6F) into HA cryogels in immune competent mice 5 days after administration and 5, 16, and 21 days following BMT.
  • FIGS.7A-7B shows data from immune lineage depletion studies.
  • FIG.7A depicts flow cytometry representations of peripheral blood stained before and after immune depletion treatments.
  • FIG.7B shows ratio of CD45 + CD11b + F4-80 + CD80 + CD86 + CD206- pro- inflammatory (M1) and CD45 + CD11b + F4-80 + CD80-CD86-CD206 + anti-inflammatory (M2) macrophages associated with HA cryogels excised 1 day and 10 days after implant.
  • FIG.7C shows a graph comparing the ratio of M1:M2 macrophages associated with HA cryogels excised 1 day and 10 days after implant.
  • FIGS.8A-8B depicts data from bone marrow transplant studies.
  • FIG.8A shows flow cytometry plots representing fraction of cells Lineage- before and after hematopoietic stem cell enrichment.
  • FIG.8B shows fraction of CD45 + CD11b + CD115-Ly6-G + neutrophil to total CD45 + CD11b + myeloid cells before and after bone marrow transplant.
  • FIG.9A shows an experimental overview of G-CSF mediated CD45 + CD11b + CD115-Ly6-G + neutrophil reconstitution study.
  • FIG.9B shows reconstitution of peripheral blood CD45 + CD11b + CD115-Ly6-G + neutrophils compiled from FACS quantification.
  • FIGS.10A-10B show data from granulocyte colony stimulating (G-CSF) factor bone marrow transplant studies. FIG.10A shows death resulting from twice daily intraperitoneal 100ng G-CSF.
  • G-CSF granulocyte colony stimulating
  • FIG.10B shows reconstitution of peripheral blood CD45 + CD11b + CD115- Ly6-G + neutrophils tracked up to 29 days after bone marrow transplant.
  • FIG.11 shows endotoxin levels of HA-Tz and Cy5-HA-Nb were quantified to be less than 5 endotoxin units/kg, the threshold pyrogenic dose for preclinical species.
  • FIGS.12A-12I depict production and characterization of Cy5-HA cryogel.
  • FIG. 12A shows a schematic for tetrazine (Tz) and norbornene (Nb) functionalization of HA, Cy5 functionalization of Nb functionalized HA (Cy5-HA-Nb) and crosslinking of Tz functionalized HA with Cy5-HA-Nb.
  • FIG.12B depicts a schematic for producing Cy5-HA cryogels.
  • FIG.12F shows a schematic depicting workflow for in vitro Cy5-HA cryogel degradation study.
  • FIG.12G depicts measuring Cy5-HA cryogel degradation in vitro by quantifying the Cy5-signal in supernatant at pre-determined timepoints normalized to total Cy5-signal in supernatant across all timepoints.
  • FIG.12H shows a schematic depicting workflow for in vivo Cy5-HA cryogel degradation study.
  • FIGS.13A-13H depict HA cryogel materials characterization data.
  • FIG.13A shows volumetric swelling ratios for low- and high-DOS Cy5-HA cryogels.
  • FIG.13B shows aqueous weight percentage of low- and high-DOS Cy5-HA cryogels.
  • FIG.13C shows a representative SEM image depicting low-DOS Cy5-HA cryogels.
  • Top scale bar 500 ⁇ m
  • bottom scale bar 100 ⁇ m.
  • FIG.13F depicts the quantification of confocal images showing penetration of 10 ⁇ m FITC-labeled microparticles into both low- and high-DOS Cy5-HA cryogels pre- and post-injection.
  • FIG.13G shows representative confocal microscopy images of low- and high- DOS Cy5-HA cryogels after thawing, after lyophilization, and after lyophilization and rehydration.
  • FIG.13H depicts average surface pore diameter of Cy5-HA cryogels measured from confocal images.
  • Data in FIG.13D represents mean ⁇ s.d.
  • FIGS.14A-14C depict Cy5-HA cryogel degradation characterization data.
  • FIG.14A depicts measuring low-DOS Cy5-HA cryogel degradation in vitro by quantification of Cy5- signal in supernatant at pre-determined timepoints normalized to total Cy5-signal in supernatant across all timepoints.
  • FIG.14B shows representative IVIS fluorescence images of gel degradation in mice and measuring low-DOS Cy5-HA cryogel degradation in vivo by quantification of total radiant efficiency normalized to the initial day 3 timepoint.
  • FIG.14C depicts measuring Cy5-HA cryogel degradation by quantification of total radiant efficiency normalized to initial day 3 timepoint.
  • FIGS.15A-15F depict Cy5-HA cryogel degradation in immunodeficient mice.
  • IVIS Images are on the same scale and analyzed using Living Image Software.
  • FIG.15E shows hematoxylin and eosin (H&E) stained histological sections of explanted Cy5-HA cryogels from the above groups, at days 1, 5, and 10 post-injection.
  • H&E hematoxylin and eosin
  • FIG.15F shows quantification of cellular density in the sections from FIG.15E.
  • FIGS.16A-16L show Cy5-HA cryogel degradation in immunodeficient mice characterization data.
  • FIGS.16A-16B show representative IVIS fluorescence images of gel degradation and quantification by measuring total radiant efficiency normalized to initial day 3 timepoint of T cell depleted B6 mice (FIG.16A) and B cell depleted B6 mice (FIG.16B).
  • FIG.16C shows a representative flow cytometry plot of peripheral blood neutrophils pre- and post-administration of neutrophil depleted mice and FIG.16D shows peripheral blood neutrophil concentration.
  • FIG.16E shows a representative flow cytometry plot of peripheral blood monocytes pre- and post- administration clodronate liposomes to mice and FIG.16F peripheral blood monocyte concentration.
  • FIG.16G shows a representative flow cytometry plot of peripheral blood T cells blood pre- and post- administration of anti-CD4 and anti-CD8 antibody treatment to mice and FIG.16H shows peripheral blood T cell concentration.
  • FIG. 16I shows a representative flow cytometry plot of peripheral blood B cells blood pre- and post- administration of anti-B220 antibody treatment to B6 mice and FIG.16J shows peripheral blood B cell concentration.
  • FIG.16K shows an overlay of normalized total radiant efficiency curves and time to 50% fluorescence intensity of untreated B6, neutrophil depleted, macrophage depleted, T cell depleted, and B cell depleted mice.
  • FIG.16L shows a photograph of a Cy5-HA cryogel retrieved from NSG mice 3 months post-injection.
  • FIGS.17A-17B depict a histomorphometric analysis of Cy5-HA cryogels retrieved from T- and B- cell depleted mice.
  • FIG.17A shows a hematoxylin and eosin (H&E) stain of explanted Cy5-HA cryogels from T cell depleted and B cell depleted mice at days 1, 5, and 10.
  • Scale bar left 800 ⁇ m
  • scale bar right 100 ⁇ m.
  • FIGS.18A-18G show an assessment of innate immune cell infiltration into Cy5-HA cryogels.
  • FIG.18A depicts a schematic for the quantification of innate immune cell content in Cy5-HA cryogels.
  • FIG.18B shows representative flow cytometry plots depicting gating strategy to determine cellular identity of CD45 + CD11b + F4/80 + (macrophage) cells, CD45 + CD11b + F4/80- Ly6G + (neutrophil) cells, and CD45 + CD11b + F4/80- Ly6G- CD115 + (monocyte) cells in untreated B6 mice, anti-Ly6G and anti-rat ⁇ immunoglobulin light chain antibody treated B6 mice, clodronate liposome treated B6 mice, and NSG mice.
  • FIGS.18C- 18E show quantification of total number of CD45 + CD11b + (myeloid) cells (FIG.18C), macrophages (FIG.18D), and neutrophils (FIG.18E) infiltrating HA cryogels in untreated B6 mice, anti-Ly6G and anti-rat ⁇ immunoglobulin light chain antibody treated B6 mice, clodronate liposome treated B6 mice, and NSG mice.
  • FIG.18F shows a schematic depicting workflow for in vivo Cy5-HA cryogel degradation study with gp91 phox- mice.
  • FIG.18G shows representative IVIS fluorescence images of gel degradation and quantification by measuring total radiant efficiency normalized to initial day 3 timepoint of gp91 phox- mice and B6 mice.
  • IVIS Images are on the same scale and analyzed using Living Image Software.
  • FIGS.19A-19H depict a supplementary analysis of myeloid cell infiltration of Cy5- HA cryogels retrieved from immunodeficient mice.
  • FIG.19A shows representative flow cytometry plots gated to determine cellular identity of CD45 + CD11b + F4/80 + (macrophage) cells, CD45 + CD11b + F4/80- Ly6G + (neutrophil) cells, and CD45 + CD11b + F4/80- Ly6G- CD115 + (monocyte) cells T cell depleted and B cell depleted mice.
  • FIG.19B shows percent of AnnexinV- (live) cells within Cy5-HA cryogels one and ten days after implant from flow cytometry analysis.
  • FIGS.19C-19D depict the quantification of total number of myeloid cells (FIG.19C) and macrophages (FIG.19D) infiltrating Cy5-HA cryogels in untreated B6 mice, T cell depleted mice, and B cell depleted mice.
  • FIG.19E shows representative flow cytometry plots from neutrophil depleted mice with and without intracellular Ly6G staining. Plotted data assessing neutrophils as a percentage of total myeloid cells (CD45 + CD11b + ) with and without intracellular Ly6G staining.
  • FIG.19F shows the quantification of total number of neutrophils infiltrating Cy5-HA cryogels in untreated B6 mice, T cell depleted mice, and B cell depleted mice.
  • FIGS.19G-19H depicts the quantification of total number of monocytes (FIG.19G) and infiltrating immune cell lineages (FIG.19H) plotted as a percentage of myeloid cells in untreated, neutrophil depleted, macrophage depleted, T cell depleted, B cell depleted, and NSG mice.
  • FIGS.20A-20C depict immunohistochemical staining of Cy5-HA cryogels retrieved from untreated B6 and NSG mice.
  • IHC immunohistochemistry
  • FIGS.21A-21G shows supplementary data for degradation kinetics of HA cryogels post-HSCT.
  • FIG.21A shows representative flow cytometry plots of bone marrow before and after lineage depletion.
  • FIG.21B shows time to 50% fluorescence intensity of Cy5-HA cryogels in non-irradiated and post-HSCT mice.
  • FIG.21C shows percent of AnnexinV- (live) cells within Cy5-HA cryogels 5- and 16-days post injection in non-irradiated mice and 5-, 16-, 21-, and 26-days post-injection in post-HSCT mice.
  • FIG.21D depicts infiltrating immune cell lineages plotted as a percentage of myeloid cells in non-irradiated and post- HSCT mice.
  • FIG.21E shows a schematic for tetrazine (Tz) and norbornene (Nb) functionalization of oxidized alginate (OxAlg), Cy5 functionalization of Nb functionalized OxAlg, and crosslinking of Tz functionalized HA with Cy5 functionalized OxAlg.
  • FIG.21F depicts measuring Cy5-OxAlg cryogel degradation in vitro by quantifying the Cy5-signal in supernatant at pre-determined timepoints normalized to total Cy5-signal in supernatant across all timepoints.
  • FIG.21G shows representative in vivo imaging system (IVIS) fluorescence images of gel degradation in mice and measuring Cy5-tagged 40% oxidized alginate cryogel degradation in vivo by quantification of total radiant efficiency normalized to initial 2-hour timepoint.
  • IVIS Images are on the same scale and analyzed using Living Image Software.
  • Data in FIG.21G represents mean ⁇ s.e.m.
  • FIGS.22A-22F shows that degradation kinetics of HA cryogels is impaired during transient immunodeficiency following HSCT.
  • FIG.22A shows a schematic depicting workflow for quantification of Cy5-HA cryogel degradation and innate immune cell infiltration in control (non-irradiated mice that do not receive a transplant) and post-HSCT B6 mice.
  • FIG.22B shows representative IVIS fluorescence images of gel degradation in non-irradiated and post-HSCT mice. Tracking gel degradation by quantification of total radiant efficiency normalized to initial day 3 timepoint.
  • FIG.22C shows a photograph of Cy5-HA cryogels in non-irradiated mice 5- and 16-days post-injection and post-HSCT mice on days 5, 16, 21, and 26.
  • FIGS.22D-22F show cell infiltration of CD45 + CD11b + (myeloid) cells (FIG.22D), CD45 + CD11b + F4/80 + (macrophage) cells (FIG.22E), and CD45 + CD11b + F4/80- Ly6G + (neutrophil) cells (FIG.22F) into HA cryogels in non-irradiated mice 5- and 16-days post-injection and 5-, 16-, 21-, and 26- days post-HSCT.
  • Data in FIG.22B were compared using two-way ANOVA with Bonferroni’s multiple comparison test.
  • Data in FIGS.22D-22F were compared using student’s t-test. Part of FIG.22A was created with BioRender.com.
  • FIGS.23A-23E shows enhanced reconstitution of peripheral blood neutrophil cells.
  • FIG.23A shows a schematic depicting outline of study to quantify Cy5 G-CSF release from HA cryogels in non-irradiated, non-transplanted B6 mice and post-HSCT B6 mice.
  • FIG.23B shows representative IVIS fluorescence images of Cy5 G-CSF release from HA cryogels and quantification by measuring total radiant efficiency normalized to initial 8-hour timepoint. IVIS Images are on the same scale and analyzed using Living Image Software.
  • FIG.23C shows a schematic depicting outline of study to quantify neutrophil reconstitution rate and Cy5-HA cryogel degradation rate in post-HSCT mice using G-CSF encapsulated Cy5-HA cryogels.
  • FIG.23D depicts peripheral blood reconstitution of neutrophils in post-HSCT mice.
  • FIG.23E shows representative in vivo imaging system (IVIS) fluorescence images of gel degradation in mice and measuring Cy5-HA cryogel degradation in vivo by quantification of total radiant efficiency normalized to initial day 3 timepoint.
  • IVIS Images are on the same scale and analyzed using Living Image Software.
  • FIGS.23B, 23E were compared using two-way ANOVA with Bonferroni’s multiple comparison test.
  • Data in FIG.23D were compared using mixed-effect regression model with random intercepts. Parts of FIG.23A, 23C were created with BioRender.com.
  • FIG.24 shows data for G-CSF release from HA cryogels. Time to 50% fluorescence intensity for Cy5 G-CSF encapsulated within HA cryogels in non-irradiated and post-HSCT mice. Data was compared using students t-test.
  • FIG.25 is a table summarizing various methods and durability of immune cell depletion. DETAILED DESCRIPTION OF THE INVENTION I.
  • modulate or “alter” are intended to include stimulation, activation, and/or enhancement (e.g., increasing or upregulating a particular response, level, or activity) and inhibition (e.g., decreasing or downregulating a particular response, level, or activity).
  • enhancement e.g., increasing or upregulating a particular response, level, or activity
  • inhibition e.g., decreasing or downregulating a particular response, level, or activity
  • “Therapeutically effective amount,” as used herein, is intended to include the amount of an active agent or composition that, when administered to a patient for treating a subject having a subject suffering from or susceptible to a disease, disorder, and/or condition, is sufficient to treat, alleviate, ameliorate, relieve, alleviate symptoms of, prevent, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of the disease, disorder, or condition, or one or more symptoms of the disease, disorder, or condition, or its related comorbidities). Determination of an effective amount is well within the capability of those skilled in the art.
  • an effective dose of an active agent described herein is an amount sufficient to produce at least some desired therapeutic effect in a subject.
  • the amount is a therapeutically effective amount.
  • agent active agent
  • therapeutic agent is defined as any chemical entity that has certain function or activity.
  • An active agent includes, but is not limited to an atom, a chemical group, a small molecule organic compound, an inorganic compound, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protein, a fusion protein, or a protein complex.
  • the agent may be detected by methods known in the art.
  • an agent or moiety may be chemiluminescent or fluorescent and can be detected by any suitable detection assays known in the art.
  • the function or activity of an agent may include any physical, chemical, biological, or physiological function or activity.
  • the term “active agent” or “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, prophylactic, and/or diagnostic effect and elicits a desired biological and/or pharmacological effect.
  • treatment or “treating” is defined as the application or administration of an active agent to a subject, or application or administration of an active agent to an isolated tissue or cell line from a subject, said subject having a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
  • treating can include suppressing, inhibiting, preventing, treating, or a combination thereof.
  • Treating refers, inter alia, to increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.
  • “Suppressing” or “inhibiting”, refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.
  • the symptoms are primary, while in another embodiment, symptoms are secondary.
  • Primary refers to a symptom that is a direct result of a disorder, e.g., diabetes, while, secondary refers to a symptom that is derived from or consequent to a primary cause. Symptoms may be any manifestation of a disease or pathological condition.
  • treatment includes any administration of a composition described herein and includes: (i) preventing the disease from occurring in a subject which may be predisposed to the disease but does not yet experience or display the pathology or symptomatology of the disease; (ii) inhibiting the disease in an subject that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., arresting further development of the pathology and/or symptomatology); or (iii) ameliorating the disease in a subject that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., reversing the pathology and/or symptomatology).
  • the efficacy of compositions as described herein in, e.g., the treatment of a condition described herein can be determined by the skilled clinician.
  • a treatment is considered "effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein.
  • Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g., absolute neutrophil count.
  • Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein.
  • successful treatment can be determined, for example, by measurement of absolute counts for individual blood cell types (e.g., white blood cells, red blood cells, and/or platelets) in the peripheral blood, reaching a number of cells accepted by those of skill in the art as within the normal range for the subject.
  • individual blood cell types e.g., white blood cells, red blood cells, and/or platelets
  • a differential leukocyte count e.g., including counts of each type of white blood cell, e.g., neutrophils, eosinophils, basophils, monocytes, and lymphocytes
  • platelet counts are known to those skilled in the art.
  • the blood can be collected at regular intervals in a tube containing an anti-coagulant like the EDTA, the cells can be counted using an automated blood count analyzer or manually using a hemocytometer.
  • Neutrophils are a type of white blood cell that are a marker of engraftment; the absolute neutrophil count (ANC) must be at least within the typical normal range for the treatment to be effective.
  • treatment means delaying or preventing the onset of such a disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing down or stopping the progression, aggravation or deterioration the progression or severity of a condition associated with such a disease or disorder.
  • the symptoms of a disease or disorder are alleviated by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% or more.
  • “preventing” or “prevention” refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). Efficacy of treatment is determined in association with any known method for diagnosing the disorder. Alleviation of one or more symptoms of the disorder indicates that the composition confers a clinical benefit. Any of the therapeutic methods described to above can be applied to any suitable subject including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • “Suppressing,” “reducing,” or “inhibiting,” refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.
  • the symptoms are primary, while in another embodiment, symptoms are secondary.
  • Primary refers to a symptom that is a direct result of a disorder, e.g., diabetes, while, secondary refers to a symptom that is derived from or consequent to a primary cause. Symptoms may be any manifestation of a disease or pathological condition.
  • the terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount.
  • the terms “reduced”, “reduction”, “decrease”, or “inhibit” can mean a decrease by at least 5% as compared to a reference level, for example a decrease by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% or more as compared to a reference level.
  • the terms can represent a 100% decrease, i.e.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 5% as compared to a reference level, for example an increase of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% or more.
  • the terms can represent an increase up to and including a 100% increase as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • an "increase" is a statistically significant increase in such level.
  • immune responsive degradation generally refers to a material that degrades or dissolves in response to changes in the immune system of a subject.
  • changes in the immune system of a subject include, but are not limited to, changes in the number and/or function of an immune cell.
  • changes in the immune system of a subject effect degradation of hylaronic acid (HA), for example, by myeloid cells through enzymatic action and by neutrophil-mediated oxidation.
  • immune responsive degradation of the material can include myeloid responsive degradation and/or neutrophil-responsive degradation to sustain or control the release of an agent from the material.
  • controlled release refers to an agent-containing formulation, such as a composition or scaffold as described herein, in which release of the agent is not immediate, i.e., with a “controlled release” formulation, administration does not result in immediate release of the agent.
  • controlled release refers to “sustained release” rather than to “delayed release” formulations.
  • the controlled release of the agent from a material is based, at least in part, on the immune responsive degradation of the material.
  • the immune responsive degradation of the material includes myeloid responsive degradation and/or neutrophil-responsive degradation to control the release of an agent from the material.
  • sustained release (synonymous with “extended release”) is used in its conventional sense to refer to a formulation, such as a composition or scaffold as described herein, that provides for gradual release of an agent over an extended period of time.
  • the sustained release of the agent from a material is based, at least in part, on the immune responsive degradation of the material.
  • the immune responsive degradation of the material includes myeloid responsive degradation and/or neutrophil- responsive degradation to sustain the release of an agent from the material.
  • delayed release is used in its conventional sense to refer to a formulation, such as a composition or scaffold as described herein, in which there is a time delay between administration of the formulation and the release of the agent therefrom.
  • “Delayed release” may or may not involve gradual release of agent over an extended period of time, and thus may or may not be “sustained release.”
  • the delayed release of the agent from a material is based, at least in part, on the immune responsive degradation of the material.
  • the immune responsive degradation of the material includes myeloid responsive degradation and/or neutrophil-responsive degradation to sustain or control the release of an agent from the material.
  • the active agents are delivered in a formulation to provide delayed release at a pre-determined time following administration. In some embodiments, the active agents are delivered in a formulation to provide delayed release based, at least in part, on the immune responsive degradation of the material following administration.
  • the active agents are delivered in a formulation to provide delayed release based, at least in part, on the number and/or function of neutrophils of and the material following administration.
  • the delay may be up to about 6 hours, about 12 hours, about 1 days, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months or more.
  • the delay is about 1 days, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 1 week, or about 2 weeks.
  • an agent such as a granulocyte colony stimulating factor (G-CSF)
  • G-CSF granulocyte colony stimulating factor
  • the present invention advantageously utilizes neutrophil-responsive degradation to sustain the release of a G-CSF from hylaronic acid (HA) hydrogels or HA cryogels.
  • the delayed release period provides sufficient time for immune recovery in a subject, during which hematopoietic stem cells can reestablish blood cell production in the subject whose bone marrow or immune system is damaged or defective.
  • the "percent release" of an agent is generally described as a percentage of the agent released for a material, such as a composition or scaffold as described herein, versus the total amount of agent encapsulated in the material. In some instances, the percent release of the agent from a material may increase with an increase in the immune responsive degradation of the material. In some instances, the immune responsive degradation of the material includes myeloid responsive degradation and/or neutrophil-responsive degradation to sustain or control the release of an agent from the material.
  • the term “immune system” refers to the ensemble, or to any one or more of the components, molecules, substances, structures (e.g. cells, tissue, and organs) and physiologic processes involved in the immune response.
  • hematopoietic stem cells or “HSC” refers to stem cells that can differentiate into the hematopoietic lineage and give rise to all blood cell types such as white blood cells and red blood cells, including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B- cells, K-cells).
  • myeloid e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • Stem cells are defined by their ability to form multiple cell types (multipotency) and their ability to self-renew.
  • Hematopoietic stem cells can be identified, for example, by cell surface markers such as CD34-, CD133+, CD48-, CD150+, CD244-, cKit+, Scal+, and lack of lineage markers (negative for B220, CD3, CD4, CD8, Macl, Grl, and Terl l9, among others).
  • Stem cell transplants are used to restore the stem cell reservoir when the bone marrow has been destroyed by disease, chemotherapy (chemo), or radiation. Depending on the source of the stem cells, this procedure may be called a bone marrow transplant, a peripheral blood stem cell transplant, or a cord blood transplant.
  • hematopoietic stem cell transplants HSCT
  • HSC Hematopoietic stem cells
  • progenitors are commonly used to replace the hematopoietic system in patients with hematopoietic malignancies, or patients undergoing high dose chemotherapy.
  • Hematopoietic reconstitution after transplantation encompasses the recovery of optimal numbers of hematopoietic stem cells and hematopoietic cells of both the myeloid and lymphoid lineages and their functions, thereby restoring a functional bone marrow.
  • Methods to determine successful transplant and/or hematopoietic reconstitution are known in the art.
  • the long term repopulating ability of candidate hematopoietic stem cells can be evaluated, e.g., in an in vivo sheep model or an in vivo NOD-SCID mouse model for human HSC.
  • the NOD/SCID mouse is an immunodeficient recipient, which allows the introduction of human, NHP or mouse cells and the determination of stem cell functionality through engraftment, proliferation and differentiation into at least two distinct lineages (typically myeloid and lymphoid).
  • This in vivo reconstitution assay is typically known as the Competitive Repopulating Unit (CRU) or SCID Repopulating Cell (SRC) assay.
  • CRU Competitive Repopulating Unit
  • SRC SCID Repopulating Cell
  • successful hematopoietic reconstitution can be determined, by measurement of absolute counts for individual blood cell types (white blood cells, red blood cells and platelets) in the peripheral blood, reaching a number of cells accepted by those of skill in the art as within the normal range for the subject.
  • Methods of conducting a complete blood count, differential leukocyte count, i.e., including counts of each type of white blood cell, for e.g., neutrophils, eosinophils, basophils, monocytes, and lymphocytes, and platelet counts are known to those skilled in the art.
  • the blood can be collected at regular intervals in a tube containing an anti- coagulant like the EDTA, the cells can be counted using an automated blood count analyzer or manually using a hemocytometer.
  • Neutrophils as defined below, are a type of white blood cell that are a marker of engraftment; the absolute neutrophil count (ANC) must be at least 500 for three days in a row to say that engraftment has occurred. This can occur as soon as 10 days after transplant, although 15 to 20 days is common for patients who are given bone marrow or peripheral blood cells.
  • Umbilical cord blood recipients usually require between 21 and 35 days for neutrophil engraftment. Platelet counts are also used to determine when engraftment has occurred.
  • the platelet count must be between 20,000 and 50,000 (without a recent platelet transfusion). This usually occurs at the same time or soon after neutrophil engraftment, but can take as long as eight weeks and even longer in some instances for people who are given umbilical cord blood.
  • hematopoietic progenitor cells encompasses pluripotent cells which are committed to the hematopoietic cell lineage, generally do not self-renew, and are capable of differentiating into several cell types of the hematopoietic system, such as granulocytes, monocytes, erythrocytes, megakaryocytes, B-cells and T-cells, including, but not limited to, short term hematopoietic stem cells (ST-HSCs), multi-potent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), granulocyte-monocyte progenitor cells (GMPs), megakaryocyte-erythrocyte progenitor cells (MEPs), and committed lymphoid progenitor cells (CLPs).
  • ST-HSCs short term hematopoietic stem cells
  • MPPs multi-potent progenitor cells
  • CMPs common myeloid progenitor cells
  • the presence of hematopoietic progenitor cells can be determined functionally as colony forming unit cells (CFU-Cs) in complete methylcellulose assays, or phenotypically through the detection of cell surface markers (e.g., CD45-, CD34+, Terl l9-, CD16/32, CD127, cKit, Seal) using assays known to those of skill in the art.
  • CFU-Cs colony forming unit cells
  • cell surface markers e.g., CD45-, CD34+, Terl l9-, CD16/32, CD127, cKit, Seal
  • functional innate leukocytes such as neutrophils
  • a functional innate leukocyte or a functional neutrophil can have the normal functions of the cell in vivo.
  • Cell function is generally determined by the combination of the lineage of the cell, the proliferation status of the cell, and the activation status of the cell.
  • the lineage status, proliferation status, and activation status can all be detected, for example, by detecting gene expression, protein expression, or a combination of gene and protein expression.
  • the lineage status of a cell can often be determined by the surface receptors on a cell, which allows for affinity based cell sorting.
  • functional neutrophils refers to neutrophils characterized by the ability to produce superoxide to perform oxidative burst.
  • Exemplary mouse models that may be useful for assessing neutrophil function associated with oxidative burst are known in the art and include, without limitation, the gp91-phox knockout mice that have neutrophils which lack this ability to produce superoxide to perform oxidative burst. See, e.g., https://www.jax.org/strain/002365; Pollock, J.D. et al. Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet 9, 202-209 (1995); and Banerjee, E.R. & Henderson, W.R., Jr. Role of T cells in a gp91phox knockout murine model of acute allergic asthma.
  • the term “subject” includes any subject who may benefit from being administered a hydrogel, cryogel, or an implantable drug delivery device of the invention comprising, for example, a hyaluronic acid (HA).
  • HA hyaluronic acid
  • the subject may benefit from being administered an HA hydrogel or an HA cryogel.
  • the term “subject” includes animals, e.g., vertebrates, amphibians, fish, mammals, non-human animals, including humans and primates, such as chimpanzees, monkeys and the like. In one embodiment of the invention, the subject is a human.
  • subject also includes agriculturally productive livestock, for example, cattle, sheep, goats, horses, pigs, donkeys, camels, buffalo, rabbits, chickens, turkeys, ducks, geese and bees; and domestic pets, for example, dogs, cats, caged birds and aquarium fish, and also so-called test animals, for example, hamsters, guinea pigs, rats and mice.
  • farmly productive livestock for example, cattle, sheep, goats, horses, pigs, donkeys, camels, buffalo, rabbits, chickens, turkeys, ducks, geese and bees
  • domestic pets for example, dogs, cats, caged birds and aquarium fish
  • test animals for example, hamsters, guinea pigs, rats and mice.
  • neutral neutrophils or "polymorphonuclear neutrophils (PMNs)" as used herein, refers to the most abundant type of white blood cells in mammals, which form an essential part of the innate immune system.
  • neutrophils are one of the first-responders of inflammatory cells to migrate toward the site of inflammation. They migrate through the blood vessels, then through interstitial tissue, following chemical signals such as interleukin- 8 (1L-8) and CSa in a process called chemotaxis, the directed motion of a motile cell or part along a chemical concentration gradient toward environmental conditions it deems attractive and/or away from surroundings it finds repellent.
  • chemical signals such as interleukin- 8 (1L-8) and CSa in a process called chemotaxis, the directed motion of a motile cell or part along a chemical concentration gradient toward environmental conditions it deems attractive and/or away from surroundings it finds repellent.
  • a variety of methods can be used to assess neutrophil function.
  • neutrophil function can be determined by assessing the phagocytic ability of neutrophils or assessing their ability to perform other important neutrophil functions such as oxidative burst and degranulation. Additionally, specific biochemical effects of neutrophil activation, such as reactive oxidant release, or hypochlorous acid production following myeloperoxidase (MPO) activation by a population of neutrophils may be evaluated.
  • neutrophil activation such as reactive oxidant release, or hypochlorous acid production following myeloperoxidase (MPO) activation by a population of neutrophils may be evaluated.
  • MPO myeloperoxidase activation
  • Neutropenia itself may be the result of disease, genetic disorders, drugs, toxins, and radiation as well as many therapeutic treatments, such as high dose chemotherapy (HDC) and conventional oncology therapy.
  • HDC high dose chemotherapy
  • many cancers have been found to be sensitive to extremely high doses of radiation or anti-neoplastic (anti-cancer) drugs, such intensive HDC is not widely used because it not only kills cancerous cells, but also frequently destroys the cells of the hematopoietic system that are responsible for generating the army of neutrophils that are necessary to maintain a functioning immune system. Complete destruction of neutrophil progenitor and precursor cells eliminates the patient's short-term capacity to generate mature neutrophils, thereby severely compromising the patient's ability to combat infection.
  • a neutropenic condition is diagnosed based on the absolute neutrophil count (ANC), which is determined by multiplying the percentage of bands and neutrophils on a differential by the total white blood cell count.
  • ANC absolute neutrophil count
  • Typical accepted reference range for absolute neutrophil count (ANC) in adults is about 1500 to about 8000 cells per microliter ( ⁇ l) of blood.
  • ⁇ l microliter
  • the severity of neutropenia is categorized as mild for an ANC of about 1000 to about 1500 cells per ml, moderate for an ANC of about 500 to about 1000 cells per ml, and severe for an ANC of fewer than about 500 cells per ml.
  • administering includes dispensing, delivering or applying a composition as described herein to a subject by any suitable route for delivery of the composition to the subject, including delivery by injection. Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion.
  • “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.
  • the compositions are administered by injection, e.g., subcutaneous injection.
  • COMPOSITIONS FOR MODULATING INNATE LEUKOCYTES The present invention features compositions and methods for delivering an active agent to a subject.
  • compositions and methods disclosed herein provide a means to treat and/or prevent diseases associated with a deficiency in levels of functional innate leukocytes, such as neutrophils.
  • the compositions of the present invention include a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent.
  • functional innate leukocytes such as neutrophils
  • neutrophils can mediate essential host defense against pathogens and are among the earliest responders in tissue injury 1-3 .
  • Neutrophil deficiency termed neutropenia, contributes to opportunistic infections and can impair tissue regeneration in affected individuals 4-7 .
  • hematopoietic stem cell transplantation pre-conditioning myelosuppressive regimens can contribute to a marked transient post-therapy impairment of neutrophils and render recipients susceptible to immune deficiency-associated complications for up to several weeks 6, 8-11 .
  • Post-HSCT neutrophil regeneration follows successful bone marrow engraftment of transplanted hematopoietic cells 12, 13 , facilitated by granulocyte colony stimulating factor (G- CSF)-mediated granulopoiesis of hematopoietic cells 14-16 .
  • G- CSF granulocyte colony stimulating factor
  • Neutropenia is typically treated as an emergency and, in a subset of patients, the risk of neutropenia may be prophylactically addressed with post-HSCT subcutaneous injection of recombinant human G-CSF (filgrastim) to facilitate recovery 6, 14, 17, 18 .
  • Daily injections are often used as G-CSF has a half-life of only about 3 to about 4 hours, which can be extended, for example, by conjugating G-CSF with polyethylene glycol (PEGylation) 19, 20 .
  • PEGylation polyethylene glycol
  • immune responses against PEG have been demonstrated to enhance clearance of PEG-G-CSF in an antibody-dependent manner 21 . As multiple cycles of PEG-G-CSF treatment are common, long-term treatment can be rendered ineffective.
  • the present invention provides compositions and sustained release methods for delivering active agents, including G-CSF, while avoiding immune responses against PEG, and for concurrently assessing innate leukocyte, including neutrophil, function to improve the current standard-of-care.
  • the present invention provides compositions and methods which can improve post-HSCT recovery of functional innate leukocytes, including functional neutrophils, and for concurrently assessing innate leukocyte, including neutrophil, function and recovery.
  • the scaffold composition can comprise a biodegradable depot which can be used to prophylactically deliver an active agent, such as G-CSF, to post- HSCT recipients.
  • the depot can comprise a porous injectable scaffold made by low- temperature crosslinking, termed cryogelation, of hyaluronic acid (HA), an easily sourced and readily derivatized anionic glycosaminoglycan, also referred to herein as “HA cryogel.”
  • HA hyaluronic acid
  • endogenous HA is a substrate for degradation by myeloid cells through enzymatic action and by neutrophil-mediated oxidation 22-24 .
  • the invention is based, at least in part, on the discovery that the degradation profile of the scaffold compositions, described herein, comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, is responsive to functional innate leukocyte recovery.
  • composition of the present invention comprise a scaffold, e.g., a polymer scaffold, comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and, optionally, an active agent.
  • the scaffold e.g., a polymer scaffold, comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof, can have immune responsive degradation behavior.
  • the scaffold can have myeloid responsive, e.g., neutrophil responsive, degradation behavior that can be configured to achieve controlled, sustained, and/or delayed release of the active agent upon administration to the subject.
  • the immune responsive degradation of the scaffold and the release of the active agent modulates the activity of an innate leukocyte; (ii) restores levels of functional innate leukocytes, e.g., neutrophils; (iii) reconstitutes levels of functional innate leukocytes; (iv) enhances the regeneration of functional innate leukocytes in the subject; (v) enhances immunity of the subject in need thereof; and/or (vi) treats or prevents a disorder in the subject, wherein the disorder is caused by or associated with a deficiency in functional innate leukocytes.
  • the scaffold can comprise one or more biomaterials.
  • the biomaterial is a biocompatible material that is non-toxic and/or non-immunogenic.
  • biocompatible material refers to any material that does not induce a significant immune response or deleterious tissue reaction, e.g., toxic reaction or significant irritation, over time when implanted into or placed adjacent to the biological tissue of a subject.
  • the scaffold can comprise biomaterials that are non-biodegradable or biodegradable.
  • the biomaterial can be a non-biodegradable material.
  • Exemplary non-biodegradable materials include, but are not limited to, metal, plastic polymer, or silk polymer.
  • the polymer scaffold comprises a biodegradable material.
  • the biodegradable material may be degraded by physical or chemical action, e.g., level of hydration, heat, oxidation, or ion exchange or by cellular action, e.g., elaboration of enzyme, peptides, or other compounds by nearby or resident cells.
  • the polymer scaffold comprises a biodegradable material that is a substrate for an endogenous enzyme.
  • the polymer scaffold comprises a biodegradable material that is a substrate for an endogenous enzyme that catalyzes the degradation of a hyaluronic acid (HA).
  • HA hyaluronic acid
  • the polymer scaffold comprises a biodegradable material that is a substrate for a hyaluronoglucosidase, such as a hyaluronidase. In certain embodiments, the polymer scaffold comprises a biodegradable material that is a substrate for a hyaluronidase. In certain embodiments, the polymer scaffold comprises both non- degradable and degradable materials. In some ebodiments, the scaffold composition degrades upon exposure to functional innate leukocytes, e.g., neutrophils.
  • the innate leukocyte (i) is selected from the group consisting of a natural killer cell, a mast cell, an eosinophil, a basophil, a macrophage, a dendritic cell, a gamma-delta ( ⁇ ) T cell, and a neutrophil; and/or (ii) is not a dendritic cell.
  • the innate leukocyte comprises a neutrophil.
  • the scaffold composition can degrade at a predetermined rate based on a physical parameter selected from the group consisting of temperature, pH, hydration status, porosity, the cross-link density, type, and chemistry or the susceptibility of main chain linkages to degradation.
  • the scaffold composition can degrade at a rate based on a level of immune deficiency and/or a level of functional innate leukocytes, including a level of activated neutrophils, in a subject.
  • a scaffold composition comprising hyaluronic acid (HA), as described herein, can degrade in a matter of days, e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more days, or can degrade in a matter of weeks, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more weeks.
  • the scaffold composition degrades at a predetermined rate based on a ratio of chemical polymers.
  • a high molecular weight polymer comprised of solely lactide degrades over a period of years, e.g., 1-2 years
  • a low molecular weight polymer comprised of a 50:50 mixture of lactide and glycolide degrades in a matter of weeks, e.g., 1, 2, 3, 4, 6, or 10 weeks.
  • a calcium cross-linked gels composed of high molecular weight, high guluronic acid alginate degrade over several months (1, 2, 4, 6, 8, 10, or 12 months) to years (1, 2, or 5 years) in vivo, while a gel comprised of low molecular weight alginate, and/or alginate that has been partially oxidized, will degrade in a matter of weeks.
  • one or more active agents, disclosed herein are covalently or non-covalently linked or attached to the scaffold composition.
  • one or more active agents disclosed herein is incorporated on, into, or present within the structure or pores of, the scaffold composition.
  • one or more compounds or proteins e.g., the growth factors, the differentiation factors, and the homing factors
  • the scaffold comprises biomaterials that are modified, e.g., oxidized or reduced.
  • the degree of modification can be varied from about 1% to about 100%.
  • the degree of modification means the molar percentage of the sites on the biomaterial that are modified with a functional group.
  • the degree of modification can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%
  • modified biomaterials include, but are not limited to, oxidized hyaluronic acid, reduced- alginate, oxidized alginate, MA-alginate (methacrylated alginate) or MA-gelatin.
  • a modified biomaterial e.g., hydrogels or cryogels
  • the use of a modified biomaterials, e.g., hydrogels or cryogels may accelerate degradation of the polymer.
  • biomaterials suitable for use as scaffolds in the present invention include glycosaminoglycan, silk, fibrin, MATRIGEL®, poly-ethyleneglycol (PEG), polyhydroxy ethyl methacrylate, polyacrylamide, poly (N-vinyl pyrolidone), (PGA), poly lactic-co- glycolic acid (PLGA), poly e-carpolactone (PCL), polyethylene oxide, poly propylene fumarate (PPF), poly acrylic acid (PAA), polyhydroxybutyric acid, hydrolysed polyacrylonitrile, polymethacrylic acid, polyethylene amine, esters of alginic acid; pectinic acid; and alginate, fully or partially oxidized alginate, hyaluronic acid, carboxy methyl cellulose, heparin, heparin sulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan, xanthan, collagen, gelatin, carboxymethyl star
  • the biomaterial is selected from the group consisting of alginate, fully or partially oxidized alginate, and combinations thereof.
  • the scaffolds of the present invention may comprise an external surface. Alternatively, or in addition, the scaffolds may comprise an internal surface. External or internal surfaces of the scaffolds of the present invention may be solid or porous. Pore size of the scaffolds can be less than about 10 nm, between about 100 nm-20 ⁇ m, or greater than about 20 ⁇ m, e.g., up to and including 1000 ⁇ m in diameter.
  • the pores may be nanoporous, microporous, or macroporous.
  • the diameter of nanopores is less than about 10 nm; the diameter of micropores is in the range of about 100 nm-20 ⁇ m; and, the diameter of macropores is greater than about 20 ⁇ m, e.g., greater than about 50 ⁇ m, e.g., greater than about 100 ⁇ m, e.g., greater than about 400 ⁇ m, e.g., greater than 600 ⁇ m or greater than 800 ⁇ m. In some embodiment the diameter of the pore is between about 50 ⁇ m and about 80 ⁇ m.
  • the scaffolds of the present invention are organized in a variety of geometric shapes (e.g., discs, beads, pellets), niches, planar layers (e.g., thin sheets).
  • discs of about 0.1-200 millimeters in diameter may be implanted subcutaneously.
  • the disc may have a thickness of 0.1 to 10 millimeters, e.g., 1, 2, or 5 millimeters.
  • the discs are readily compressed or lyophilized for administration to a patient.
  • An exemplary disc for subcutaneous administration has the following dimensions: 8 millimeters in diameter and 1 millimeter in thickness.
  • the scaffolds may comprise multiple components and/or compartments.
  • a multiple compartment device is assembled in vivo by applying sequential layers of similarly or differentially doped gel or other scaffold material to the target site.
  • the device is formed by sequentially injecting the next, inner layer into the center of the previously injected material using a needle, thereby forming concentric spheroids.
  • non-concentric compartments are formed by injecting material into different locations in a previously injected layer.
  • a multi- headed injection device extrudes compartments in parallel and simultaneously.
  • the layers are made of similar or different biomaterials differentially doped with pharmaceutical compositions.
  • compartments self-organize based on their hydro-philic/phobic characteristics or on secondary interactions within each compartment.
  • multicomponent scaffolds are optionally constructed in concentric layers each of which is characterized by different physical qualities such as the percentage of polymer, the percentage of crosslinking of polymer, chemical composition of the hydrogel, pore size, porosity, and pore architecture, stiffness, toughness, ductility, viscoelasticity, the growth factors, the differentiation factors, and/or homing factors incorporated therein and/or any other compositions incorporated therein.
  • Hydrogel and Cryogel Scaffolds In certain embodiments, the scaffolds of present invention comprise one or more hydrogels.
  • a hydrogel is a polymer gel comprising a network of crosslinked polymer chains.
  • a hydrogel is usually a composition comprising polymer chains that are hydrophilic.
  • hydrogels allow them to absorb significant amounts of water. Some hydrogels are highly stretchable and elastic; others are viscoelastic. Hydrogel are sometimes found as a colloidal gel in which water is the dispersion medium. In certain embodiments, hydrogels are highly absorbent (they can contain over 99% water (v/v)) natural or synthetic polymers that possess a degree of flexibility very similar to natural tissue, due to their significant water content. In certain embodiments, a hydrogel may have a property that, when an appropriate shear stress is applied, the deformable hydrogel is dramatically and reversibly compressed (up to 95% of its volume), resulting in injectable macroporous preformed scaffolds.
  • Hydrogels have been used for therapeutic applications, e.g., as vehicles for in vivo delivery of therapeutic agents, such as small molecules, cells and biologics.
  • Hydrogels are commonly produced from polysaccharides, such as alginates.
  • the polysaccharides may be chemically manipulated to modulate their properties and properties of the resulting hydrogels.
  • the hydrogels of the present invention may be either porous or non-porous.
  • the compositions of the invention are formed of porous hydrogels.
  • the hydrogels may be nanoporous wherein the diameter of the pores is less than about 10 nm; microporous wherein the diameter of the pores is preferably in the range of about 100 nm-20 ⁇ m; or macroporous wherein the diameter of the pores is greater than about 20 ⁇ m, more preferably greater than about 100 ⁇ m and even more preferably greater than about 400 ⁇ m.
  • the hydrogel is macroporous with pores of about 50-80 ⁇ m in diameter.
  • the hydrogel is macroporous with aligned pores of about 400-500 ⁇ m in diameter.
  • the hydrogel may be constructed out of a number of different rigid, semi-rigid, flexible, gel, self-assembling, liquid crystalline, or fluid compositions such as peptide polymers, polysaccharides, synthetic polymers, hydrogel materials, ceramics (e.g., calcium phosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans, metals and metal alloys.
  • the compositions are assembled into hydrogels using methods known in the art, e.g., injection molding, lyophilization of preformed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, gas foaming, fiber forming/processing, particulate leaching or a combination thereof.
  • the assembled devices are then implanted or administered to the body of an individual to be treated.
  • the composition comprising a hydrogel may be assembled in vivo in several ways.
  • the hydrogel is made from a gelling material, which is introduced into the body in its ungelled form where it gels in situ.
  • Exemplary methods of delivering components of the composition to a site at which assembly occurs include injection through a needle or other extrusion tool, spraying, painting, or methods of deposit at a tissue site, e.g., delivery using an application device inserted through a cannula.
  • the ungelled or unformed hydrogel material is mixed with at least one pharmaceutical composition prior to introduction into the body or while it is introduced.
  • the resultant in vivo/in situ assembled device e.g., hydrogel
  • In situ assembly of the hydrogel may occur as a result of spontaneous association of polymers or from synergistically or chemically catalyzed polymerization.
  • Synergistic or chemical catalysis is initiated by a number of endogenous factors or conditions at or near the assembly site, e.g., body temperature, ions or pH in the body, or by exogenous factors or conditions supplied by the operator to the assembly site, e.g., photons, heat, electrical, sound, or other radiation directed at the ungelled material after it has been introduced.
  • the energy is directed at the hydrogel material by a radiation beam or through a heat or light conductor, such as a wire or fiber optic cable or an ultrasonic transducer.
  • a shear-thinning material such as an amphiphile, is used which re-cross links after the shear force exerted upon it, for example by its passage through a needle, has been relieved.
  • the hydrogel may be assembled ex vivo.
  • the hydrogel is injectable.
  • the hydrogels are created outside of the body as macroporous scaffolds. Upon injection into the body, the pores collapse causing the gel to become very small and allowing it to fit through a needle.
  • hydrogels for both in vivo and ex vivo assembly of hydrogel devices are well known in the art and described, e.g., in Lee et al., 2001, Chem. Rev.7:1869-1879.
  • the peptide amphiphile approach to self-assembly assembly is described, e.g., in Hartgerink et al., 2002, Proc. Natl. Acad. Sci. USA 99:5133-5138.
  • exemplary hydrogels are comprised of materials that are compatible with encapsulation of materials including polymers, nanoparticles, polypeptides, and cells.
  • Exemplary hydrogels are fabricated from alginate, polyethylene glycol (PEG), PEG-acrylate, agarose, hyaluronic acid, or synthetic protein (e.g., collagen or engineered proteins (i.e., self-assembly peptide-based hydrogels)).
  • PEG polyethylene glycol
  • PEG-acrylate e.g., agarose, hyaluronic acid, or synthetic protein (e.g., collagen or engineered proteins (i.e., self-assembly peptide-based hydrogels)
  • synthetic protein e.g., collagen or engineered proteins (i.e., self-assembly peptide-based hydrogels)
  • BDTM PuraMatrixTM BDTM PuraMatrixTM.
  • BDTM PuraMatrixTM Peptide Hydrogel is a synthetic matrix that is used to create defined three dimensional (3D) micro-environments for cell culture.
  • the hydrogel is a biocompatible polymer matrix that is biodegradable in whole or in part.
  • Examples of materials which can form hydrogels include alginates and alginate derivatives, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA) polymers, gelatin, collagen, agarose, hyaluronic acid, hyaluronic acid derivative, natural and synthetic polysaccharides, polyamino acids such as polypeptides particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon.- caprolactone, polyanhydrides; polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone), and copolymers of the above, including graft copolymers.
  • Synthetic polymers and naturally-occurring polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels may also be used.
  • derivative refers to a compound that is derived from a similar compound by a chemical reaction.
  • oxidized alginate which is derived from alginate through oxidization reaction, is a derivative of alginate
  • the implantable composition can have virtually any regular or irregular shape including, but not limited to, spheroid, cubic, polyhedron, prism, cylinder, rod, disc, or other geometric shape.
  • the implant is of cylindrical form from about 0.5 to about 10 mm in diameter and from about 0.5 to about 10 cm in length. Preferably, its diameter is from about 1 to about 5 mm and its length from about 1 to about 5 cm.
  • the compositions of the invention are of spherical form. When the composition is in a spherical form, its diameter can range, in some embodiments, from about 0.5 to about 50 mm in diameter. In some embodiments, a spherical implant’s diameter is from about 5 to about 30 mm. In an exemplary embodiment, the diameter is from about 10 to about 25 mm.
  • the scaffold comprises click-hydrogels and/or click-cryogels.
  • a click hydrogel or cryogel is a gel in which cross-linking between hydrogel or cryogel polymers is facilitated by click reactions between the polymers.
  • Each polymer may contain one of more functional groups useful in a click reaction. Given the high level of specificity of the functional group pairs in a click reaction, active compounds can be added to the preformed device prior to or contemporaneously with formation of the hydrogel device by click chemistry.
  • Non-limiting examples of click reactions that may be used to form click- hydrogels include Copper I catalyzed azide-alkyne cycloaddition, strain-promoted assize- alkyne cycloaddition, thiol-ene photocoupling, Diels-Alder reactions, inverse electron demand Diels-Alder reactions, tetrazole-alkene photo-click reactions, oxime reactions, thiol- Michael addition, and aldehyde-hydrazide coupling.
  • Non-limiting aspects of click hydrogels are described in Jiang et al., 2014, Biomaterials, 35:4969-4985, the entire content of which is incorporated herein by reference.
  • a click alginate is utilized (see, e.g., PCT International Patent Application Publication No. WO 2015/154078 published October 8, 2015, hereby incorporated by reference in its entirety).
  • a hydrogel e.g., cryogel
  • a growth factor such as BMP-2
  • a differentiation factor such as a DLL-4
  • a space for cells e.g., stem cells such as hematopoietic stem cells (HSC) infiltration and trafficking.
  • HSC hematopoietic stem cells
  • the hydrogel system according to the present invention delivers BMP-2, which acts as a hematopoietic stem cell (HSC) and/or hematopoietic progenitor cell enhancement/recruitment factor, and DLL-4 as a differentiation factor, which facilitates T cell lineage specification of hematopoietic stem cell and/or hematopoietic progenitor cells.
  • BMP-2 acts as a hematopoietic stem cell (HSC) and/or hematopoietic progenitor cell enhancement/recruitment factor
  • DLL-4 as a differentiation factor, which facilitates T cell lineage specification of hematopoietic stem cell and/or hematopoietic progenitor cells.
  • a cryogel composition e.g., formed of MA-alginate, can function as a delivering platform by creating a local niche, such as a specific niche for enhancing T-lineage specification.
  • the cryogel creates a local niche in which the encounter of cells, such as recruited stem cells or progenitor cells, and various exemplary agent of the invention, such as the growth factor and/or differentiation factor can be controlled.
  • the cells and the exemplary agents of the present invention are localized into a small volume, and the contacting of the cells and the agents can be quantitatively controlled in space and time.
  • the hydrogel e.g., cryogel
  • the hydrogel can be engineered to coordinate the delivery of an active agent in space and time, potentially enhancing overall immune modulation performance by adjusting the differentiation and/or specification of recruited cells, such as hematopoietic stem cells or progenitor cells.
  • the cells and growth factor/differentiation factor are localized into a small volume, and the delivery of factors in space and time can be quantitatively controlled. As the growth/differentiation factors are released locally, few systemic effects are anticipated, in contrast to systemically delivered agents, such as growth factors.
  • Examples of polymer compositions from which the cryogel or hydrogel is fabricated are described throughout the present disclosure, and include alginate, hyaluronic acid, gelatin, heparin, dextran, carob gum, PEG, PEG derivatives including PEG-co-PGA and PEG-peptide conjugates.
  • the techniques can be applied to any biocompatible polymers, e.g., collagen, chitosan, carboxymethylcellulose, pullulan, polyvinyl alcohol (PVA), Poly(2-hydroxyethyl methacrylate) (PHEMA), Poly(N-isopropylacrylamide) (PNIPAAm), or Poly(acrylic acid) (PAAc).
  • the composition comprises an alginate- based hydrogel/cryogel.
  • the scaffold comprises a gelatin-based hydrogel/cryogel.
  • Cryogels are a class of materials with a highly porous interconnected structure that are produced using a cryotropic gelation (or cryogelation) technique. Cryogels also have a highly porous structure.
  • cryogel devices are characterized by high porosity, e.g., at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% pores with thin pore walls that are characterized by high density of polymer crosslinking.
  • porosity refers to the percentage of the volume of pores to the volume of the scaffold. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • the walls of cryogels are typically dense and highly cross-linked, enabling them to be compressed through a needle into a subject without permanent deformation or substantial structural damage.
  • the pore walls comprise at least about 10, 15, 20, 25, 30, 35, or 40% (w/v) polymer. It is intended that values and ranges intermediate to the recited values are part of this invention. In other embodiments, the pore walls comprise about 10-40% polymer. In some embodiments, a polymer concentration of about 0.5-4% (w/v) (before the cryogelation) is used, and the concentration increases substantially upon completion of cryogelation. Non-limiting aspects of cryogel gelation and the increase of polymer concentration after cryogelation are discussed in Beduer et al., 2015 Advanced Healthcare Materials 4.2: 301-312, the entire content of which is incorporated herein by reference.
  • cryogelation comprises a technique in which polymerization- crosslinking reactions are conducted in quasi-frozen reaction solution.
  • Non-limiting examples of cryogelation techniques are described in U.S. Patent Application Publication No. 20140227327, published August 14, 2014, the entire content of which is incorporated herein by reference.
  • An advantage of cryogels compared to conventional macroporous hydrogels obtained by phase separation is their high reversible deformability. Cryogels may be extremely soft but can be deformed and reform their shape.
  • cryogels can be very tough, can withstand high levels of deformations, such as elongation and torsion and can also be squeezed under mechanical force to drain out their solvent content.
  • the improved deformability properties of alginate cryogels originate from the high crosslinking density of the unfrozen liquid channels of the reaction system.
  • the macromonomer e.g., methacrylated alginate
  • the macromonomers and initiator system e.g., APS/TEMED
  • APS/TEMED APS/TEMED
  • a porous material is produced whose microstructure is a negative replica of the ice formed. Ice crystals act as porogens. Desired pore size is achieved, in part, by altering the temperature of the cryogelation process.
  • the cryogelation process is typically carried out by quickly freezing the solution at -20 °C. Lowering the temperature to, e.g., -80° C , would result in more ice crystals and lead to smaller pores.
  • the cryogel is produced by cryo-polymerization of at least methacrylated (MA)-alginate and MA-PEG.
  • the cryogel is produced by cryo-polymerization of at least MA-alginate, the growth factor, the differentiation factor, and MA-PEG.
  • the invention also features gelatin scaffolds, e.g., gelatin hydrogels such as gelatin cryogels, which are a cell-responsive platform for biomaterial- based therapy.
  • Gelatin is a mixture of polypeptides that is derived from collagen by partial hydrolysis. These gelatin scaffolds have distinct advantages over other types of scaffolds and hydrogels/cryogels.
  • the gelatin scaffolds of the invention support attachment, proliferation, and survival of cells and are degraded by cells, e.g., by the action of enzymes such as matrix metalloproteinases (MMPs) (e.g., recombinant matrix metalloproteinase-2 and -9).
  • MMPs matrix metalloproteinases
  • prefabricated gelatin cryogels rapidly reassume their approximately original shape ("shape memory") when injected subcutaneously into a subject (e.g., a mammal such as a human, dog, cat, pig, or horse) and elicit little or no harmful host immune response (e.g., immune rejection) following injection.
  • the hydrogel e.g., cryogel
  • the hydrogel comprises polymers that are modified, e.g., sites on the polymer molecule are modified with a methacrylic acid group (methacrylate (MA)) or an acrylic acid group (acrylate).
  • exemplary modified hydrogels/cryogels are MA- alginate (methacrylated alginate) or MA-gelatin.
  • the degree of methacrylation of alginate or gelatin 50% corresponds to the degree of methacrylation of alginate or gelatin. This means that every other repeat unit contains a methacrylated group.
  • the degree of methacrylation can be varied from about 1% to about 100%. Preferably, the degree of methacrylation varies from about 1% to about 90%.
  • polymers can also be modified with acrylated groups instead of methacrylated groups. The product would then be referred to as an acrylated-polymer.
  • the degree of methacrylation (or acrylation) can be varied for most polymers. However, some polymers (e.g., PEG) maintain their water-solubility properties even at 100% chemical modification.
  • cross-linking efficiency refers to the percentage of macromonomers that are covalently linked.
  • the polymers in the hydrogel are 50-100% crosslinked (covalent bonds).
  • the extent of crosslinking correlates with the durability of the hydrogel.
  • a high level of crosslinking (90-100%) of the modified polymers is desirable.
  • the highly crosslinked hydrogel/cryogel polymer composition is characterized by at least about 50% polymer crosslinking (e.g., about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%; it is intended that values and ranges intermediate to the recited values are part of this invention.).
  • the high level of crosslinking confers mechanical robustness to the structure.
  • the percentage of crosslinking is less than about 100%.
  • the composition is formed using a free radical polymerization process and a cryogelation process.
  • the cryogel is formed by cryopolymerization of methacrylated gelatin, methacrylated alginate, or methacrylated hyaluronic acid.
  • the cryogel comprises a methacrylated gelatin macro monomer or a methacrylated alginate macromonomer at concentration of about 1.5% (w/v) or less (e.g., about 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or less; it is intended that values and ranges intermediate to the recited values are part of this invention.).
  • the methacrylated gelatin or alginate macromonomer concentration is about 1% (w/v).
  • the cryogel comprises at least about 75% (v/v) pores, e.g., about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (v/v) or more pores. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • the pores are interconnected. Interconnectivity is important to the function of the hydrogel and/or cryogel, as without interconnectivity, water would become trapped within the gel. Interconnectivity of the pores permits passage of water (and other compositions such as cells and compounds) in and out of the structure.
  • the hydrogel in a fully hydrated state, comprises at least about 90% water (volume of water / volume of the scaffold) (e.g., between about 90-99%, at least about 92%, 95%, 97%, 99%, or more).
  • at least about 90% (e.g., at least about 92%, 95%, 97%, 99%, or more) of the volume of the cryogel is made of liquid (e.g., water) contained in the pores. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • the cryogel in a compressed or dehydrated hydrogel, up to about 50%, 60%, 70% of that water is absent, e.g., the cryogel comprises less than about 25% (e.g., about 20%, 15%, 10%, 5% or less) water.
  • the cryogels of the invention comprise pores large enough for a cell to travel through.
  • the cryogel contains pores of about 20-500 ⁇ m in diameter, e.g., about 20-30 ⁇ m, about 30-150 ⁇ m, about 50-500 ⁇ m, about 50-450 ⁇ m, about 100-400 ⁇ m, about 200-500 ⁇ m.
  • the hydrated pore size is about 1- 500 ⁇ m (e.g., about 10-400 ⁇ m, about 20-300 ⁇ m, about 50-250 ⁇ m).
  • the cryogel contains pores about 50-80 ⁇ m in diameter.
  • injectable hydrogels or cryogels are further functionalized by addition of a functional group selected from the group consisting of: amino, vinyl, aldehyde, thiol, silane, carboxyl, azide, or alkyne.
  • the cryogel is further functionalized by the addition of a further cross-linker agent (e.g., multiple arms polymers, salts, aldehydes, etc.).
  • the solvent can be aqueous, and in particular, acidic or alkaline.
  • the aqueous solvent can comprise a water-miscible solvent (e.g., methanol, ethanol, DMF, DMSO, acetone, dioxane, etc).
  • a water-miscible solvent e.g., methanol, ethanol, DMF, DMSO, acetone, dioxane, etc.
  • the cryo-crosslinking may take place in a mold and the cryogels (which may be injected) can be degradable.
  • the pore size can be controlled by the selection of the main solvent used, the incorporation of a porogen, the freezing temperature and rate applied, the crosslinking conditions (e.g. polymer concentration), and also the type and molecule weight of the polymer used.
  • the shape of the cryogel may be dictated by a mold and can thus take on any shape desired by the fabricator, e.g., various sizes and shapes (disc, cylinders, squares, strings, etc.) are prepared by cryogenic polymerization.
  • Injectable cryogels can be prepared in the micrometer-scale to centimeter-scale. Exemplary volumes vary from a few hundred ⁇ m 3 (e.g., about 100-500 ⁇ m 3 ) to about 10 cm 3 .
  • an exemplary scaffold composition is between about 100 ⁇ m 3 to 100 mm 3 in size.
  • the scaffold is between about 10 mm 3 to about 100 mm 3 in size.
  • the scaffold is about 30 mm 3 in size.
  • the cryogels are hydrated, loaded with compounds and loaded into a syringe or other delivery apparatus.
  • the syringes are prefilled and refrigerated until use.
  • the cryogel is dehydrated, e.g., lyophilized, optionally with a compound (such as a growth factor or differentiation factor) loaded in the gel and stored dry or refrigerated.
  • a cryogel-loaded syringe or apparatus may be contacted with a solution containing compounds to be delivered.
  • the barrel of the cryogel pre-loaded syringe is filled with a physiologically- compatible solution, e.g., phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the cryogel may be administered to a desired anatomical site followed by administration of the physiologically- compatible solution, optionally containing other ingredients, e.g., a growth factor and/or a differentiation factor or together with one or more compounds disclosed herein.
  • the cryogel is then rehydrated and regains its shape integrity in situ.
  • the volume of PBS or other physiologic solution administered following cryogel placement is generally about 10 times the volume of the cryogel itself.
  • the cryogel also has the advantage that, upon compression, the cryogel composition maintains structural integrity and shape memory properties.
  • the cryogel is injectable through a hollow needle.
  • the cryogel returns to its approximately original geometry after traveling through a needle (e.g., a 16 gauge (G) needle, e.g., having a 1.65 mm inner diameter).
  • a needle e.g., a 16 gauge (G) needle, e.g., having a 1.65 mm inner diameter.
  • Other exemplary needle sizes are 16-gauge, an 18-gauge, a 20- gauge, a 22- gauge, a 24-gauge, a 26-gauge, a 28-gauge, a 30-gauge, a 32-gauge, or a 34- gauge needle.
  • Injectable cryogels have been designed to pass through a hollow structure, e.g., very fine needles, such as 18-30 G needles.
  • the cryogel returns to its approximately original geometry after traveling through a needle in a short period of time, such as less than about 10 seconds, less than about 5 seconds, less than about 2 seconds, or less than about 1 second.
  • the cryogels may be injected to a subject using any suitable injection device.
  • the cryogels may be injected using syringe through a needle.
  • a syringe may include a plunger, a needle, and a reservoir that comprises compositions of the present invention.
  • the injectable cryogels may also be injected to a subject using a catheter, a cannula, or a stent.
  • the injectable cryogels may be molded to a desired shape, in the form of rods, square, disc, spheres, cubes, fibers, foams.
  • the cryogel is in the shape of a disc, cylinder, square, rectangle, or string.
  • the cryogel composition is between about 100 ⁇ m 3 to 10 cm 3 in size, e.g., between 10 mm 3 to 100 mm 3 in size.
  • the cryogel composition is between about 1 mm in diameter to about 50 mm in diameter (e.g., about 5 mm).
  • the thickness of the cryogel is between about 0.2 mm to about 50 mm (e.g., about 2 mm).
  • Three exemplary cryogel materials systems are described below.
  • the base material is click alginate (PCT International Patent Application Publication No. WO 2015/154078 published October 8, 2015, hereby incorporated by reference in its entirety).
  • the base material contains laponite (commercially available silicate clay used in many consumer products such as cosmetics).
  • Laponite has a large surface area and highly negative charge density which allows it to adsorb positively charged moieties on a variety of proteins and other biologically active molecules by an electrostatic interaction, thereby allowing drug loading. When placed in an environment with a low concentration of drug, adsorbed drug releases from the laponite in a sustained manner.
  • Various embodiments of the present subject matter include delivery vehicles comprising a pore-forming scaffold composition.
  • pores such as macropores
  • a pore-forming scaffold composition For example, pores (such as macropores) are formed in situ within a hydrogel following hydrogel injection into a subject. Pores that are formed in situ via degradation of a sacrificial porogen hydrogel within the surrounding hydrogel (bulk hydrogel) facilitate recruitment and trafficking of cells, as well as the release of any composition or agent of the present invention, for example, a growth factor, such as BMP-2, a differentiation factor , or a homing factor, or any combination thereof.
  • a growth factor such as BMP-2, a differentiation factor , or a homing factor, or any combination thereof.
  • the sacrificial porogen hydrogel, the bulk hydrogel, or both the sacrificial porogen hydrogel and the bulk hydrogel may comprise any composition or agent of the present invention, for example, a growth factor, a differentiation factor, and/or, a homing factor, or any combination thereof.
  • the pore-forming composition becomes macroporous over time when resident in the body of a recipient animal such as a mammalian subject.
  • the pore-forming composition may comprise a sacrificial porogen hydrogel and a bulk hydrogel, wherein the sacrificial porogen hydrogel degrades at least about 10% faster (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% faster) than the bulk hydrogel. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • the sacrificial porogen hydrogel may degrade leaving macropores in its place. In certain embodiments, the macropores are open interconnected macropores.
  • the sacrificial porogen hydrogel may degrade more rapidly than the bulk hydrogel, because the sacrificial porogen hydrogel (i) is more soluble in water (comprises a lower solubility index), (ii) is cross-linked to protease-mediated degradation motifs as described in U.S.
  • Patent Application Publication No.2005-0119762 published June 2, 2005 (incorporated herein by reference in its entirety), (iii) comprises a shorter polymer that degrades more quickly compared to that of a longer bulk hydrogel polymer, (iv) is modified to render it more hydrolytically degradable than the bulk hydrogel (e.g., by oxidation), and/or (v) is more enzymatically degradable compared to the bulk hydrogel.
  • a scaffold is loaded (e.g., soaked with) with one or more active compounds after polymerization.
  • device or scaffold polymer forming material is mixed with one or more active compounds before polymerization.
  • a device or scaffold polymer forming material is mixed with one or more active compounds before polymerization, and then is loaded with more of the same or one or more additional active compounds after polymerization.
  • pore size or total pore volume of a composition or scaffold is selected to influence the release of compounds from the device or scaffold. Exemplary porosities (e.g., nanoporous, microporous, and macroporous scaffolds and devices) and total pore volumes (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more of the volume of the scaffold) are described herein. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • a pore size or total pore volume is selected to increase the speed at which active ingredients exit the composition or scaffold.
  • an active ingredient may be incorporated into the scaffold material of a hydrogel or cryogel, e.g., to achieve continuous release of the active ingredient from the scaffold or device over a longer period of time compared to active ingredient that may diffuse from a pore cavity. Porosity influences recruitment of the cells into devices and scaffolds and the release of substances from devices and scaffolds. Pores may be, e.g., nanoporous, microporous, or macroporous.
  • the diameter of nanopores is less than about 10 nm.
  • Micropores are in the range of about 100 nm to about 20 ⁇ m in diameter.
  • Macropores are greater than about 20 ⁇ m (e.g., greater than about 100 ⁇ m or greater than about 400 ⁇ m) in diameter.
  • Exemplary macropore sizes include about 50 ⁇ m, about 100 ⁇ m, about 150 ⁇ m, about 200 ⁇ m, about 250 ⁇ m, about 300 ⁇ m, about 350 ⁇ m, about 400 ⁇ m, about 450 ⁇ m, about 500 ⁇ m, about 550 ⁇ m, and about 600 ⁇ m in diameter. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • Macropores are those of a size that permit a eukaryotic cell to traverse into or out of the composition.
  • a macroporous composition has pores of about 400 ⁇ m to about 500 ⁇ m in diameter. The preferred pore size depends on the application. In certain embodiments, the pores have a diameter of about 50 ⁇ m to about 80 ⁇ m.
  • the composition is manufactured in one stage in which one layer or compartment is made and infused or coated with one or more compounds.
  • Exemplary bioactive compositions comprise polypeptides or polynucleotides.
  • the composition is manufactured in two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) stages in which one layer or compartment is made and infused or coated with one or more compounds followed by the construction of second, third, fourth or more layers, which are in turn infused or coated with one or more compounds in sequence.
  • each layer or compartment is identical to the others or distinguished from one another by the number or mixture of bioactive compositions as well as distinct chemical, physical and biological properties.
  • Polymers may be formulated for specific applications by controlling the molecular weight, rate of degradation, and method of scaffold formation. Coupling reactions can be used to covalently attach bioactive agent, such as the differentiation factor to the polymer backbone.
  • one or more compounds is added to the scaffold compositions using a known method including surface absorption, physical immobilization, e.g., using a phase change to entrap the substance in the scaffold material.
  • a growth factor is mixed with the scaffold composition while it is in an aqueous or liquid phase, and after a change in environmental conditions (e.g., pH, temperature, ion concentration), the liquid gels or solidifies thereby entrapping the bioactive substance.
  • covalent coupling e.g., using alkylating or acylating agents, is used to provide a stable, long term presentation of a compound on the scaffold in a defined conformation.
  • the composition of the present invention comprises a hyaluronic acid hydrogel or cryogel.
  • Hyaluronic acid HA; conjugate base hyaluronate
  • HA conjugate base hyaluronate
  • Hyaluronic acid is a polymer of disaccharides, composed of D-glucuronic acid and N- acetyl-D-glucosamine, linked via alternating ⁇ -(1 ⁇ 4) and ⁇ -(1 ⁇ 3) glycosidic bonds.
  • Hyaluronic acid can be 25,000 disaccharide repeats in length.
  • Polymers of hyaluronic acid can range in size from 5,000 to 20,000,000 Da.
  • Hyaluronic acid can also contain silicon.
  • Hyaluronic acid is energetically stable, in part because of the stereochemistry of its component disaccharides. Bulky groups on each sugar molecule are in sterically favored positions, whereas the smaller hydrogens assume the less-favorable axial positions.
  • Hyaluronic acid can be degraded by a family of enzymes called hyaluronidases, which are present in many mammals, e.g., a human. Hyaluronic acid can also be degraded via non-enzymatic reactions. These include acidic and alkaline hydrolysis, ultrasonic disintegration, thermal decomposition, and degradation by oxidants.
  • Hyaluronic acid is used to form hydrogels, e.g., cryogels, as a biomaterial scaffold in tissue engineering research.
  • Hyaluronic acid hydrogels are formed through crosslinking.
  • Hyaluronic acid can form a hydrogel, e.g., cryogel, into a desired shape to deliver therapeutic molecules into a host.
  • Hyaluronic acids for use in the present compositions, can be crosslinked by attaching thiols, methacrylates, hexadecylamides, and tyramines.
  • Hyaluronic acids can also be crosslinked directly with formaldehyde or with divinylsulfone.
  • hyaluronic acid includes unmodified hyaluronic acid or modified hyaluronic acid. Modified hyaluronic acid includes, but is not limited to, oxidized hyaluronic acid and/or reduced hyaluronic acid.
  • hyaluronic acid or hyaluronic acid polymers may also include hyaluronic acid, e.g., unmodified hyaluronic acid, oxidized hyaluronic acid or reduced hyaluronic acid, or methacrylated hyaluronic acid or acrylated hyaluronic acid.
  • Hyaluronic acid may also refer to any number of derivatives of hyaluronic acid.
  • the composition of the invention comprises an alginate hydrogel.
  • Alginates are versatile polysaccharide based polymers that may be formulated for specific applications by controlling the molecular weight, rate of degradation and method of scaffold formation.
  • Alginate polymers are comprised of two different monomeric units, (1- 4)-linked ⁇ -D-mannuronic acid (M units) and ⁇ L-guluronic acid (G units) monomers, which can vary in proportion and sequential distribution along the polymer chain.
  • Alginate polymers are polyelectrolyte systems which have a strong affinity for divalent cations (e.g., Ca +2 , Mg +2 , Ba +2 ) and form stable hydrogels when exposed to these molecules.
  • the alginate polymers useful in the context of the present invention can have an average molecular weight from about 20 kDa to about 500 kDa, e.g., from about 20 kDa to about 40 kDa, from about 30 kDa to about 70 kDa, from about 50 kDa to about 150 kDa, from about 130 kDa to about 300 kDa, from about 230 kDa to about 400 kDa, from about 300 kDa to about 450 kDa, or from about 320 kDa to about 500 kDa.
  • the alginate polymers useful in the present invention may have an average molecular weight of about 32 kDa. In another example, the alginate polymers useful in the present invention may have an average molecular weight of about 265 kDa. In some embodiments, the alginate polymer has a molecular weight of less than about 1000 kDa, e.g., less than about 900 KDa, less than about 800 kDa, less than about 700 kDa, less than about 600 kDa, less than about 500 kDa, less than about 400 kDa, less than about 300 kDa, less than about 200 kDa, less than about 100 kDa, less than about 50 kDa, less than about 40 kDa, less than about 30 kDa or less than about 25 kDa.
  • kDa e.g., less than about 900 KDa, less than about 800 kDa, less than about 700 kDa, less than about 600
  • the alginate polymer has a molecular weight of about 1000 kDa, e.g., about 900 kDa, about 800 kDa, about 700 kDa, about 600 kDa, about 500 kDa, about 400 kDa, about 300 kDa, about 200 kDa, about 100 kDa, about 50 kDa, about 40 kDa, about 30 kDa or about 25 kDa.
  • the molecular weight of the alginate polymers is about 20 kDa.
  • Coupling reactions can be used to covalently attach bioactive agent, such as an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protein, or a protein complex, to the polymer backbone.
  • bioactive agent such as an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protein, or a protein complex
  • bioactive agent such as an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protein, or a protein complex
  • alginate used interchangeably with the term “al
  • oxidized alginate comprises alginate comprising one or more aldehyde groups, or alginate comprising one or more carboxylate groups. In other embodiments, oxidized alginate comprises highly oxidized alginate, e.g., comprising one or more algoxalate units.
  • Oxidized alginate may also comprise a relatively small number of aldehyde groups (e.g., less than 15%, e.g., 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% aldehyde groups or oxidation on a molar basis). It is intended that values and ranges intermediate to the recited values are part of this invention.
  • alginate or “alginate polymers” may also include alginate, e.g., unmodified alginate, oxidized alginate or reduced alginate, or methacrylated alginate or acrylated alginate.
  • Alginate may also refer to any number of derivatives of alginic acid (e.g., calcium, sodium or potassium salts, or propylene glycol alginate ). See, e.g., WO1998012228A1, hereby incorporated by reference.
  • Porous and Pore-forming Scaffolds The scaffolds of the present invention may be nonporous or porous. In certain embodiments, the scaffolds of the present invention are porous. Porosity of the scaffold composition influences migration of the cells through the device.
  • Pores may be nanoporous, microporous, or macroporous.
  • the diameter of nanopores is less than about 10 nm.
  • Micropores are in the range of about 100 nm to about 20 ⁇ m in diameter.
  • Macropores are greater than about 20 ⁇ m (e.g., greater than about 100 ⁇ m or greater than about 400 ⁇ m) in diameter.
  • Exemplary macropore sizes include about 50 ⁇ m, 100 ⁇ m, 150 ⁇ m, 200 ⁇ m, 250 ⁇ m, 300 ⁇ m, 350 ⁇ m, 400 ⁇ m, 450 ⁇ m, 500 ⁇ m, 550 ⁇ m, and 600 ⁇ m in diameter. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • Macropores are of a size that permits a eukaryotic cell to traverse into or out of the composition.
  • a macroporous composition has pores of about 400 ⁇ m to 500 ⁇ m in diameter. The size of pores may be adjusted for different purpose. For example, for cell recruitment and cell release, the pore diameter may be greater than 50 ⁇ m.
  • a macroporous composition has pores of about 50 ⁇ m – about 80 ⁇ m in diameter.
  • the scaffolds contain pores before the administration into a subject.
  • the scaffolds comprise a pore-forming scaffold composition. Pore-forming scaffolds and the methods for making pore-forming scaffolds are known in the art. See, e.g., U.S.
  • the pore-forming scaffolds are not initially porous, but become macroporous over time resident in the body of a recipient animal such as a mammalian subject.
  • the pore-forming scaffolds are hydrogel scaffolds. The pore may be formed at different time, e.g., after about 12 hours, or 1, 3, 5, 7, or 10 days or more after administration, i.e., resident in the body of the subject.
  • the pore-forming scaffolds comprise a first hydrogel and a second hydrogel, wherein the first hydrogel degrades at least about 10% faster (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% faster, at least about 2 times faster, or at least about 5 times faster) than the second hydrogel. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • the first hydrogel comprises a porogen that degrades leaving a pore in its place.
  • the first hydrogel is a porogen and the resulting pore after degradation in situ is within 25% of the size of the initial porogen, e.g., within 20%, within 15%, or within 10% of the size of the initial porogen. Preferably, the resulting pore is within 5% of the size of the initial porogen. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • the first hydrogel may degrade faster than the second hydrogel due to the difference in their physical, chemical, and/or biological properties. In certain embodiments, the first hydrogel degrades more rapidly than the second hydrogel, because the first hydrogel is more soluble in water (comprises a lower solubility index).
  • the first hydrogel degrades more rapidly because it is cross-linked to protease-mediated degradation motifs as described in U.S. Patent Publication US2005/0119762A1, the content of which is incorporated herein by reference.
  • the molecular mass of the polymers used to form the first hydrogel composition is approximately 50 kilodaltons (kDa)
  • the molecular mass of the polymers used to form the second hydrogel composition is approximately 250 kDa.
  • a shorter polymer e.g., that of a porogen degrades more quickly compared to that of a longer polymer (e.g., that of the bulk composition).
  • a composition is modified to render it more hydrolytically degradable by virtue of the presence of sugar groups (e.g., approximately 3-10% sugar of an alginate composition).
  • the porogen hydrogel is chemically modified, such as oxidized, to render it more susceptible to degradation.
  • the porogen hydrogel is more enzymatically degradable compared to the bulk hydrogel.
  • the composite (first and second hydrogel) composition is permeable to bodily fluids, e.g., containing an enzyme which is exposed to the composition and degrades the porogen hydrogel.
  • the second hydrogel is cross-linked around the first hydrogel, i.e., the porogens (first hydrogel) are completely physically entrapped in the bulk (second) hydrogel.
  • the click reagents disclosed herein can be provided in the bulk hydrogel or the porogen hydrogel.
  • the click reagents e.g., polymers or nanoparticles, are provided in the bulk hydrogel.
  • hydrogel micro-beads (“porogens”) are formed. Porogens are encapsulated into a “bulk” hydrogel that is either non-degradable or which degrades at a slower rate compared to the porogens.
  • the composite material Immediately after hydrogel formation, or injection into the desired site in vivo, the composite material lacks pores. Subsequently, porogen degradation causes pores to form in situ. The size and distribution of pores are controlled during porogen formation, and mixing with the polymers which form the bulk hydrogel.
  • the polymer utilized in the pore-forming scaffolds is naturally- occurring or synthetically made.
  • both the porogens and bulk hydrogels are formed from alginate.
  • the alginate polymers suitable for porogen formation have a molecular weight from 5,000 to 500,000 Daltons.
  • the polymers are optionally further modified (e.g., by oxidation with sodium periodate, (Bouhadir et al., 2001, Biotech. Prog.
  • the polymers are crosslinked by extrusion through a nebulizer with co-axial airflow into a bath of divalent cation (for example, Ca 2+ or Ba 2+ ) to form hydrogel micro- beads. Higher airflow rate leads to lower the porogen diameter.
  • the porogen hydrogel microbeads contain oxidized alginate.
  • the porogen hydrogel can contain about 1-50% (w/v) oxidized alginate.
  • the porogen hydrogel can contain about 1-10% oxidized alginate.
  • the porogen hydrogel contains about 7.5% oxidized alginate.
  • the concentration of divalent ions used to form porogens may vary from about 5 to about 500 mM, and the concentration of polymer from about 1% to about 5% by weight/volume.
  • Porogen chemistry can further be manipulated to produce porogens that interact with host proteins and/or cells, or inhibit interactions with host proteins and/or cells.
  • the alginate polymers suitable for formation of the bulk hydrogel have a molecular weight from about 5,000 to about 500,000 Da.
  • the polymers may be further modified (for example, by oxidation with sodium periodate), to facilitate degradation, as long as the bulk hydrogel degrades more slowly than the porogen.
  • the polymers may also be modified to present biological cues to control cell responses (e.g., integrin binding adhesion peptides such as RGD).
  • Either the porogens or the bulk hydrogel may also encapsulate bioactive factors such as oligonucleotides, growth factors or drugs to further control cell responses.
  • the concentration of divalent ions used to form the bulk hydrogel may vary from about 5 to about 500 mM, and the concentration of polymer from about 1% to about 5% by weight/volume.
  • the elastic modulus of the bulk polymer is tailored for its purpose, e.g., to recruit stem cells or progenitor cells. Methods relevant to generating the hydrogels described herein include the following.
  • Bouhadir et al., 1999, Polymer, 40: 3575-84 (incorporated herein by reference in its entirety) describes the oxidation of alginate with sodium periodate, and characterizes the reaction.
  • Bouhadir et al., 2001, Biotechnol. Prog., 17: 945-50 (incorporated herein by reference in its entirety) describes oxidation of high molecular weight alginate to form alginate dialdehyde (alginate dialdehyde is high molecular weight (M w ) alginate in which a certain percent, e.g., 5%, of sugars in alginate are oxidized to form aldehydes), and application to make hydrogels degrade rapidly.
  • alginate dialdehyde is high molecular weight (M w ) alginate in which a certain percent, e.g., 5%, of sugars in alginate are oxidized to form aldehydes
  • Kong et al., 2002, Polymer, 43: 6239-46 shows that binary combinations of high Mw, GA rich alginate with irradiated, low Mw, high GA alginate crosslinks with calcium to form rigid hydrogels, but which degrade more rapidly and also have lower solution viscosity than hydrogels made from the same overall weight concentration of only high Mw, GA rich alginate.
  • Alsberg et al., 2003, J Dent Res, 82(11): 903-8 (incorporated herein by reference in its entirety) describes degradation profiles of hydrogels made from irradiated, low Mw, GA- rich alginate, with application in bone tissue engineering. Kong et al., 2004, Adv.
  • Mater, 16(21): 1917-21 describes control of hydrogel degradation profile by combining gamma irradiation procedure with oxidation reaction, and application to cartilage engineering.
  • Techniques to control degradation of hydrogen biomaterials are well known in the art.
  • Lutolf MP et al., 2003, Nat Biotechnol., 21: 513-8 describes poly(ethylene glycol) based materials engineered to degrade via mammalian enzymes (MMPs).
  • MMPs mammalian enzymes
  • Bryant SJ et al., 2007, Biomaterials, 28(19): 2978-86 US 7,192,693 B2; incorporated herein by reference in its entirety
  • a pore template (e.g., poly- methylmethacrylate beads) is encapsulated within a bulk hydrogel, and then acetone and methanol are used to extract the porogen while leaving the bulk hydrogel intact.
  • Silva et al., 2008, Proc. Natl. Acad. Sci USA, 105(38): 14347-52 (incorporated herein by reference in its entirety; US 2008/0044900) describes deployment of endothelial progenitor cells from alginate sponges. The sponges are made by forming alginate hydrogels and then freeze- drying them (ice crystals form the pores).
  • the scaffold composition comprises open interconnected macropores.
  • the scaffold composition comprises a pore-forming scaffold composition.
  • the pore-forming scaffold composition may comprise a sacrificial porogen hydrogel and a bulk hydrogel, wherein the pore-forming scaffold composition lacks macropores.
  • the sacrificial porogen hydrogel may degrade at least 10% faster than the bulk hydrogel leaving macropores in its place following administration of said pore-forming scaffold into a subject.
  • the sacrificial porogen hydrogel is in the form of porogens that degrade to form said macropores.
  • the macropores may comprise pores having a diameter of, e.g., about 10-400 ⁇ m.
  • active agent refers to an agent that is capable of modulating the activity of an innate leukocyte, restoring levels of functional innate leukocytes, reconstituting levels of functional innate leukocytes, enhancing the regeneration of functional innate leukocytes, enhancing immunity, and/or treating or preventing a disease or disorder in a subject, such as a disorder that is caused by or associated with a deficiency in functional innate leukocytes.
  • the active agent comprises an amino acid, a peptide, a protein, a nucleic acid, an oligonucleotide, a small molecule, or a combination thereof.
  • the active agent comprises a protein, optionally, wherein the protein is an isolated protein and/or a recombinant protein. In certain embodiments, the active agent comprises a cytokine, a chemokine, and/or a growth factor. In certain embodiments, the active agent comprises a cytokine, optionally, a pro-inflammatory cytokine and/or an anti- inflammatory cytokine.
  • the active agent is selected from the group consisting of a granulocyte colony stimulating factor (G-CSF), a granulocyte-macrophage colony- stimulating factor (GM-CSF), a bone morphogenetic protein 2 (BMP2), a stromal cell- derived factor (SDF), a platelet activating factor (PAF), a tumor necrosis factor ⁇ (TNF ⁇ ), an interleukin-1 (IL-1), an interleukin-1 ⁇ (IL-1 ⁇ ), an interleukin-1 ⁇ (IL-1 ⁇ ), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin- 6 (IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin-10 (IL-10), an interleukin-11 (IL-11), an interleukin-12 (IL-12), an interleukin-9
  • the active agent comprises a G-CSF.
  • the active agent comprises a polyethylene glycol conjugated granulocyte colony-stimulating factor (PEG-G-CSF).
  • PEG-G-CSF polyethylene glycol conjugated granulocyte colony-stimulating factor
  • the active agent is G-CSF.
  • the inclusion of such active agents in the compositions of the present invention promotes the infiltration of functional innate leukocytes, e.g., neutrophils, to the scaffold composition administered to the subject.
  • the active agents are encapsulated in the material.
  • the active agents are released from the material over an extended period of time (e.g., about 7-30 days or longer, about 17- 18 days).
  • the degradation profile of the scaffold compositions, described herein, comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent is responsive to functional innate leukocyte recovery.
  • immune deficiency and/or the level of functional innate leukocytes, including the level of activated neutrophils, in a subject can impact the rate of degradation of the scaffold compositions and the release of encapsulated active agent.
  • the immune-responsiveness of the scaffold compositions, described herein can facilitate the controlled, sustained, and/or delayed release of an encapsulated active agent, such as G-CSF.
  • the active agents retain their bioactivity over an extended period of time.
  • the bioactivity of the active agent may be measured by any appropriate means.
  • the active agents retain at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or more of their bioactivity for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days at least 20 days, or at least 30 days after the incorporation of the active agent into the scaffold.
  • the active agent may be present at between about 0.001 nmol and about 1000 nmol per scaffold, or about 0.001 and about 100 nmol per scaffold, or about 0.001 nmol and about 1 nmol per scaffold. In some embodiments, the active agent may be present at between about 1 ng to 1000 micrograms per scaffold. For example, the active agent may be present at an amount between about 1 ⁇ g and about 1000 ⁇ g, between about 1 ⁇ g and 500 ⁇ g, between about 1 ⁇ g and about 200 ⁇ g, between about 1 ⁇ g and about 100 ⁇ g, between about 1 ⁇ g and about 50 ⁇ g, or between about 1 ⁇ g and 10 ⁇ g.
  • the composition of the present invention comprises nanogram quantities of active agent (e.g., about 1 ng to about 1000 ng of active agent).
  • the active agent may be present at an amount between about 5 ng and about 500 ng, between about 5 ng and about 250 ng, between about 5 ng and about 200 ng, between about 10 ng and about 200 ng, between about 25 ng and about 200 ng, between about 50 ng and 200 ng, between about 100 ng and 200 ng, and about 200 ng.
  • Nanogram quantities of the active agent are also released in a controlled or sustained manner.
  • the nanogram quantities of the active agent and/or the controlled or sustained release can contribute to reduced toxicity of the compositions and methods of the present invention as compared to other delivery system, which uses high dose of active agent and has suboptimal release kinetics.
  • the amount of active agent present in a scaffold may vary according to the size of the scaffold.
  • the growth factor may be present at about 0.03 ng/mm 3 (the ratio of the amount of active agent in weight to the volume of the scaffold) to about 350 ng/mm 3 , such as between about 0.1 ng/mm 3 and about 300 ng/mm 3 , between about 0.5 ng/mm 3 and about 250 ng/mm 3 , between about 1 ng/mm 3 and about 200 ng/mm 3 , between about 2 ng/mm 3 and about 150 ng/mm 3 , between about 3 ng/mm 3 and about 100 ng/mm 3 , between about 4 ng/mm 3 and about 50 ng/mm 3 , between about 5 ng/mm 3 and 25 ng/mm 3 , between about 6 ng/mm 3 and about 10 ng/mm 3 , or between about 6.5 ng/mm 3 and about 7.0 ng/mm 3 .
  • the amount of active agent may be present at between about 300 ng/mm 3 and about 350 ⁇ g/mm 3 , such as between about 400 ng/mm 3 and between about 300 ⁇ g/mm 3 , between about 500 ng/mm 3 and about 200 ⁇ g/mm 3 , between about 1 ⁇ g/mm 3 and about 100 ⁇ g/mm 3 , between about 5 ⁇ g/mm 3 and about 50 ⁇ g/mm 3 , between about 10 ⁇ g/mm 3 and about 25 ⁇ g/mm 3 .
  • the active agent is covalently or non-covalently linked or attached to the scaffold composition.
  • one or more active agents disclosed herein is incorporated on, into, or present within the structure or pores of, the scaffold composition.
  • the active agent is covalently linked to the scaffold.
  • the active agent is covalently linked to the scaffold utilizing click chemistry.
  • the active agent is covalently linked to the scaffold utilizing N- hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or di-sulfide chemistry.
  • the composition of the present invention does not comprise a growth factor.
  • growth factor refers to an agent that is capable of stimulating cellular growth, proliferation, healing, and/or cellular differentiation.
  • growth factors are polypeptides. Growth factor polypeptides typically act as signaling molecules.
  • the growth factor polypeptides are cytokines.
  • the growth factor can recruit a cell to the scaffold following the administration of the composition to a subject.
  • the recruited cell may be autologous.
  • the recruited cell may be a stromal cell from the subject.
  • the autologous cell may be a stem cell (e.g., umbilical cord stem cells) of the subject.
  • the recruited cell may also be syngeneic, allogeneic or xenogeneic.
  • syngeneic refers to genetically identical, or sufficiently identical and immunologically compatible as to allow for transplantation.
  • syngeneic cells may include transplanted cells obtained from an identical twin.
  • allogeneic refers to cells that are genetically dissimilar, although from individuals of the same species.
  • xenogeneic refers to cells derived from a different species and therefore genetically different.
  • the recruited cell may be a donor cell in a transplantation.
  • the transplantation is a hematopoietic stem cell transplantation (HSCT).
  • HSCT hematopoietic stem cell transplantation
  • HSCT refers to the transplantation of multipotent hematopoietic stem cells or hematopoietic progenitor cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood.
  • HSCT may be autologous (the patient's own stem cells or progenitor cells are used), allogeneic (the stem cells or progenitor cells come from a donor), syngeneic (from an identical twin) or xenogenic (from different species).
  • the growth factors of the present invention may induce the formation of a tissue or organ within or around the administered composition.
  • the tissue or organ is a bony tissue or hematopoietic tissue.
  • the tissue formation may be restricted to the scaffold of the composition.
  • polypeptides e.g., growth factor polypeptides
  • methods of incorporating polypeptides are known in the art. See, US Patent Nos.: 8,728,456; 8,067,237; and 10,045,947; US Patent Publication No.: US20140079752; International Patent Publication No.: WO2017/136837; incorporated herein by reference in their entirety.
  • the release of the growth factor polypeptides may be controlled.
  • the methods of controlled release of polypeptides are known in the art. See, US Patent Nos.: 8,728,456; 8,067,237; 10,045,946, incorporated by reference in their entirety.
  • the growth factors may be released over an extended period of time, such as 7-30 days or longer.
  • the controlled release of the growth factors may affect the timing of the formation of the tissue or organ within the scaffold.
  • the release of the growth factors is controlled with the goal of creating a functional, active bone nodule or tissue within one to two weeks after subcutaneous injection of the compositions of the present invention.
  • the growth factors retain their bioactivity over an extended period of time.
  • bioactivity refers to the beneficial or adverse effects of an agent, such as a growth factor.
  • the bioactivity of the growth factor may be measured by any appropriate means.
  • the bioactivity of BMP-2 may be measured by its capacity to induce the formation of bone nodule or tissue and/or recruit cells into the scaffold.
  • the growth factors retain their bioactivity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after the incorporation of the growth factors into the scaffold.
  • Exemplary growth factors include, but are not limited to, bone morphogenetic proteins (BMP), epidermal growth factor (EGF), transforming growth factor beta (TGF- ⁇ ), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophins, Platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and interleukins.
  • BMP bone morphogenetic proteins
  • EGF epidermal growth factor
  • TGF- ⁇ transforming growth factor beta
  • G-CSF granulocyte-colony stimulating
  • the growth factor comprises a protein belonging to the transforming growth factor beta (TGF- ⁇ ) superfamily.
  • TGF- ⁇ superfamily is a large group of structurally related cell regulatory proteins.
  • TGF- ⁇ superfamily includes four major subfamilies: the TGF- ⁇ subfamily, the bone morphogenetic proteins and the growth differentiation factors, the activing and inhibin subfamilies, and a group encompassing various divergent members. Proteins from the TGF- ⁇ superfamily are active as homo- or heterodimer, the two chains being linked by a single disulfide bond.
  • TGF- ⁇ superfamily proteins interact with a conserved family of cell surface serine/threonine-specific protein kinase receptors, and generate intracellular signals using a conserved family of proteins called SMADs. TGF- ⁇ superfamily proteins play important roles in the regulation of basic biological processes such as growth, development, tissue homeostasis and regulation of the immune system.
  • TGF- ⁇ superfamily proteins include, but are not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, MSTN, NODAL, NRTN, PSPN, TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, and TGF- ⁇ 4.
  • the growth factor is BMP2.
  • the growth factor comprises a bone morphogenetic protein (BMP).
  • BMP bone morphogenetic protein
  • a BMP is a protein belonging to a group of growth factors also known as cytokines and as metabologens. BMPs can induce the formation of bone and cartilage and constitute a group of important morphogenetic signals, orchestrating tissue architecture throughout the body. Absence or deficiency of BMP signaling may be an important factor in diseases or disorders.
  • the BMP is selected from a group consisting of a BMP-2, a BMP-4, a BMP-6, a BMP-7, a BMP-12, a BMP-14, and any combination thereof.
  • the BMP is BMP-2.
  • BMP-2 plays an important role in the development of bone and cartilage. BMP-2 can potently induce osteoblast differentiation in a variety of cell types.
  • the growth factor comprises a TGF- ⁇ subfamily protein.
  • TGF- ⁇ subfamily protein or TGF- ⁇ is a multifunctional cytokine that includes four different isoforms (TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, and TGF- ⁇ 4).
  • TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, and TGF- ⁇ 4 Activated TGF- ⁇ complexes with other factors to form a serine/threonine kinase complex that binds to TGF- ⁇ receptors, which is composed of both type 1 and type 2 receptor subunits.
  • the type 2 receptor kinase phosphorylates and activates the type 1 receptor kinase that activates a signaling cascade. This leads to the activation of different downstream substrates and regulatory proteins, inducing transcription of different target genes that function in differentiation, chemotaxis, proliferation, and activation of many immune cells.
  • the growth factor comprises a TGF- ⁇ 1.
  • TGF- ⁇ 1 plays a role in the induction from CD4+ T cells of both induced Tregs (iTregs), which have a regulatory function, and Th17 cells, which secrete pro-inflammatory cytokines. TGF- ⁇ 1 alone precipitates the expression of Foxp3 and Treg differentiation from activated T helper cells.
  • the growth factors may be isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous growth factor polypeptides may be isolated from healthy human tissue. Synthetic growth factor polypeptides are synthesized in vivo following transfection or transformation of template DNA into a host organism or cell, e.g., a mammalian or human cell line. Alternatively, synthetic growth factor polypeptides are synthesized in vitro by cell free translation or other art-recognized methods Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual.
  • growth factor e.g., BMP-2 or TGF- ⁇ 1 polypeptides may be recombinant.
  • growth factor polypeptides are humanized derivatives of mammalian growth factor polypeptides. Exemplary mammalian species from which growth factor polypeptides are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate.
  • the growth factor is a recombinant human protein .
  • the growth factor is a recombinant murine (mouse) protein.
  • the growth factor is a humanized derivative of a recombinant mouse protein.
  • the growth factor polypeptides may be modified to increase protein stability in vivo.
  • the growth factor polypeptides may be engineered to be more or less immunogenic.
  • immunogenic and “immunogenicity” refer to the ability of a particular substance, such as a protein, an antigen, or an epitope, to provoke an immune response in the body of a human and other animal.
  • the growth factors may be present at between about 0.001 nmol and about 1000 nmol per scaffold, or about 0.001 and about 100 nmol per scaffold, or about 0.001 nmol and about 1 nmol per scaffold.
  • the growth factors may be present at between about 1 ng to 1000 micrograms per scaffold.
  • the growth factors may be present at an amount between about 1 ⁇ g and about 1000 ⁇ g, between about 1 ⁇ g and 500 ⁇ g, between about 1 ⁇ g and about 200 ⁇ g, between about 1 ⁇ g and about 100 ⁇ g, between about 1 ⁇ g and about 50 ⁇ g, or between about 1 ⁇ g and 10 ⁇ g.
  • the composition of the present invention comprises nanogram quantities of growth factors (e.g., about 1 ng to about 1000 ng of BMP-2).
  • the growth factors may be present at an amount between about 5 ng and about 500 ng, between about 5 ng and about 250 ng, between about 5 ng and about 200 ng, between about 10 ng and about 200 ng, between about 25 ng and about 200 ng, between about 50 ng and 200 ng, between about 100 ng and 200 ng, and about 200 ng.
  • Nanogram quantities of the growth factor are also released in a controlled manner.
  • the nanogram quantities of the growth factors and/or the controlled release can contribute to reduced toxicity of the compositions and methods of the present invention as compared to other delivery system, which uses high dose of growth factors and has suboptimal release kinetics.
  • the amount of growth factors present in a scaffold may vary according to the size of the scaffold.
  • the growth factor may be present at about 0.03 ng/mm 3 (the ratio of the amount of growth factors in weight to the volume of the scaffold) to about 350 ng/mm 3 , such as between about 0.1 ng/mm 3 and about 300 ng/mm 3 , between about 0.5 ng/mm 3 and about 250 ng/mm 3 , between about 1 ng/mm 3 and about 200 ng/mm 3 , between about 2 ng/mm 3 and about 150 ng/mm 3 , between about 3 ng/mm 3 and about 100 ng/mm 3 , between about 4 ng/mm 3 and about 50 ng/mm 3 , between about 5 ng/mm 3 and 25 ng/mm 3 , between about 6 ng/mm 3 and about 10 ng/mm 3 , or between about 6.5 ng/mm 3 and about 7.0 ng/mm 3 .
  • the amount of growth factors may be present at between about 300 ng/mm 3 and about 350 ⁇ g/mm 3 , such as between about 400 ng/mm 3 and between about 300 ⁇ g/mm 3 , between about 500 ng/mm 3 and about 200 ⁇ g/mm 3 , between about 1 ⁇ g/mm 3 and about 100 ⁇ g/mm 3 , between about 5 ⁇ g/mm 3 and about 50 ⁇ g/mm 3 , between about 10 ⁇ g/mm 3 and about 25 ⁇ g/mm 3 .
  • DIFFERENTIATION FACTORS The composition of the present invention can comprise a differentiation factor.
  • the composition of the present invention does not comprise a differentiation factor, for example, that induces the differentiation of a hematopoietic stem cell into a T cell progenitor cell.
  • a differentiation factor is an agent that can induce the differentiation of a cell, for example, a recruited cell.
  • the differentiation factor is a polypeptide.
  • “differentiation,” “cell differentiation,” “cellular differentiation,” or other similar terms refer to the process where a cell changes from one cell type to another. In certain embodiments, the cell changes to a more specialized type, e.g., from a stem cell or a progenitor cell to a T cell progenitor cell.
  • Differentiation occurs numerous times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Differentiation may change a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes may be due to highly controlled modifications in gene expression. Among dividing cells, there are multiple levels of cell potency, the cell's ability to differentiate into other cell types. A greater potency indicates a larger number of cell types that can be derived. A cell that can differentiate into all cell types, including the placental tissue, is known as totipotent.
  • pluripotent A cell that can differentiate into all cell types of the adult organism is known as pluripotent.
  • a pluripotent cell may include embryonic stem cells and adult pluripotent cells.
  • Induced pluripotent stem (iPS) cells may be created from fibroblasts by induced expression of certain transcription factors, e.g., Oct4, Sox2, c-Myc, and KIF4.
  • iPS Induced pluripotent stem
  • a multipotent cell is one that can differentiate into multiple different, but closely related cell types. Oligopotent cells are more restricted than multipotent, but can still differentiate into a few closely related cell types. Finally, unipotent cells can differentiate into only one cell type, but are capable of self-renewal.
  • the differentiation factors of the present invention induce the differentiation of stem cells or progenitor cells into T-cell progenitor cells.
  • T cell progenitor cell refers to a progenitor cell that ultimately can differentiate to a T lymphocyte (T cell).
  • lymphocyte refers to one of the subtypes of white blood cell in a vertebrate’s (e.g., human being) immune system. Lymphocytes include natural killer cells, T cells, and B cells.
  • Lymphocytes originate from a common lymphoid progenitor during hematopoiesis, a process during which stem cells differentiate into several kinds of blood cells within the bone marrow, before differentiating into their distinct lymphocyte types.
  • the T cell progenitor cell comprises a common lymphoid progenitor cell.
  • the term “common lymphoid progenitor cell,” as used herein, refers to the earliest lymphoid progenitor cells, which give rise to lymphocytes including T-lineage cells, B-lineage cells, and natural killer (NK) cells.
  • the T cell progenitor cell comprises a T cell competent common lymphoid progenitor cell.
  • T cell competent common lymphoid progenitor cell refers to a common lymphoid progenitor cell that differentiates into T-lineage progenitor cell.
  • a T cell competent common lymphoid progenitor is usually characterized by lacking of biomarker Ly6D.
  • the composition of the present invention can create an ectopic niche that mimics important features of bone marrow and induces the differentiation of stem cells or progenitor cells into T cell progenitor cells.
  • the lymphocytes comprise T cells.
  • the T cells are na ⁇ ve T cells.
  • a na ⁇ ve T cell is a T cell that has differentiated in bone marrow.
  • Na ⁇ ve T cells may include CD4 + T cells, CD8 + T cells, and regulatory T cells (T reg ).
  • the differentiation factors induce the differentiation of the recruited cells into T cell progenitor cells.
  • the differentiation factors induce the differentiation of the recruited cells into T cell progenitor cells through the Notch signaling pathway.
  • the Notch signaling pathway is a highly conserved cell signaling system present in many multicellular organisms. Mammals possess four different Notch receptors, referred to as Notch1, Notch2, Notch3, and Notch4. Notch signaling plays an important role in T cell lineage differentiation from common lymphoid progenitor cells.
  • the differentiation factors bind to one or more Notch receptors and activates the Notch signaling pathway.
  • the differentiation factor is selected from a group consisting of a Delta-like 1 (DLL-1), a Delta-like 2 (DLL-2), a Delta-like 3 (DLL-3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1, a Jagged 2, and any combination thereof.
  • the binding of the differentiation factor to one or more Notch receptors activates the Notch signaling pathway and induces T cell lineage differentiation.
  • the differentiation factor is a Delta-like 4 (DLL-4).
  • DLL-4 is a protein that is a homolog of the Drosophila Delta protein.
  • the Delta protein family includes Notch ligands that are characterized by a DSL domain, EGF repeats, and a transmembrane domain.
  • the differentiation factor polypeptides are isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous differentiation factor polypeptides may be isolated from healthy human tissue. Synthetic differentiation factor polypeptides are synthesized in vivo following transfection or transformation of template DNA into a host organism or cell, e.g., a mammal or cultured human cell line.
  • differentiation factor polypeptides are synthesized in vitro by cell free translation or other art-recognized methods Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol.1, 2, 3 (1989), herein incorporated by reference).
  • differentiation factor polypeptides may be recombinant.
  • the differentiation factor polypeptides are humanized derivatives of mammalian differentiation factor polypeptides. Exemplary mammalian species from which the differentiation factor polypeptides are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate.
  • the differentiation factor is a recombinant human protein . In some embodiments, the differentiation factor is a recombinant murine (mouse) protein . In some embodiments, the differentiation factor is a humanized derivative of a recombinant mouse protein. In certain embodiments, the differentiation factor polypeptides may be modified to achieve a desired activity, for example, to increase protein stability in vivo. In certain embodiments, the differentiation factor polypeptides may be engineered to be more or less immunogenic. In certain embodiments, the differentiation factor (e.g., DLL-4) may be covalently linked to the scaffold of the present invention.
  • a differentiation factor may be covalently bound to polymer backbone and retained within the composition that forms following implantation of the composition in the subject.
  • a differentiation factor By covalently binding or coupling a differentiation factor to the scaffold material, such differentiation factor will be retained within the scaffold that forms following administration of the composition to a subject, and thus will be available to promote the differentiation of stem cells or progenitor cells, as contemplated herein.
  • the differentiation factors are conjugated to the scaffold material utilizing N- hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry.
  • NHS N- hydroxysuccinimide
  • EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • the differentiation factor may be covalently linked to the scaffold utilizing click chemistry.
  • the methods of covalently binding or coupling differentiation factors include, but are not limited to, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or di-sulfide bond.
  • the differentiation factors (e.g., DLL-4) of the present invention further comprise a tether (e.g., PEG, PEG 2k ) and a methacrylate group (MA).
  • the differentiation factor is methacrylated DLL-4-PEG2k.
  • the covalent linking retains the differentiation factors within the scaffold to provide the differentiation signal to the recruited cells in the scaffold. For example, less than 1 % of the total differentiation factor is detected outside of the scaffold.
  • the bioactivity of the differentiation factor may be retained for an extended period of time, such as at least three months after incorporation to the scaffold.
  • the bioactivity of the differentiation factors may be measured by any appropriate methods, such as a colorimetric assay for DLL-4.
  • the differentiation factors may be present at between about 0.01 nmol and 1000 nmol, about 0.1 nmol and 100 nmol, or about 1 nmol and 10 nmol per scaffold. In some embodiments, the differentiation factors may be present at between about 1 ng and 1000 micrograms per scaffold.
  • the differentiation factor may be present at between about 10 ng and about 500 ⁇ g, between about 50 ng and about 250 ⁇ g, between about 100 ng and about 200 ⁇ g, between about 1 ⁇ g and about 100 ⁇ g, between about 1 ⁇ g and about 50 ⁇ g, between about 1 ⁇ g and about 25 ⁇ g, between about 1 ⁇ g and about 10 ⁇ g, between about 2 ⁇ g and about 10 ⁇ g, or about 6 ⁇ g.
  • the amount of differentiation factor present in a scaffold may vary according to the size of the scaffold.
  • the differentiation factor may be present at about 0.03 ng/mm 3 (the ratio of the amount of differentiation factor in weight to the volume of the scaffold) to about 350 ⁇ g/mm 3 , such as between about 0.1 ng/mm 3 and about 300 ⁇ g/mm 3 , between about 1 ng/mm 3 and about 250 ⁇ g/mm 3 , between about 10 ng/mm 3 and about 200 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and about 100 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and 50 about ⁇ g/mm 3 , or between about 0.1 ⁇ g/mm 3 and about 20 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and about 10 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and about 5 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and about 1 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and 0.5 ⁇ g/mm 3 ,
  • the DLL-4 may be present at about 6 ⁇ g per scaffold.
  • the composition of the present invention may further comprise a homing factor.
  • the composition of the present invention does not comprise a homing factor.
  • the term “homing factor” refers to an agent that is capable of inducing directed movement of a cell, e.g., a stem cell or a progenitor cell.
  • the homing factors of the present invention are signaling proteins that can induce directed chemotaxis in nearby responsive cells.
  • the homing factors are cytokines and/or chemokines.
  • the inclusion of such homing factors in the compositions of the present invention promotes the homing of cells (e.g., transplanted stem cells and/or progenitor cells) to the scaffold composition administered to a subject.
  • such homing factors promote the infiltration of the cells (e.g., transplanted stem cells or progenitor cells) to the scaffold composition administered to the subject.
  • the homing factors comprise stromal cell derived factor (SDF-1).
  • the homing factors are encapsulated in the material.
  • the homing factors are released from the material over an extended period of time (e.g., about 7-30 days or longer, about 17-18 days).
  • the homing factors retain their bioactivity over an extended period of time.
  • the bioactivity of the growth factor may be measured by any appropriate means.
  • the homing factors retain their bioactivity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after the incorporation of the homing factors into the scaffold.
  • the homing factors may be present at between about 0.01 nmol and 1000 nmol, about 0.1 nmol and 100 nmol, or about 1 nmol and 10 nmol per scaffold. In some embodiments, the homing factors may be present at between about 1 ng and 1000 micrograms per scaffold.
  • the homing factor may be present at between about 10 ng and about 500 ⁇ g, between about 50 ng and about 250 ⁇ g, between about 100 ng and about 200 ⁇ g, between about 1 ⁇ g and about 100 ⁇ g, between about 1 ⁇ g and about 50 ⁇ g, between about 1 ⁇ g and about 25 ⁇ g, between about 1 ⁇ g and about 10 ⁇ g, between about 2 ⁇ g and about 10 ⁇ g, or about 6 ⁇ g.
  • the amount of differentiation factor present in a scaffold may vary according to the size of the scaffold.
  • the differentiation factor may be present at about 0.03 ng/mm 3 (the ratio of the amount of differentiation factor in weight to the volume of the scaffold) to about 350 ⁇ g/mm 3 , such as between about 0.1 ng/mm 3 and about 300 ⁇ g/mm 3 , between about 1 ng/mm 3 and about 250 ⁇ g/mm 3 , between about 10 ng/mm 3 and about 200 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and about 100 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and 50 about ⁇ g/mm 3 , or between about 0.1 ⁇ g/mm 3 and about 20 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and about 10 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and about 5 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and about 1 ⁇ g/mm 3 , between about 0.1 ⁇ g/mm 3 and 0.5 ⁇ g/mm 3 ,
  • the composition of the present invention may further comprise a a detectable agent.
  • a detectable agent Incorporation of a detectable agent into a composition of the present invention can allow for the tracking, labeling, and/or visualization of the composition and its degradation after being administered to a subject.
  • active agents can also be labeled with a detectable agent, prior to being incorporated into a composition of the invention, which allows for the tracking, labeling and/or visualization of the active agent in the subject.
  • Such detectable agents may include, but are not limited to a dye, a fluorophore, a fluorescent compound, a luminescent compound, a chemiluminescent compound, a radioisotope, a paramagnetic metal ion, a nanoparticle, and a quantum dot, or a combination thereof.
  • Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like.
  • the compositions described herein, comprising a detectable agent can be administered to a subject and be used for a variety of diagnostic and/or therapeutic methods.
  • the detectable agent is for detecting the scaffold and/or active agent in vivo. In certain embodiments, the detectable agent is for assessing the level of degradation of the scaffold composition. In certain embodiments, the detectable agent is for assessing levels of functional innate leukocytes. In certain embodiments, the detectable agent can be used for assessing the level of degradation of the scaffold by detecting the level of the detectable agent and comparing to a detectable agent reference level, optionally, wherein the detectable agent reference level is the level of the detectable agent upon administration of the scaffold composition or at an earlier time point after administration of the scaffold composition to the subject.
  • the detectable agent is detectable by eye, fluorescence imaging, magnetic resonance imaging (MRI), surface-enhanced Raman scattering (SERS), multi-mode imaging, ultrasound imaging (USI), photoacoustic imaging (PAI), and/or optical coherence tomography (OCT).
  • the detectable agent is covalently or non-covalently linked or attached to the scaffold composition.
  • one or more detectable agent disclosed herein is incorporated on, into, or present within the structure or pores of, the scaffold composition.
  • the detectable agent is covalently linked to the scaffold.
  • the detectable agent is covalently linked to the scaffold utilizing click chemistry.
  • the detectable agent is covalently linked to the scaffold utilizing N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or di-sulfide chemistry.
  • NHS N-hydroxysuccinimide
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • DCC dicyclohexylcarbodiimide
  • avidin-biotin reaction azide and dibenzocycloocytne chemistry
  • tetrazine and transcyclooctene chemistry
  • compositions of the present invention may be used in combination with an additional active agent or therapy, such as one or more cancer therapies.
  • the cancer therapies can include, but are not limited to, surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, hormone therapy, or stem cell transplant. These therapies are well known in the art. See, e.g., https://www.cancer.gov/about- cancer/treatment/types.
  • compositions of the present invention may be used in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, or BGB-A317), a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody such as avelumab, atezolizumab, durvalumab, or MDX-1105), a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T cell co-inhibitor or ligand (e.g., an antibody to CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA), an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial a a
  • compositions of the present invention may be used in combination with cancer vaccines including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc.
  • cancer vaccines including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc.
  • the one or more other active agents or therapies may be administered prior to, concurrently with, or after the administration of the composition.
  • the one or more other active agents or therapies may be administered prior to the administration of the composition.
  • the one or more other active agents or therapies may be administered between 1 day and 2 years prior to the administration of the vaccine composition, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, or two years prior to the administration of the vaccine composition.
  • the one or more other active agents or therapies may be administered after the administration of the vaccine composition.
  • the one or more other active agents or therapies may be administered between 1 day and 2 years after the administration of the vaccine composition, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, or two years prior to the administration of the vaccine composition. It is intended that values and ranges intermediate to the recited values are part of this invention.
  • EXEMPLARY SCAFFOLD COMPOSITIONS The present invention provides scaffold compositions for delivering an active agent to a subject.
  • the composition and methods disclosed herein provide a means to treat and/or prevent diseases associated with a deficiency in levels of functional innate leukocytes, e.g., neutrophils.
  • compositions of the present invention include a scaffold composition, for example, a hydrogel or a cryogel, comprising a hyaluronic acid and, optionally, an active agent
  • a scaffold composition for example, a hydrogel or a cryogel
  • an active agent that increases the number of and/or modulates the activity of an innate leukocyte
  • a detectable agent that allows for assessment of degradation of the scaffold composition and/or assessment of the levels of functional innate leukocytes, e.g., neutrophils, in vivo.
  • the injectable composition comprises both the active agent and the detectable agent.
  • the innate leukocytes are neutrophils.
  • the active agent is G-CSF.
  • the present invention provides an injectable composition, comprising a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof, and at least one of (i) an active agent that increases the number of and/or modulates the activity of an innate leukocyte; and/or (ii) a detectable agent that allows for assessment of degradation of the scaffold composition and/or assessment of the levels of functional innate leukocytes, e.g., neutrophils, in vivo.
  • the injectable composition comprises both the active agent and the detectable agent.
  • the innate leukocytes are neutrophils.
  • the active agent is G-CSF.
  • the present invention provides an injectable composition, comprising a scaffold encapsulating an effective amount of an active agent that modulates the number and/or function of an innate leukocyte, wherein the scaffold is formulated to degrade in vivo by immune-responsive degradation to sustain the release of the active agent over a period of time sufficient to modulate the number and/or function of the innate leukocyte.
  • the immune-responsive degradation comprises neutrophil- responsive degradation.
  • the injectable composition further comprises a detectable agent for monitoring the immune-responsive degradation of the scaffold and/or assessing neutrophil functionality in vivo.
  • the detectable agent is selected from the group consisting of a dye, a fluorophore, a fluorescent compound, a luminescent compound, a chemiluminescent compound, a radioisotope, a paramagnetic metal ion, a nanoparticle, and a quantum dot, or a combination thereof.
  • the detectable agent comprises a dye.
  • the detectable agent is detectable by eye.
  • the detectable agent is detectable by fluorescence imaging, magnetic resonance imaging (MRI), surface-enhanced Raman scattering (SERS), multi-mode imaging, ultrasound imaging (USI), photoacoustic imaging (PAI), and/or optical coherence tomography (OCT).
  • the active agent comprises an amino acid, a nucleic acid, a small molecule, or a combination thereof.
  • the active agent comprises a protein, optionally, wherein the protein is an isolated protein and/or a recombinant protein.
  • the active agent comprises a cytokine, a chemokine, and/or a growth factor.
  • the active agent comprises a cytokine.
  • the cytokine comprises a pro-inflammatory cytokine and/or an anti-inflammatory cytokine.
  • the active agent is selected from the group consisting of a granulocyte colony stimulating factor (G-CSF), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a bone morphogenetic protein 2 (BMP2), a stromal cell-derived factor (SDF), a platelet activating factor (PAF), a tumor necrosis factor ⁇ (TNF ⁇ ), an interleukin-1 (IL-1), an interleukin-1 ⁇ (IL-1 ⁇ ), an interleukin-1 ⁇ (IL-1 ⁇ ), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-6 (IL-6), an interleukin- 7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin-10 (IL-10), an interleukin-11 (IL-11), an interleukin-12 (IL-12), an
  • the active agent comprises a G-CSF. In some embodiments, the active agent comprises a polyethylene glycol conjugated granulocyte colony-stimulating factor (PEG-G- CSF). In some embodiments, the injectable composition further comprises an additional active agent. In some embodiments, the injectable composition enhances the number and/or function of the innate leukocyte. In some embodiments, the injectable composition not comprise a differentiation factor that induces the differentiation of a hematopoietic stem cell into a T cell progenitor cell.
  • PEG-G- CSF polyethylene glycol conjugated granulocyte colony-stimulating factor
  • the innate leukocyte is selected from the group consisting of a natural killer cell, a mast cell, an eosinophil, a basophil, a macrophage, a dendritic cell, a gamma-delta ( ⁇ ) T cell, and a neutrophil, or a combination thereof.
  • the innate leukocyte is not a dendritic cell.
  • the innate leukocyte comprises a neutrophil.
  • the scaffold comprises a hydrogel, optionally, wherein the hydrogel gel comprises a cryogel. In some embodiments, the hydrogel or cryogel is biodegradable.
  • the immune-responsive degradation of the hydrogel or cryogel is facilitated by an enzyme produced by the innate leukocyte and/or by innate leukocyte-mediated oxidation.
  • the hydrogel or cryogel comprises a polymer, and wherein the polymer is modified to be attached to a moiety that is a substrate of the enzyme.
  • the enzyme comprises a hyaluronidase.
  • the immune-responsive degradation of the hydrogel or cryogel results in the release of the active agent, the detectable agent, and/or the additional active agent from the scaffold.
  • the enzyme increases the degradation of the hydrogel or cryogel in vivo by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to a reference level; and/or the enzyme increases the degradation of the hydrogel or cryogel in vivo by about 1-fold, about 2-fold, about 3-fold, or more compared to a reference level.
  • the hydrogel or cryogel comprises a crosslinked polymer, and wherein the polymer comprises a polymer selected from the group consisting of an alginate, a collagen, a gelatin, a hyaluronic acid, and a laminin, or a combination thereof.
  • the polymer comprises a hyaluronic acid.
  • the hydrogel or cryogel comprises a covalently crosslinked polymer.
  • the polymer is covalently crosslinked via click chemistry.
  • the present invention provides a scaffold composition comprising a click-functionalized hyaluronic acid and an active agent.
  • the present invention provides a scaffold composition comprising a click-functionalized HA cryogels.
  • the scaffold composition comprises a lyophilized HA cryogels.
  • the scaffold compositions is made from a polymer solution comprising about 0.1 wt% to about 10 wt% polymer.
  • the scaffold compositions is made from a polymer solution, such as an HA solution, comprising about about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt% polymer.
  • a polymer solution such as an HA solution
  • the scaffold composition comprises a high molecular weight HA (e.g., about 1.8 MDa).
  • the click-functionalized hyaluronic acid may comprise tetrazine functionalized HA (HA-Tz) and/or norbornene functionalized HA (HA-Nb), for example, prepared by reacting tetrazine amine or norbornene amine to HA using EDC/NHS carbodiimide chemistry.
  • the polymer comprises a first polymer and a second polymer; the first polymer is functionalized with a first bioorthogonal functional group; the second polymer is functionalized with a second bioorthogonal functional group; and the first and the second bioorthogonal functional groups react in a click chemistry reaction.
  • the first and the second bioorthogonal functional group are independently selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine and norbornene and variants thereof.
  • the first and the second bioorthogonal functional group is tetrazine or norbornene.
  • the hydrogel or cryogel comprises a crosslinked hyaluronic acid (HA); wherein the HA comprises a first HA and a second HA; and wherein the first HA is functionalize with a tetrazine and the second HA is functionalized with a norbornene.
  • the scaffold is macroporous.
  • the scaffold comprises pores having a diameter of about 50 microns to about 150 microns, about 80 microns to about 180 microns, or about 40 microns to about 90 microns.
  • the scaffold composition comprises a cryogel with interconnected pores.
  • the average pore size range was between about 10 ⁇ m to about 500 ⁇ m, about 20 ⁇ m to about 300 ⁇ m, about 80 ⁇ m to about 180 ⁇ m, or about 40 ⁇ m to about 90 ⁇ m.
  • the average surface pore size of HA cryogels for example, measured by scanning electron microscopy (SEM), is about 10 ⁇ m, about 20 ⁇ m, about 30 ⁇ m, about 40 ⁇ m, about 50 ⁇ m, about 60 ⁇ m, about 70 ⁇ m, about 80 ⁇ m, about 90 ⁇ m, about 100 ⁇ m, about 110 ⁇ m, about 120 ⁇ m, about 130 ⁇ m, about 140 ⁇ m, about 150 ⁇ m, about 160 ⁇ m, about 170 ⁇ m, about 180 ⁇ m, about 190 ⁇ m, about 200 ⁇ m, about 210 ⁇ m, about 220 ⁇ m, about 230 ⁇ m, about 240 ⁇ m, about 250 ⁇ m, about 260 ⁇ m, about 270 ⁇ m, about 280 ⁇ m, about 290 ⁇ m, about 300 ⁇ m, about 310 ⁇ m
  • the composition is formulated for administration to a subject intravenously, intramuscularly, or subcutaneously. In some embodiments, the composition is formulated for administration to a subject via injection. In some embodiments, the injection is intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebral, or intrasternal. III.
  • the present invention features methods of delivering an active agent to a subject in need thereof, wherein the subject has a deficiency in levels of functional innate leukocytes, e.g., neutrophils, the method comprising: administering to the subject a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby delivering the active agent to the subject.
  • a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby delivering the active agent to the subject.
  • the present invention also provides methods of modulating the activity of an innate leukocyte, restoring levels of functional innate leukocytes, e.g., neutrophils, reconstituting the levels of functional innate leukocytes and/or enhancing the regeneration of functional innate leukocytes in a subject in need thereof, wherein the subject has a deficiency in levels of functional innate leukocytes, the method comprising: administering to the subject a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby modulating the activity of an innate leukocyte, reconstituting the levels of innate leukocyte and/or enhancing the regeneration of an innate leukocyte in the subject.
  • a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby modulating the activity of an innate leukocyte, reconstituting the levels of
  • the present invention provides methods of enhancing the immunity of a subject in need thereof, wherein the subject has a deficiency in levels of functional innate leukocytes, e.g., neutrophils, the method comprising: administering to the subject a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby enhancing the immunity of the subject.
  • a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby enhancing the immunity of the subject.
  • the present invention provides methods of treating or preventing a disorder in a subject, wherein the disorder is caused by and/or is associated with a deficiency in an innate leukocyte, comprising: administering to a subject having a deficiency in an innate leukocyte or at risk of developing a deficiency in an innate leukocyte a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby treating or preventing the disorder in the subject.
  • the present invention provides methods of treating or preventing a neutropenia in a subject, comprising: administering to a subject having a neutropenia or at risk of developing a neutropenia a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent, thereby treating or preventing the neutropenia in the subject.
  • the present invention provides methods of a cancer in a subject, comprising (i) selecting the subject if a predetermined level of an innate leukocyte is present in the subject; (ii) administering to the subject a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent; (iii) determining a level of the innate leukocyte in the subject following administration of the scaffold composition; and (iv) administering a myelosuppressive therapy to the subject if the level of the innate leukocyte in the subject has attained a reference level following administration of the composition.
  • the present invention provides methods of immune-responsive degradation of a scaffold, monitoring immune recovery, and/or innate leukocyte functionality in a subject suffering from depleted or reduced innate leukocytes, comprising (i) selecting a subject if a predetermined level of an innate leukocyte is present in the subject; (ii) administering to the subject a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof and an active agent; and (iii) determining a level of a detectable agent encapsulated in the scaffold following administration of the composition, thereby monitoring immune-responsive degradation of the scaffold, immune recovery and/or innate leukocyte functionality in the subject.
  • the present invention provides a method for detecting immune recovery in a subject having a deficiency in levels of functional innate leukocytes, e.g., neutrophils, comprising: administering to the subject a scaffold composition comprising a hyaluronic acid, a hyaluronic acid derivative, or a combination thereof, assessing the level of degradation of the scaffold composition, wherein degradation of the scaffold composition is indicative of immune recovery in the subject.
  • the scaffold composition further comprises an active agent.
  • the method provides for controlled, sustained, and/or delayed release of the active agent upon administration to the subject.
  • the active agent modulates the activity of an innate leukocyte; (ii) restores levels of functional innate leukocytes, e.g., neutrophils; (iii) reconstitutes levels of functional innate leukocytes; (iv) enhances the regeneration of functional innate leukocytes in the subject; (v) enhances immunity of the subject in need thereof; and/or (vi) treats or prevents a disorder in the subject, wherein the disorder is caused by or associated with a deficiency in functional innate leukocytes.
  • the active agent comprises an amino acid, a peptide, a protein, a nucleic acid, an oligonucleotide, a small molecule, or a combination thereof.
  • the active agent comprises a protein, optionally, wherein the protein is an isolated protein and/or a recombinant protein.
  • the active agent comprises a cytokine, a chemokine, and/or a growth factor.
  • the active agent comprises a cytokine, optionally, a pro-inflammatory cytokine and/or an anti- inflammatory cytokine.
  • the active agent is selected from the group consisting of a granulocyte colony stimulating factor (G-CSF), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a bone morphogenetic protein 2 (BMP2), a stromal cell-derived factor (SDF), a platelet activating factor (PAF), a tumor necrosis factor ⁇ (TNF ⁇ ), an interleukin-1 (IL-1), an interleukin-1 ⁇ (IL-1 ⁇ ), an interleukin-1 ⁇ (IL-1 ⁇ ), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin- 6 (IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin-10 (IL-10), an interleukin-11 (IL-11), an interleukin-12 (IL-12), an
  • the active agent comprises a G- CSF. In some embodiments, the active agent comprises a polyethylene glycol conjugated granulocyte colony-stimulating factor (PEG-G-CSF). In some embodiments, the active agent active agent is G-CSF. In various embodiments of the methods described herein, the method further comprises administering an additional active agent. In some embodiments, the additional active agent is encapsulated in the scaffold. In some embodiments, the additional active agent is administered systemically. In various embodiments of the methods described herein, the subject is or has undergone therapy that depletes or reduces the innate leukocyte in the subject. In some embodiments, the therapy is a chemotherapy or a radiotherapy for the treatment of a cancer.
  • PEG-G-CSF polyethylene glycol conjugated granulocyte colony-stimulating factor
  • the active agent active agent is G-CSF.
  • the method further comprises administering an additional active agent. In some embodiments, the additional active agent is encapsulated in the scaffold. In some embodiments, the
  • the composition further comprises a detectable agent.
  • the detectable agent is attached to the active agent.
  • the detectable agent is for detecting the scaffold and/or active agent in vivo.
  • the detectable agent is for assessing the level of degradation of the scaffold composition.
  • the detectable agent is selected from the group consisting of a dye, a fluorophore, a fluorescent compound, a luminescent compound, a chemiluminescent compound, a radioisotope, a paramagnetic metal ion, a nanoparticle, and a quantum dot, or a combination thereof.
  • the detectable agent comprises a dye.
  • the detectable agent is detectable by eye, fluorescence imaging, magnetic resonance imaging (MRI), surface-enhanced Raman scattering (SERS), multi-mode imaging, ultrasound imaging (USI), photoacoustic imaging (PAI), and/or optical coherence tomography (OCT).
  • the method further comprises assessing the level of degradation of the scaffold.
  • assessing the level of degradation of the scaffold comprises detecting the level of the detectable agent and comparing to a detectable agent reference level, optionally, wherein the detectable agent reference level is the level of the detectable agent upon administration of the scaffold composition or at an earlier time point after administration of the scaffold composition.
  • degradation of the scaffold composition is indicative of modulation of the activity of an innate leukocyte; restoration of levels of functional innate leukocytes, e.g., neutrophils; reconstitution of levels of functional innate leukocytes; regeneration of functional innate leukocytes in the subject; enhanced immunity of the subject; and/or treatment or prevention of a disorder in the subject, wherein the disorder is caused by or associated with a deficiency in functional innate leukocytes.
  • functional innate leukocytes e.g., neutrophils
  • reconstitution of levels of functional innate leukocytes e.g., neutrophils
  • regeneration of functional innate leukocytes in the subject e.g., enhanced immunity of the subject
  • treatment or prevention of a disorder in the subject wherein the disorder is caused by or associated with a deficiency in functional innate leukocytes.
  • the method described herein may further comprise obtaining a measurement of (i) the level of the detectable agent and/or active agent in the scaffold prior to administration of the scaffold to the subject; (ii) the level of functional innate leukocytes, e.g., neutrophils in the subject prior to administration of the scaffold to the subject; (iii) the level of the innate leukocytes in the subject prior to administration of the scaffold to the subject; (iv) the level of the detectable agent and/or active agent in the scaffold after administration of the scaffold to the subject; (v) the level of functional innate leukocytes after administration of the scaffold to the subject; (vi) the level of the innate leukocytes in the subject prior to administration of the scaffold to the subject; (vii) the level of functional innate leukocytes in the subject before the subject receives a therapy that depletes or reduces the level of the innate leukocyte; and/or (viii) the level of functional innate leukocytes in the subject after the subject receives
  • the level of functional innate leukocytes is increased by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% or more as compared to a reference level.
  • the level of functional innate leukocytes, e.g., neutrophils is increased by at least about 1-fold, about 2-fold, or about 3-fold or more as compared to a reference level.
  • the reference level is a standardized level.
  • the reference level is the level of functional innate leukocytes, e.g., neutrophils prior to administration of the scaffold composition or at an earlier time point after administration of the scaffold composition.
  • the scaffold composition does not comprise a differentiation factor that induces the differentiation of a hematopoietic stem cell into a T cell progenitor cell.
  • the scaffold composition degrades upon exposure to an enzyme produced by the innate leukocyte.
  • the scaffold composition degrades by neutrophil-mediated oxidation.
  • the degradation of the scaffold composition results in and/or enhances the release of the active agent.
  • the degradation of the scaffold composition results in and/or enhances the release of the detectable agent.
  • the scaffold is macroporous or wherein the scaffold comprises pores having a diameter of about 50 microns to about 150 microns, about 80 microns to about 180 microns, or about 40 microns to about 90 microns.
  • the composition is administered to the subject via injection, optionally, intravenously, intramuscularly, or subcutaneously.
  • the subject is a human.
  • the subject has a disorder selected from the group consisting of primary neutropenia, acute neutropenia, severe chronic neutropenia (SCN), severe congenital neutropenia (Kostmann's syndrome), severe infantile genetic agranulocytosis, benign neutropenia, cyclic neutropenia, chronic idiopathic neutropenia, secondary neutropenia, syndrome associated neutropenia, and immune-mediated neutropenia, chronic granulomatous disease, Chediak-Higashi syndrome, Griscelli Syndrome-II, Hermansky-Pudlak Syndrome type 2, leukocyte adhesion deficiency, specific granule deficiency, x-linked neutropenia, myeloperoxidase deficiency, and hyper-IgE syndrome.
  • SCN severe chronic neutropenia
  • Kostmann's syndrome severe congenital neutropenia
  • severe infantile genetic agranulocytosis benign neutropenia
  • the subject has a neutropenia, optionally, wherein the neutropenia is caused by or associated with radiation, alcoholism, drugs, allergic disorders, aplastic anemia, autoimmune disease, T- ⁇ lymphoproliferative disease (T- ⁇ LPD), myelodysplasia, myelofibrosis, dysgammaglobulinemia, paroxysmal nocturnal hemoglobinuria, cancer, vitamin B12 deficiency, folate deficiency, viral infection, bacterial infection, spleen disorder, hemodialysis, or transplantation, toxins, bone marrow failure, Schwachman-Diamond syndrome, cartilage-hair hypoplasia, dyskeratosis congenita, glycogen storage disease type IB, splenomegaly of any cause, and intrinsic defects in myeloid cells or their precursors.
  • T- ⁇ lymphoproliferative disease T- ⁇ lymphoproliferative disease (T- ⁇ LPD)
  • myelodysplasia myelo
  • the subject has or is receiving a hematopoietic stem cell transplantation (HSCT).
  • HSCT hematopoietic stem cell transplantation
  • the administration of the composition is prior to, concurrently with, or subsequent to the HSCT.
  • the method reconstitutes the innate leukocyte within 7 days, 14 days, 21 days, or 28 days.
  • the reconstitution of the innate leukocyte results in the level of innate leukocyte to be at least about 75%, 80%, 85%, 90%, 95% or 99% of a reference level.
  • the reference level is (i) a standardized level; (ii) the level of the innate leukocyte of the subject before depletion of the level of the innate leukocyte; and/or (iii) the level of the innate leukocyte of the subject before the subject receives a therapy the depletes or reduces the level of the innate leukocyte.
  • the method results in (i) an increase in the number of the innate leukocytes in the subject; (ii) an increase in the number of functional innate leukocytes in the subject; (iii) a reduction in the incidence, severity, and/or duration of an opportunistic infection in the subject; (iv) a reduction in the incidence, severity, and/or duration of a symptom of neutropenia in the subject; (v) a reduction in the incidence, severity, and/or duration of a symptom of an immunodeficiency-associated complication in the subject; (vi) an increase in the level of tissue regeneration in the subject; (vii) an increase in the therapeutic efficacy of a therapy that depletes or reduces the innate leukocyte in the subject; (viii) an increase in the degradation of the scaffold in the subject; and/or (ix) an increase in the release of a detectable agent, an active agent, and/or an additional active agent from the scaffold in the subject.
  • the subject has or is receiving systemic G-CSF and/or PEG-G-CSF.
  • the administration of the composition is prior to, concurrently with, or subsequent to the systemic G-CSF and/or PEG-G-CSF.
  • the method further comprising administering to the subject systemic G-CSF and/or PEG-G-CSF.
  • the subject has or is at risk of developing anti-PEG antibody (APA)-mediated rapid clearance.
  • the subject has pre-existing or induced anti-PEG antibody (APA)-mediated rapid clearance.
  • the subject has failed or is at risk of failing to respond to systemic G- CSF and/or PEG-G-CSF.
  • the present invention provides a method of treating a subject for cancer in need thereof, wherein the subject is undergoing chemotherapy or radiotherapy, comprising administering to a subject suffering from depleted or reduced innate leukocytes, wherein the composition comprises a biodegradable scaffold and a cytokine that enhances the generation of the innate leukocyte; assessing the levels of the innate leukocyte in the subject; administering chemotherapy or radiotherapy to the subject wherein the levels of innate leukocyte have attained a reference level following administration of the composition.
  • the innate leukocytes are neutrophils.
  • the cytokine is G-CSF.
  • the subject is suffering from depleted or reduced innate leukocytes as a result of previous exposure to chemotherapy or radiotherapy.
  • the reference level is a standardized level, optionally, (i) wherein the reference level is the level of the innate leukocyte of the subject before depletion of the level of the innate leukocyte; and or (ii) the reference level is the level of the innate leukocyte of the subject before the subject receives a therapy the depletes or reduces the level of the innate leukocyte.
  • the scaffold comprises a hydrogel. In some embodiments, the scaffold comprises a cryogel.
  • the scaffold comprises pores having a diameter between about 1 ⁇ m and 100 ⁇ m. In some embodiments, the scaffold comprises a macropore. In some embodiments, the macropore has a diameter between about 50 ⁇ m and 80 ⁇ m. In still another embodiment, the scaffold includes macropores of different sizes. In various embodiments of the methods described herein, the scaffold comprises a click-hydrogel or click cryogel. In one embodiment, the scaffold comprises a click hyaluronic acid. In various embodiments of the methods described herein, the scaffold is between about 100 ⁇ m 3 to about 10 cm 3 in size. In one embodiment, the scaffold is between about 10 mm 3 to about 100 mm 3 in size. In another embodiment, the scaffold is about 30 mm 3 in size.
  • the active agent is present at an amount between about 1 ng to about 1000 ⁇ g. In one embodiment, the active agent is present at between about 5 ng to about 500 ng. In another embodiment, the active agent is present at between about 5 ng to about 250 ng. In yet another embodiment, the active agent is present at between about 5 ng to about 200 ng. In still another embodiment, the active agent is present at about 200 ng. In yet another embodiment, the active agent is present at about 6 ng/mm 3 to about 10 ng/mm 3 . In yet another embodiment, the active agent is present at about 6.5 ng/mm 3 to about 7.0 ng/mm 3 .
  • the active agent retains its bioactivity for at least twelve days after the active agent is incorporated into the scaffold. In some embodiments, the active agent retains its bioactivity for at three months after the active agent is incorporated into the scaffold. IV. KITS Any of the compositions described herein may be included in a kit.
  • the kit includes a scaffold composition comprising hyaluronic acid and an active agent..
  • the kit includes the composition described elsewhere herein.
  • the kit comprises a syringe or alternative injection device for administering the composition.
  • the prefilled syringe or injection device is prefilled with the composition.
  • the kit may further include reagents or instructions for administering the composition of the present invention to a subject. It may also include one or more reagents.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
  • kits of the present invention also will typically include a means for containing the compositions of the invention, e.g., the compositions for modulating immune system, and any other reagent containers in close confinement for commercial sale.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • HA cryogel an injectable, biodegradable hyaluronic acid (HA)-based scaffold, termed HA cryogel, with myeloid responsive degradation behavior is described.
  • HA cryogel an injectable, biodegradable hyaluronic acid (HA)-based scaffold, termed HA cryogel, with myeloid responsive degradation behavior.
  • HA cryogel an injectable, biodegradable hyaluronic acid (HA)-based scaffold, termed HA cryogel
  • G-CSF granulocyte colony stimulating factor
  • sustained release from HA cryogels accelerated post-HSCT neutrophil recovery, with full recovery achieved within 14 days post-HSCT, and accelerated the rate of degradation of HA cryogels.
  • the HA-cryogel is a potential off-the-shelf therapeutic approach for enhancing neutrophil regeneration in settings of immune deficiency and reducing the risk of immune- deficiency related complications.
  • Neutrophils are one of the most abundant cell types of the innate immune system in mammals and mediate essential host defense against invading pathogens.
  • HSCT hematopoietic stem cell transplantation
  • high dose chemotherapy there is a marked transient post-therapy deficiency in neutrophils in the peripheral blood, which renders patients susceptible to opportunistic infections for up to several weeks, despite the application of prophylactic antibiotics and supportive therapy (4, 5).
  • Complications due to infection are a leading cause of morbidity and mortality, with up to 55% of patients developing post-HSCT blood-borne bacterial infections (3, 6, 7).
  • Post-HSCT neutrophil regeneration follows successful engraftment of hematopoietic progenitors in the bone marrow (8, 9).
  • the generation of neutrophils relies on the engraftment of adequate number of progenitors and endogenous production of granulocyte colony stimulating factor (G-CSF).
  • G-CSF promotes neutrophil recovery by binding to its cognate receptor (G-CSFR) on hematopoietic stem cells (HSCs), activating the downstream JAK- STAT signaling through STAT3 nuclear migration, and finally contributing to granulopoiesis by promoting gene expression pertinent to cell cycle arrest, an essential prelude to myeloid differentiation (10, 11).
  • the clinical standard for enhancing neutrophil regeneration is daily administration of recombinant human (G-CSF) or a single high dose of pegylated G-CSF (PEG G-CSF) 24 hours after the conditioning regimen (3, 12, 13). Therapy is typically discontinued after the absolute neutrophil count reaches ⁇ 2,000-3,000 cells/ ⁇ L (14). G-CSF has a half-life of 3.5 hours and therefore the post-injection concentration in circulating blood rapidly returns to a normal homeostatic concentration. However, side effects and adverse events often pose a greater risk to patients than neutropenia (15).
  • G-CSF hematopoietic progenitors
  • HA hyaluronic acid
  • the process of cryogelation relies on a sub-zero crosslinking environment in which the formation of ice crystals during the gelation process creates macro- porosity throughout the HA cryogels (FIG.1C) (22-24).0.8% and 7% degrees of substitution Tz-HA was synthesized and mixed with Nb-HA to make HA cryogels with different density of crosslinking. Scanning electron microscopy (SEM) of lyophilized HA cryogels was used to visualize the interconnected pore structure of HA cryogels with varying cross-linking density (FIG.1D).
  • SEM scanning electron microscopy
  • Average cross-sectional pore area from these SEM images was measured and it was determined that pore size was influenced by the degree of crosslinking of the HA cryogels.
  • the average cross-sectional pore diameter was 133.0 ⁇ 52.1 and 62.8 ⁇ 22.7 micron on the surface HA cryogels made from 0.8% and 7% Tz-HA respectively (FIG.1).
  • Analysis of confocal microscopy images determined that both pore morphology and relative pore size distribution are maintained after rehydration of lyophilized HA cryogels (FIGS.2A, 2B). Additionally, HA cryogels maintained shape and structure after injection through a 16-gauge needle intact.
  • HYAL2 hyaluronidase-2
  • HYAL2 degradation of HA results in cleavage of internal beta-N- acetyl-D-glucosaminidic linkages resulting in fragmenting on the HA cryogels throughout the degradation process (25).
  • Cy5 a fluorescent tag, Cy5
  • Cy5-HA cryogels In vitro degradation of Cy5 labeled HA cryogels (Cy5-HA cryogels) indicated that there was a strong correlation between crosslinking density and degradation kinetics as the Cy5-HA cryogels synthesized with 7% DOS Tz-HA took longer to degrade (FIG.3C).
  • Cy5-HA cryogels were injected into the subcutaneous space of mice and degradation was tracked using in vivo imaging system (IVIS) fluorescence spectroscopy (FIG.3D).
  • IVIS in vivo imaging system fluorescence spectroscopy
  • Cy5-HA cryogels were injected into the subcutaneous space of untreated mice (FIG.4A) and the degradation kinetics were compared to degradation in mouse models of T-cell depletion (FIG.4B), B-cell depletion (FIG.4C), macrophage depletion (FIG.4D), and the immune deficient NSG mouse strain (FIG.4E).
  • HA cryogel half-life quantified as the time to achieve a 50% reduction in fluorescence intensity, was 9 ⁇ 2 days (FIG.4A).
  • the half-life in the macrophage and T-cell deficient mice were 8 ⁇ 1 days and 10 ⁇ 3 days respectively (FIGS.4B, 4C).
  • CD45 + CD11b + myeloid cells into HA cryogels one day post-HSCT was similar in the untreated (1.7 x 10 5 ⁇ 5.7 x 10 4 cells), T-cell depleted (1.8 x 10 5 ⁇ 4 x 10 4 cells), and macrophage depleted mice (1.5 x 10 5 ⁇ 1.2 x 10 5 cells).
  • the infiltration of CD45 + CD11b + myeloid cells was approximately 60% lower (6 x 10 4 ⁇ 4 x 10 4 cells) in the B-cell depleted mice.
  • the number of CD45 + CD11b + myeloid cells within the HA cryogels in the immune competent and T-cell depleted mice increased 3.2-fold (5.4 x 10 5 ⁇ 3.3 x 10 5 cells ) and 3.5-fold (6.3 x 10 5 ⁇ 2.8 x 10 5 cells) respectively whereas the CD45 + CD11b + myeloid infiltration in B-cell depleted mice was unchanged (5.8 x 10 4 ⁇ 2.6 x 10 4 cells).
  • CD45 + CD11b + F4-80 + macrophage infiltration was similar to that of CD45 + CD11b + myeloid cells in the immune competent and T-cell depleted mice.
  • CD45 + CD11b + F4-80 + macrophage infiltration had increased 2.7-fold to 1.8 x 10 5 ⁇ 1.2 x 10 5 cells in the immune competent mice and 3.5-fold to 2.7 x 10 5 ⁇ 1.2 x 10 5 cells in the T-cell depleted mice.
  • the B-cell depleted mice had 2.4 x 10 4 ⁇ 1.3 x 10 4 infiltrating CD45 + CD11b + F4-80 + macrophages with HA cryogels, a similar number of infiltrating cells when compared to the immune competent and T-cell depleted mice, but had only increased 45% to 3.5 x 10 4 ⁇ 1.3 x 10 4 infiltrating cells by day ten.
  • mice had few infiltrating CD45 + CD11b + F4-80 + macrophages, with 92% less than untreated mice with 5.2 x 10 3 ⁇ 4.4 x 10 3 infiltrating cells after one day which increased 2.3-fold to 1.2 x 10 4 ⁇ 6.2 x 10 3 cells after ten days (FIG.5D).
  • CD45 + CD11b + F4-80 + macrophage polarization in the HA cryogel was quantified by comparing CD45 + CD11b + F4-80 + CD80 + CD86 + CD206- pro-inflammatory and CD45 + CD11b + F4-80 + CD80-CD86-CD206 + anti-inflammatory macrophages.1-day post- HSCT, a higher M1:M2 ratio in the T-cell depleted (7.4:1) and B-cell depleted mice (4.1:1) was quantified compared with the untreated mice (2.6:1). At day ten, the M1:M2 ratio was approximately 1:1 in all the groups (FIG.7).
  • FIG.6A injection of lineage depleted hematopoietic stem cells (2 x 10 5 cells; 87.5% depleted; FIG.8A) isolated from bone marrow of syngeneic donor mice concurrent to subcutaneous HA cryogel administration (FIG.6A).
  • Cy5-tagged HA cryogels were used to track degradation in mice that received L-TBI/HSCT and degradation rate was compared to non-irradiated control mice (FIG.6B).
  • the fluorescence intensity was constant until day 20 in post-BMT mice, after which the HA cryogel proceeded to degrade at a rate comparable to that in non-irradiated mice.
  • CD45 + CD11b + F4-80 + macrophage infiltration into HA cryogels was delayed in the mice that received a BMT. 5 days after HA cryogel administration, the number of infiltrating CD45 + CD11b + F4-80 + macrophage cells in excised HA cryogels from the non-irradiated mice was 1.4 x 10 5 ⁇ 2.1 x 10 4 . In post-HSCT mice, 5.1 x 10 3 ⁇ 3.6 x 10 3 infiltrating CD45 + CD11b + F4-80 + macrophage cells (96% decrease) were quantified in excised HA cryogels 5 days after administration.
  • the HA cryogel was developed as a depot for mediating the sustained release of G-CSF and enhance CD45 + CD11b + CD115-Ly6-G + neutrophil recovery in mice after syngeneic HSCT (FIG.9A).
  • CD45 + CD11b + CD115-Ly6-G + neutrophil cells were quantified by flow cytometry analysis of peripheral blood.
  • CD45 + CD11b + CD115-Ly6-G + neutrophil reconstitution was enhanced in mice that received either bolus IP injection of 2 ⁇ g PEG G- CSF or administration of two HA cryogels containing 1 ⁇ g of G-CSF each as compared to mice that received either bolus IP injection of 2 ⁇ g of G-CSF or untreated mice (FIG.9B).
  • mice that received bolus PEG G-CSF and G-CSF encapsulated within HA cryogels had recovered 6.3 ⁇ 1.1% and 6.4 ⁇ 0.7% of neutrophils respectively whereas the mice that received bolus G-CSF and untreated had recovered 3.5 ⁇ 0.6% and 3.9 ⁇ 1.3%, respectively (FIG.9C).
  • HA was chosen as the backbone of this device as it is ubiquitous in the extracellular matrix and has an extensive history of clinical use as a biomaterial in both biomedical and cosmetic applications (27-32).
  • the process of cryogelation was utilized during the cross- linking process in order to provide enhanced mechanical stability and provide shape memory properties upon injection (33, 34).
  • other degradable polymers such as poly (lactic-co-glycolic acid) or oxidized alginate typically degrade by hydrolysis, independent of myeloid lineage functionality (35-38).
  • Tz and Nb click-chemistry was used as the functional groups are biorthogonal and the reaction proceeds without the need for addition of external energy, such as by increasing temperature, or catalysts (39, 40).
  • a polymer mixture was formulated in which the DOS of Nb was greater than that of Tz. Through this approach the cross-linking density was modified by changing the DOS of Tz on HA prior to cryogelation.
  • HA cryogel represents a simple-to-administer and safe system that can enhance neutrophil regeneration in myelosuppressive treatments, such as HSCT, with neutropenia as a side-effect and thereby improve treatment outcomes in bone marrow transplant and other cancer therapies.
  • tetrazine amine (4-(1,2,4,5-tetrzain- 3-yl)phenyl)methanamine (tetrazine amine) was purchased from Kerafast.1- bicyclo[2.2.1]hept-5-en-2-ylmethanamine (norbornene amine) was purchased from Matrix Scientific. Cy5-tetrazine amine was purchased from Click Chemistry Tools. Click functionalization of HA Tetrazine functionalized HA (Tz-HA) or norbornene functionalized HA (Nb-HA) were prepared by reacting tetrazine amine or norbornene amine to HA using EDC/NHS carbodiimide chemistry.
  • Sodium hyaluronate was dissolved in a buffer solution (0.2% wt/vol, pH ⁇ 6.5) of 100mM MES buffer.
  • the solution was stirred at room temperature for 24 hours and transferred to a 12,000Da MW cutoff dialysis sack (Sigma Aldrich) and dialyzed in 4L of NaCl solutions of decreasing molarity (0.125M, 0.100M, 0.075M, 0.050M, 0.025M, 0M, 0M, 0M, 0M) for 8 hours per solution. After dialysis, solutions containing Tz-HA or Nb- HA were frozen overnight and lyophilized (Labconco Freezone 4.5) for 48 hours.
  • Cy5 conjugated Nb-HA was synthesized following a previously described technique with some modifications.0.8mg of cy5-tetrazine was reacted with 100mg of Nb-HA at 0.2 wt/vol in DI water for 24 hours at 37 oC and purified by dialysis in DI water using a 12,000Da MW cutoff dialysis sack for 48 hours. Dialysis water bath was changed once every 8 hours. Cy5 conjugated Nb-HA was then frozen overnight and lyophilized for 48 hours. HA cryogel fabrication and pore quantification HA cryogels were made following a previously described technique with some modifications(24) .
  • Aqueous solutions of 0.6% wt/vol Tz-HA and Nb-HA were prepared by dissolving lyophilized polymers into deionized water.
  • Aqueous Tz-HA and Nb-HA solutions were mixed at a 1:1 volume ratio, pipetted into 30 ⁇ L Teflon molds, and transferred to a -20 oC freezer to allow for overnight cryogelation.
  • Synthesis of cy5-HA cryogels follows the same protocol as above, except substituting cy5 conjugated Nb-HA for Nb-HA. For scanning electron microscopy (SEM), frozen HA cryogels were lyophilized for 24 hours.
  • SEM scanning electron microscopy
  • Lyophilized HA cryogels were collected in Petri dishes and adhered onto sample stubs using carbon table and coated with iridium in a sputter coater. Samples were imaged using secondary electron detection on a FEI Quanta 250 field emission SEM. Fluorescence images of cy5-HA cryogels were acquired using an Upright Zeiss LSM 710. Pore size quantification of SEM images and relative distribution of pore sizes of confocal images was doing using FIJI image processing package (50). For biological assays, cryogels were thawed and collected in Petri dishes.
  • B-cell lineage depletion in B6 mice was induced using anti-mouse B220/CD45R antibodies (RA3.31/6.1, Bio X Cell).
  • T-cell lineage depletion in B6 mice was induced using anti-mouse CD4 antibodies (GK1.5, Bio X Cell) and anti-mouse CD8 ⁇ antibodies (2.43, Bio X Cell).
  • Macrophage depletion in B6 mice was induced by clodronate liposomes (Liposoma).
  • mice received intraperitoneal injections of 0.1mL (400 ⁇ g) of antibodies or 0.1mL of clodronate liposome solution 3 days before subcutaneous HA cryogel or cy5-HA cryogel injection.
  • Antibodies or clodronate liposomes were administered twice per week until complete gel degradation or until mice were euthanized and gels removed for further analysis.
  • Transplant models All total-body irradiation experiments were performed with a cesium-137 gamma- radiation source: SL: TBI (B6 recipients) with one dose of 2 x 10 5 lineage-depleted bone marrow cells from B6 mice. Bone marrow cells for transplantation or analysis were harvested by crushing all limps.
  • Crushed tissue and cells were filtered through a 70 ⁇ m mesh.
  • a single- cell suspension was prepared by passing the cells once through a 20-gauge needle. Total cellularity was determined by counting cells using a hemacytometer. Bone marrow cells were depleted of mature immune cells (expressing CD3 ⁇ , CD45R/B220, Ter-119, CD11b, or Gr-1) by magnetic selection (BD Biosciences). Cells were incubated with a mix of Pacific Blue- conjugated lineage specific antibodies (antibodies to CD3, NK1.1, Gr-1, CD11b, CD19, CD4 and CD8) and with Sca-1 and cKit-specific antibodies.
  • Hematopoietic stem cells (Lin-Sca- 1 hi c-kit hi ) were isolated using a FACSAria cell sorter (BD). Sorted cells were ⁇ 87% pure. Lineage-depleted bone marrow cells were suspended in 100 ⁇ L of sterile 1x PBS and administered to mice via a single i.v. injection. While mice were anesthetized, received a subcutaneous injection of a single HA cryogel or cy5-HA cryogel, which was suspended in 200 ⁇ L of sterile 1x PBS, into the dorsal flank by means of a 16-gauge needle. The site of injection was shaved and wiped with a sterile alcohol pad prior to gel injection.
  • BD FACSAria cell sorter
  • Cy5-HA cryogels synthesized with 0.2%, 0.8%, and 7% DOS Tz-HA and placed into individual 1.5mL microcentrifuge tubes (Thermo Scientific) with 1mL of 100U/mL HYAL2 (Sigma Aldrich) in 1x PBS. Degradation studies were conducted in tissue culture incubators at 37 oC. Supernatant from samples were collected every 24 – 72 hours by centrifuging the samples at room temperature at 2,000G for 5 minutes and removing 0.9mL of supernatant. Cy5-HA cryogels were resuspended by adding 0.9mL of freshly made 100U/mL HYAL2 in 1x PBS.
  • a single cy5-HA cryogel is administered into the dorsal flank of an anesthetized mouse and the fluorescent intensity of the cy5-HA cryogel was quantified using an IVIS spectrometer (PerkinElmer) at predetermined timepoints and analyzed using LivingImage software (PerkinElmer). At each timepoint, mice were anesthetized and the area around the subcutaneous cy5-HA cryogel was shaved to reduce fluorescence signal attenuation. Fluorescence radiant efficiency, the ratio of fluorescence emission to excitation, was measured longitudinally as a metric to quantify fluorescence from subcutaneous cy5-HA cryogels. These values were normalized to the to the measured initial value at day 3.
  • T-, B- and myeloid cells in peripheral blood blood was first collected from the tail vein of mice into EDTA coated tubes (BD). Samples then underwent lysis of red blood cells and were stained with appropriate antibodies corresponding to cell populations of interest. To quantify infiltrating immune cells within HA cryogels, mice were sacrificed, gels removed, and HA cryogels crushed against a 70-micron filter screen before antibody staining. Absolute numbers of cells were calculated using flow cytometry frequency.
  • Flow cytometry was analyzed using FlowJo (BD) software. Histology After euthanasia, HA cryogels were explanted and fixed in 4% paraformaldehyde (PFA) for 24 hours. The fixed HA cryogels were then transferred to 70% ethanol solution and embedded in paraffin wax. Sections (5 ⁇ m) of the samples were stained with routine H&E stain. Quantification of histology slides Digital copies of H&E slides were made using an Aperio AT2. Digital slides were rendered in ImageScope and the positive pixel algorithm was used to quantify pixels associated with cell nuclei. The average number of pixels per cell nuclei was determined to be ⁇ 55.
  • PFA paraformaldehyde
  • Neutrophils are essential effector cells for mediating rapid host defense and their insufficiency arising from therapy-induced side-effects, termed neutropenia, can lead to immunodeficiency-associated complications.
  • neutropenia is a complication that limits therapeutic efficacy.
  • HA biodegradable hyaluronic acid
  • HA cryogels are a potential approach for enhancing neutrophils and concurrently assessing immune recovery in neutropenic hosts.
  • Neutrophil deficiency, termed neutropenia contributes to opportunistic infections and could impair tissue regeneration in affected individuals 4-7 .
  • HSCT autologous hematopoietic stem cell transplantation
  • pre-conditioning myelosuppressive regimens can contribute to a marked transient post-therapy impairment of neutrophils and render recipients susceptible to immune deficiency-associated complications for up to several weeks 6, 8-11 .
  • Post-HSCT neutrophil regeneration follows successful bone marrow engraftment of transplanted hematopoietic cells 12, 13 , facilitated by granulocyte colony stimulating factor (G- CSF)-mediated granulopoiesis of hematopoietic cells 14-16 .
  • G- CSF granulocyte colony stimulating factor
  • Neutropenia is typically treated as an emergency and, in a subset of patients, the risk of neutropenia may be prophylactically addressed with post-HSCT subcutaneous injection of recombinant human G-CSF (filgrastim) to facilitate recovery 6, 14, 17, 18 .
  • Daily injections are used as G-CSF has a half-life of a 3 – 4 hours, which can be extended by conjugating G-CSF with polyethylene glycol (PEGylation) 19, 20 .
  • PEGylation polyethylene glycol
  • the depot comprised a porous injectable scaffold made by low-temperature crosslinking, termed cryogelation, of hyaluronic acid (HA), an easily sourced and readily derivatized anionic glycosaminoglycan, termed ‘HA cryogel.’
  • cryogelation hyaluronic acid
  • HA cryogel an easily sourced and readily derivatized anionic glycosaminoglycan
  • Tz amine-functionalized HA was prepared at 7% degree of substitution (termed high-DOS).0.8% DOS HA-Tz (termed low DOS) was also prepared for comparison.
  • Endotoxin levels of HA-Tz and Cy5-HA-Nb were quantified to be less than 5 endotoxin units/kg, the threshold pyrogenic dose for preclinical species 26 (FIG.11).
  • 0.6% w/v aqueous solutions of HA-Tz and Cy5-HA-Nb, pre-cooled to 4oC, were well mixed in a 1:1 (v/v) ratio by vortexing (FIG.12A).
  • the aqueous mass composition was 76.3 ⁇ 4.0% and 71.9 ⁇ 2.6% in low- and high-DOS Cy5-HA cryogels respectively (FIG.13B).
  • SEM scanning electron microscopy
  • the surface pore structure images were used to measure the average pore diameter using FIJI, which were between 80-180 ⁇ m and 40-90 ⁇ m for low- and high-DOS Cy5-HA cryogels respectively (FIG.13D).
  • Cy5-HA cryogel pore structure To characterize interconnectedness of the Cy5-HA cryogel pore structure, fully hydrated low- and high-DOS Cy5-HA cryogels were incubated with Fluorescein isothiocyanate (FITC)-labeled 10 ⁇ m diameter melamine resin particles and imaged using a confocal microscope (FIG.12E, FIG. 13E). Since the route of administration of the Cy5-HA cryogels is through a needle, this experiment was repeated with Cy5-HA cryogels after injection and observed similar penetration of the FITC-labeled 10 ⁇ m particles (FIG.12E, FIG.13E).
  • FITC Fluorescein isothiocyanate
  • HYAL2 cleaves internal beta-N-acetyl-D-glucosaminidic linkages resulting in fragmentation of HA 27 .
  • HYAL2 degraded HA cryogels and high DOS Cy5-HA cryogels degraded at a slower rate compared to the low DOS Cy5-HA cryogels in vitro (FIG.12G, FIG.14A).
  • low- and high-DOS Cy5-HA cryogels were injected in subcutaneously in the hind flank of mice and degradation was measured using in vivo imaging system (IVIS) fluorescence spectroscopy (FIG.12H).
  • Cy5-HA cryogels were subcutaneously injected into the hind flank of untreated C57Bl/6J (B6) mice (FIG.15A) and the degradation profile was compared to that in B6 mice receiving (i) anti-Ly6G antibodies and anti-rat ⁇ immunoglobulin light chain to deplete neutrophils (FIG.15B), (ii) clodronate liposomes to deplete macrophages (FIG.15C), (iii) anti-CD4 and anti-CD8 antibodies to deplete T cells (FIG.16A), (iv) anti-B220 to deplete B-cells (FIG.16B) and immune deficient NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice (FIG.15D).
  • Cy5-HA cryogels were explanted from the above groups at 1-, 5-, and 10-days post-injection and stained using haemotoxylin and eosin (H&E).
  • H&E haemotoxylin and eosin
  • Macrophage depletion did not affect the initial neutrophil infiltration 1-day post-injection compared with untreated B6 mice but reduced the number of infiltrating neutrophils by 65% compared with untreated B6 mice by day 10.
  • HA cryogels retrieved from NSG mice had 50% fewer neutrophils than those from untreated B6 mice on day 1 and very few to none were found by day 10 post-injection (FIG.18E).
  • neutrophils constituted over 90% of total myeloid cells on day 1 but decreased to about 8% by day 10 (FIG.18E). This observation along with minimal Cy5-HA cryogel degradation in NSG mice (FIG.15D), supported a key role of functional neutrophils in mediating degradation.
  • Cy5- HA cryogels were injected in gp91 phox- mice and B6 mice and degradation was quantified using IVIS (FIG.18F). Cy5-HA cryogels did not degrade appreciably in the gp91 phox- over the course of the two-month study whereas the Cy5-HA cryogels in B6 mice degraded within 4 weeks, as expected (FIG.18G).
  • B6 recipients and control mice (B6, non-irradiated that do not receive a transplant) were injected subcutaneously with Cy5-HA cryogels, and the degradation rate was compared (FIG.22A).
  • a steady fluorescence signal was quantified for about 20 days in post-HSCT mice after which it decreased, corresponding to HA cryogel degradation, at a rate comparable to that in non-irradiated mice.
  • the time interval to 50% of the initial fluorescence intensity was approximately 30 days in post-HSCT mice whereas in non-irradiated mice, a comparable decrease was achieved by day 13 (FIG.22B, FIG.21B).
  • Cy5-HA cryogels were excised on days 5 and 16 post-injection in non-irradiated mice and excised on days 5, 16, 21, and 26 in post- HSCT mice (FIG.22C). Viability of infiltrating cells, quantified by negative AnnexinV staining, was initially lower 5- and 16-days post-injection, and increased by day 21 (FIG. 21C). In non-irradiated B6 mice, the number of infiltrating myeloid cells decreased by 96% from days 5 to 16 post-injection (FIG.22D), mirroring near-complete Cy5-HA cryogel degradation (FIG.22B, FIG.21B).
  • infiltrating macrophages in some post-HSCT mice were quantified but remained significantly lower than macrophage infiltration on day 5 in non-irradiated mice (FIG.22E).
  • Neutrophils constituted a majority of the myeloid cells in Cy5-HA cryogels in non- irradiated mice on day 5, but not by day 16 (FIG.22F) when the majority of myeloid cells were macrophages (FIG.21D).
  • very few cells were in HA cryogels retrieved on day 5 in post-HSCT mice with a near absence of neutrophils, in contrast with non-irradiated mice at the same timepoint.
  • Cy5-OxAlg cryogels injected in B6 and post-HSCT B6 mice degraded rapidly at a comparable rate, with approximately 70% reduction in fluorescence signal within 24 hours post-injection (FIG.21G).
  • HA cryogels sustain G-CSF delivery and enhance post-HSCT reconstitution of neutrophils
  • the delay in post-HSCT degradation of HA cryogels was leveraged to mediate G- CSF release and enhance neutrophil recovery.
  • 1 ⁇ g of Cy5-labeled G-CSF (Cy5 G-CSF) was encapsulated in HA cryogels and one cryogel was injected either in 1-day post-HSCT or in non-irradiated B6 mice.
  • Encapsulated Cy5 G-CSF was quantified using IVIS and normalized to the initial 8-hour timepoint fluorescence signal (FIG.23A).
  • Cy5 G-CSF release assessed by fluorescence attenuation, from non-irradiated mice proceeded in a sustained manner immediately post-injection with over 80% released after approximately 12-days post- injection.
  • 20% Cy5 G-CSF released after approximately 12-days post- injection and subsequently released in a sustained manner (FIG.23B).
  • the time to 50% fluorescence intensity in non-irradiated mice was 5.9 ⁇ 3.0 days compared to 15.5 ⁇ 5.9 days in post-HSCT mice (FIG.24). Then, the effect of G-CSF delivery on peripheral blood neutrophil recovery and acceleration of Cy5-HA cryogel degradation was assessed.
  • mice receiving either two blank Cy5-HA cryogels or two G-CSF-encapsulated HA-cryogels were compared and, as a positive control, mice with blank Cy5-HA cryogels injected systemically with 2 ⁇ g pegylated (PEG) G-CSF (FIG.23C) were included, corresponding to the clinical- equivalent dose for mice 33, 34 .
  • Mice were bled at pre-determined timepoints, and peripheral blood neutrophil concentration, quantified by flow cytometry, was consistently higher when G-CSF from Cy5-HA cryogels was delivered, and comparable with PEG G-CSF treatment than in mice which received blank Cy5-HA cryogels (FIG.23D).
  • HA was selected as the primary constituent polymer as it is ubiquitous in the extracellular matrix and has a long history of clinical use as a biodegradable material in a range of biomedical applications 43-48 .
  • commercially purchased HA was derivatized with bioorthogonal Tz and Nb groups to facilitate crosslinking without the need for external energy input or addition of external agents such as stabilizers and catalysts 49, 50 , which can make it challenging to purify the final product.
  • HA-Tz and HA-Nb also facilitated cryogelation at a slower rate, compared to free-radical polymerization methods, and consequently provided enhanced control over the crosslinking process 51, 52 .
  • other common cross-linking strategies that directly target the carboxylic acid or hydroxyl side chains groups and unreacted agents may inadvertently react with encapsulated proteins 53-56 .
  • Tz can be quantified spectroscopically and the DOS was readily assessed 57, 58 . Consistent with prior work, these results show that DOS affected the rate of HA cryogel degradation by enzymatic cleavage 22, 62 .
  • De novo anti-PEG antibody induction may not require T cell activation 76 and therefore could also be induced in post-HSCT immunodeficient hosts.
  • PEG G-CSF may therefore be less effective with pre-existing or induced APA 77 .
  • Therapy-induced neutropenia substantially limits the applicability of therapies that could be life-saving.
  • HA cryogels not only deliver G-CSF in a sustained manner to enhance neutrophil regeneration, while avoiding the potential of APA-mediated enhanced clearance, but also show a responsive degradation behavior. Collectively, these findings support that the HA cryogels might be leveraged to enhance and functionally assess neutrophil functionality and aid in treatment-related decisions for recipients of myelosuppressive therapy. Materials and Methods The following Materials and Methods were used in Example 2.
  • MES morpholinoethanesulfonic acid
  • NaCl sodium chloride
  • NaOH sodium hydroxide
  • NHS N-hydroxysuccinimide
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • AB ammonia borane
  • HA Tetrazine functionalized HA (HA-Tz) or norbornene functionalized HA (HA-Nb) were prepared by reacting tetrazine amine or norbornene amine to HA using EDC/NHS carbodiimide chemistry.
  • EDC/NHS carbodiimide chemistry e.g., EDC/NHS carbodiimide chemistry.
  • Sodium hyaluronate was dissolved in a buffer solution (0.75% wt/vol, pH ⁇ 6.5) of 100mM MES buffer.
  • NHS and EDC were added to the mixture to activate the carboxylic acid groups on the HA backbone followed by either tetrazine amine or norbornene amine.
  • HA was assumed to be 1.8 MDa for purposes of conjugation reactions.
  • the molar ratios of HA:EDC:NHS:tetrazine are 1:25000:25000:2500.
  • the molar ratios of HA:EDC:NHS:tetrazine are 1:2860:2860:286.
  • the molar ratios of HA:EDC:NHS:norbornene are 1:25000:25000:2500.
  • Cy5 conjugated HA-Nb (Cy5-HA-Nb) was synthesized following a previously described technique with some modifications 78 .0.8mg of Cy5-Tz was reacted with 100mg of HA-Nb at 0.2 wt/vol in DI water for 24 hours at 37 oC and purified by dialysis in DI water using a 12,000Da MW cutoff dialysis sack for 48 hours. Dialysis water bath was changed once every 8 hours. Cy5-HA-Nb was then frozen overnight and lyophilized for 48 hours.
  • oxidized alginate Alginate was oxidized by mixing a 1% wt/vol solution of sodium alginate in DI water with an aqueous solution of 23 mM sodium periodate (Sigma Aldrich) to achieve a 1:586 molar ratio of alginate: periodate. The reaction was stirred in the dark at room temperature overnight. Sodium chloride (1.8 grams/gram of alginate) was added to solution to achieve a 0.3 M solution, followed by purification via tangential flow filtration (TFF) using a mPES 1kDa molecular weight cutoff (MWCO) membrane (Spectrum) and sequential solvent exchanges with 0.15 M – 0.10 M – 0.05 M and 0.0 M sodium chloride in DI water.
  • THF tangential flow filtration
  • MWCO molecular weight cutoff
  • the resulting solution was treated with ammonia borane (AB) complex (Sigma Aldrich) at 1:4 alginate:AB molar ratio and stirred at room temperature overnight.
  • AB ammonia borane
  • Sodium chloride (1.8 grams/gram of alginate) was added to solution to achieve a 0.3 M solution, followed by purification via TFF using a 1kDa MWCO mPES membrane and sequential solvent exchanges with 0.15 M – 0.10 M – 0.05 M and 0.0 M sodium chloride in DI water.
  • the resulting solution was lyophilized to dryness.
  • oxidized alginate prepared as described above, was solubilized in 0.1 M MES buffer,0.3 M sodium chloride, pH 6.5 at 1%wt/vol. NHS and EDC were added to the mixture followed by either tetrazine or norbornene.
  • the molar ratio of oxidized alginate:NHS:EDC:tetrazine or norbornene was 1:5000:5000:1000. The reaction was stirred in the dark at room temperature overnight.
  • the resulting solution was centrifuged at 4700 rpm for 15 minutes and filtered through a 0.2-micron filter.
  • the solution was purified via TFF using a mPES 1kDa molecular weight cutoff (MWCO) membrane and sequential solvent exchanges with 0.15 M – 0.10 M – 0.05 M and 0.0 M sodium chloride in DI water.
  • the purified solution was treated with activated charcoal (1 gram / gram of alginate) for 20 minutes at room temperature.
  • the slurry was filtered through 0.2-micron filter and the filtrate was lyophilized to dryness.
  • Endotoxin Testing of high-DOS HA-Tz and Cy5-HA-Nb were conducted using a commercially available endotoxin testing kit (88282, Thermo Fisher Scientific, lot: VH310729) and following manufacturer’s instructions. High-DOS HA-Tz and Cy5-HA-Nb were solubilized at 0.6 wt% in endotoxin free water and samples were tested in technical triplicates.
  • aqueous solutions of 0.6% wt/vol HA-Tz and HA-Nb or OxAlg- Tz and OxAlg-Nb were prepared by dissolving lyophilized polymers into deionized water and left on a rocker at room temperature for a minimum of 8 hours to allow for dissolution. The aqueous solutions were then pre-cooled to 4oC before cross-linking to slow reaction kinetics.
  • HA-Tz and HA- Nb or OxAlg- Tz and OxAlg-Nb solutions were mixed at a 1:1 volume ratio, pipetted into 30 ⁇ L Teflon molds which were pre-cooled to -20 oC, and quickly transferred to a -20 oC freezer to allow for overnight cryogelation.
  • Synthesis of Cy5-HA or Cy5-OxAlg cryogels followed the same protocol as above, substituting Cy5-HA-Nb for HA-Nb or Cy5-OxAlg-Nb for OxAlg-Nb.
  • Lyophilized HA cryogels were adhered onto sample stubs using carbon tape and coated with iridium in a sputter coater. Samples were imaged using secondary electron detection on a FEI Quanta 250 field emission SEM in the Nano3 user facility at UC San Diego. Fluorescence images of Cy5-HA cryogels were acquired using a Leica SP8 All experiments were performed at the UC San Diego School of Medicine Microscopy Core. Pore size quantification of SEM images and relative distribution of pore sizes of confocal images was done using FIJI image processing package 81 .
  • Cy5-HA cryogels were synthesized with low- and high-DOS HA-Tz and incubated in 1mL of FITC-labeled 10 ⁇ M diameter melamine resin micro particles (Sigma Aldrich) at 0.29mg/mL concentration on a rocker at room temperature overnight. Fluorescence images of Cy5-HA cryogels with FITC-labeled microparticles were acquired using a Leica SP8 confocal.
  • Interconnectedness of the HA cryogels was determined by generating 3D renderings of confocal z-stacks using FIJI imaging processing package and assessing fluorescence intensity of both the Cy5 and FITC channels with depth starting from the top of the HA cryogel. To determine the effect of injection on pore interconnectedness, HA cryogels were injected through a 16G needle prior to incubation in FITC-labeled microparticle solution. All experiments were performed at the UC San Diego School of Medicine Microscopy Core.
  • Cy5-HA cryogels synthesized with low- and high-DOS HA-Tz and placed into individual 1.5mL microcentrifuge tubes (Thermo Scientific) with 1mL of 100U/mL Hyaluronidase from sheep testes Type II (HYAL2, H2126, Sigma Aldrich, lot: SLBZ9984) in 1x PBS. Degradation studies were conducted in tissue culture incubators at 37 oC. Supernatant from samples were collected every 24 – 72 hours by centrifuging the samples at room temperature at 2,000G for 5 minutes and removing 0.9mL of supernatant.
  • Cy5-HA cryogels were resuspended by adding 0.9mL of freshly made 100U/mL HYAL2 in 1x PBS. Fluorescence measurements were conducted using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific) and these values were normalized to sum of the fluorescence values over the course of the experiment. All experiments were performed at UC San Diego. In vitro degradation of Cy5-OxAlg Cryogels Cy5-OxAlg cryogels were placed into individual 1.5mL microcentrifuge tube with 1mL of 1x PBS. Degradation studies were conducted in tissue culture incubators at 37oC.
  • mice Female C57BL/6J (B6, Jax # 000664) and NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG, Jax # 005557) mice were 6-8 weeks at the start of the experiments.
  • mice Neutrophil depletion in B6 mice was achieved by following the previously established protocol 28, 82 . Briefly, 25 ⁇ L of anti-mouse Ly6G antibody (1A8, Bio X Cell, lot: 737719A2)) was administered i.p. every day for the first week. Concurrently, 50 ⁇ L of anti-rat ⁇ immunoglobulin light chain antibody (MAR 18.5, Bio X Cell, lot: 752020J2) was administered every other day starting on the second day of depletion.
  • anti-mouse Ly6G antibody (1A8, Bio X Cell, lot: 737719A2
  • MAR 18.5, Bio X Cell, lot: 752020J2 was administered every other day starting on the second day of depletion.
  • Macrophage depletion in B6 mice was induced by i.p. administration of 100uL of clodronate liposomes (Liposoma, lot: C44J0920) every 3-days.
  • B cell lineage depletion in B6 mice was induced by i.p. administration of 400 ⁇ g of anti-mouse B220/CD45R antibody (RA3.31/6.1, Bio X Cell, lot: 754420N1) once every 3-days.
  • T cell lineage depletion in B6 mice was induced by i.p.
  • mice received intraperitoneal injections of 0.1mL (400 ⁇ g) of antibodies or 0.1mL of clodronate liposome solution 3 days before subcutaneous HA cryogel or Cy5-HA cryogel injection. Depletion started 3-days prior to Cy5-HA cryogel administration to mice and continued until complete cryogel degradation or until mice were euthanized and cryogels retrieved for analysis.
  • FIG.25 further provides a table in which the methods and durability of immune cell depletion are described.
  • Transplant models Irradiations were performed with a Cesium-137 gamma-radiation source irradiator (J.L. Shepherd & Co.).
  • Syngeneic HSCT B6 recipients consisted of 1 dose of 1,000 cGy + 1 x 10 5 lineage-depleted bone marrow cells from syngeneic B6 donors.
  • Bone marrow cells for transplantation (from donors) or analysis were harvested by crushing all limbs with a mortar and pestle, diluted in 1x PBS, filtering the tissue homogenate through a 70 ⁇ m mesh and preparing a single-cell suspension by passing the cells in the flowthrough once through a 20- gauge needle. Total cellularity was determined by counting cells using a hemacytometer. Bone marrow cells were depleted of immune cells (expressing CD3 ⁇ , CD45R/B220, Ter-119, CD11b, or Gr-1) by magnetic selection using a Mouse Hematopoietic Progenitor Cell Enrichment Set (BD Biosciences # 558451, lot: 0114777).
  • Subcutaneous cryogel administration While mice were anesthetized, a subset received a subcutaneous injection of HA cryogel or OxAlg cryogel, which was suspended in 200 ⁇ L of sterile 1x PBS, into the dorsal flank by means of a 16G needle positioned approximately midway between the hind- and forelimbs. The site of injection was shaved and wiped with a sterile alcohol pad prior to gel injection.
  • Cy5-HA cryogel degradation was performed with Cy5-HA cryogels synthesized with low- and high-DOS Tz-HA in untreated B6 mice, immune deficient B6 mice, NSG mice, and gp91 phox- mice.
  • Anti-mouse antibodies to CD45 (30-F11, lot: B280746), CD11b (M1/70, lot: B322056), CD4 (RM4-5, lot: B240051), CD8 ⁇ (53-6.7, lot: B266721), B220 (RA3-6B2, lot: B298555), Ly6-G/Gr-1 (1A8, lot: B259670), lineage cocktail (17A2/RB6-8C5/RA3-6B2/Ter- 119/M1/70, lot: B266946), Ly-6A/E/Sca-1 (D7, lot: B249343), and CD117/cKit (2B8, lot: B272462) were purchased from Biolegend.
  • Anti-mouse F4/80 (BM8, lot: 2229150) and was purchased from eBioscience. All cells were gated based on forward and side scatter characteristics to limit debris, including dead cells. AnnexinV (Biolegend, lot: B300974) stain was used to separate live and dead cells. Antibodies were diluted according to the manufacturer’s suggestions. Cells were gated based on fluorescence-minus-one controls, and the frequencies of cells staining positive for each marker were recorded. To quantify T cells, B cells, monocytes, and neutrophils in peripheral blood, blood was first collected from the tail vein of mice into EDTA coated tubes (BD). Samples then underwent lysis of red blood cells and were stained with appropriate antibodies corresponding to cell populations of interest.
  • BD EDTA coated tubes
  • mice were sacrificed, cryogels removed, and HA cryogels crushed against a 70-micron filter screen before antibody staining. Absolute numbers of cells were calculated using flow cytometry frequency.
  • Flow cytometry was analyzed using FlowJo (BD) software. All flow cytometry experiments were performed using a Attune ® NxT Acoustic Focusing cytometer analyzer (A24858) at UC San Diego. Histology After euthanasia, HA cryogels were explanted and fixed in 4% paraformaldehyde (PFA) for 24 hours. The fixed HA cryogels were then transferred to 70% ethanol solution.
  • PFA paraformaldehyde
  • antigen retrieval was conducted using Unmasking solution (Citrate based, pH 6) (Vector Laboratories, H-3300) at 95 oC for 30 minutes. Staining was performed using Intellipath Automated IHC Stainer (Biocare). A peroxidase block, Bloxall (Vector Laboratories, SP-6000) was performed for 10 minutes, followed by 2x washes in 1x tris-buffered saline with 0.1% Tween 20 (TBST, Sigma Aldrich), and a blocking step using 3% Donkey Serum for 10 minutes. Samples were stained using anti- Ly6G primary antibody (Rabbit, Cell Signaling Technology, 87048S) at 1:100 concentration for 1 hr.
  • G-CSF encapsulation To quantify G-CSF release from HA cryogels, recombinant human G-CSF (300-23, Peprotech, lots: 041777 and 041877) was reacted with sulfo-Cy5 NHS ester (13320, Lumiprobe, lot 7FM7C) at a 1:25 molar ratio of G-CSF:sulfo-Cy5 NHS ester in MES buffer to form Cy5 G-CSF.
  • sulfo-Cy5 NHS ester 13320, Lumiprobe, lot 7FM7C
  • the reaction rate can be calculated as: The rate of reaction was calculated to be about 40 ⁇ M/s from equation 1 and the time to completion to be about 46.3 minutes at 21oC. In this system, the initial temperature is 4oC and therefore the actual time for completion of the reaction would be significantly longer. As the solution cools and freezes during the crosslinking process, the freezing time was estimated. First, the energy required to freeze 30uL of HA solution starting from 4oC and ending at 0oC was determined using: The energy required to freeze 30 ⁇ L HA solution from 4oC to 0oC is 10.5J 3 . Since the HA solution is very dilute (0.6 wt%), the enthalpy of formation and density was approximated to that of water.
  • the rate of energy extraction from the HA solution was estimated. Since the teflon cryomold is pre-cooled to -20oC and rests on a metal shelf in the freezer, the rate of freezing was estimated using the thermal conductivity of teflon, thickness of teflon (25mm), and conductive heat transfer area (5.75mm) using equation 3. To simplify the analysis, it was assumed that convective heat loss at air-cryogel interface is negligible. The rate of heat transfer is calculated to be 0.010J/s and therefore the time to reach 0oC is ⁇ 16.8 minutes.
  • Granulocyte Colony-Stimulating Factor Use after Autologous Peripheral Blood Stem Cell Transplantation Comparison of Two Practices. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 24, 288-293 (2016). 19. Ashrafi, F. & Salmasi, M. Comparison of the effects of pegylated granulocyte-colony stimulating factor and granulocyte-colony stimulating factor on cytopenia induced by dose-dense chemotherapy in breast cancer patients. Journal of research in medical sciences : the official journal of Isfahan University of Medical Sciences 23, 73 (2016). 20. Molineux, G.
  • pegfilgrastim PEG-rmetHuG-CSF, Neulasta
  • Pegfilgrastim (PEG-G-CSF) induces anti-PEG IgM in a dose dependent manner and causes the accelerated blood clearance (ABC) phenomenon upon repeated administration in mice.
  • Pegfilgrastim after high-dose chemotherapy and autologous peripheral blood stem cell transplant phase II study. Bone Marrow Transplantation 35, 1165-1169 (2005). 34. Martino, M. et al. Pegfilgrastim compared with filgrastim after high-dose melphalan and autologous hematopoietic peripheral blood stem cell transplantation in multiple myeloma patients. European journal of haematology 77, 410-415 (2006). 35. Barr, S., Hill, E.W. & Bayat, A. Functional biocompatibility testing of silicone breast implants and a novel classification system based on surface roughness. Journal of the Mechanical Behavior of Biomedical Materials 75, 75-81 (2017). 36.
  • Methylprednisolone acetate induces, and ⁇ 7-dafachronic acid suppresses, Strongyloides stercoralis hyperinfection in NSG mice. Proceedings of the National Academy of Sciences of the United States of America 115, 204-209 (2016). 69. Pavl ⁇ , J. et al. Analysis of hematopoietic recovery after autologous transplantation as method of quality control for long-term progenitor cell cryopreservation. Bone Marrow Transplant 52, 1599-1601 (2017). 70. Ramaprasad, C., Pouch, S. & Pitrak, D.L. Neutrophil function after bone marrow and hematopoietic stem cell transplant.

Abstract

La présente invention concerne des compositions et des méthodes qui améliorent la récupération des neutrophiles chez un sujet et/ou qui permettent l'administration d'agents thérapeutiques.
PCT/US2021/058359 2020-11-06 2021-11-05 Échafaudages pour amélioration des neutrophiles et utilisations associées WO2022099093A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100174346A1 (en) * 2007-08-17 2010-07-08 Boyden Edward S System, devices, and methods including actively-controllable sterilizing excitation delivery implants
US20170368169A1 (en) * 2016-03-21 2017-12-28 Elstar Therapeutics, Inc. Multispecific and multifunctional molecules and uses thereof
US20190290696A1 (en) * 2016-06-08 2019-09-26 Lysat Pharma GmbH (Lysatpharma) Human platelet lysate derived extracellular vesicles for use in medicine
US20200024339A1 (en) * 2012-11-06 2020-01-23 Scholar Rock, Inc. Compositions and methods for modulating cell signaling

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100174346A1 (en) * 2007-08-17 2010-07-08 Boyden Edward S System, devices, and methods including actively-controllable sterilizing excitation delivery implants
US20200024339A1 (en) * 2012-11-06 2020-01-23 Scholar Rock, Inc. Compositions and methods for modulating cell signaling
US20170368169A1 (en) * 2016-03-21 2017-12-28 Elstar Therapeutics, Inc. Multispecific and multifunctional molecules and uses thereof
US20190290696A1 (en) * 2016-06-08 2019-09-26 Lysat Pharma GmbH (Lysatpharma) Human platelet lysate derived extracellular vesicles for use in medicine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TITAN ASHLEY, SCHÄR MICHAEL, HUTCHINSON IAN, DEMANGE MARCO, CHEN TONY, RODEO SCOTT: "Growth Factor Delivery to a Cartilage-Cartilage Interface Using Platelet-Rich Concentrates on a Hyaluronic Acid Scaffold", ARTHROSCOPY., RAVEN PRESS, NEW YORK, NY., US, vol. 36, no. 5, 1 May 2020 (2020-05-01), US , pages 1431 - 1440, XP055938738, ISSN: 0749-8063, DOI: 10.1016/j.arthro.2019.12.004 *

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