(358b) Cell-Responsive Biodegradable Scaffolds for Enhancing Immune Regeneration

Introduction: Neutrophils are one of the most abundant cell types of the innate immune system and mediate essential host defense against a broad range of invading pathogens. Dysfunction and deficiency in their function and number is implicated in multidrug-resistant bacterial, fungal and viral infections in patients with immune deficiency or therapy-related neutropenia. In patients treated with hematopoietic stem cell transplantation (HSCT) and high dose chemotherapy, there is a marked transient 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. The standard clinical treatment to enhance post-HSCT neutrophil regeneration is by using recombinant granulocyte colony stimulating factor (G-CSF), which accelerates neutrophil recovery. However, daily administration of G-CSF is burdensome and associated with splenic rupture and exposure to high-dose PEG G-CSF may cause hematopoietic stem cell quiescence and loss of long-term repopulating activity. We hypothesized that tunable delivery of low dose G-CSF would be able to mediate accelerated neutrophil recovery while minimizing toxicities associated with high-dose PEG G-CSF. To test the hypothesis, we developed an in vivo depot of G-CSF to mediate its sustained release to accelerate post-HSCT neutrophil recovery. We characterized the degradation of a hyaluronic acid (HA) depot, termed as a HA cryogel and therapeutic delivery in settings of immune deficiency.

Methods and Materials: Injectable macro-porous HA cryogels were synthesized by low-temperature polymerization of tetrazine (Tz)-functionalized HA (Tz-HA) and norbornene (Nb)-functionalized HA (Nb-HA). Characterization of subcutaneous HA cryogel degradation was conducted by tagging HA polymer with Cy5 fluorophore and tracking fluorescence intensity using in vivo imaging system (IVIS) microscopy. Degradation of HA cryogels was characterized in C57BL/6 (B6) mice with single immune lineage depletion, B6 mice following lethal irradiation and HSCT, and in Nod Scid Gamma (NSG) mice. Innate immune cell infiltration into HA cryogel was monitored by excising HA cryogels at pre-determined timepoints. Excised gels were either fixed for histology or crushed to isolate cells for FACS analysis. G-CSF was tagged with Cy5 to characterize in vivo release from HA cryogels using IVIS microscopy. G-CSF mediated recovery of innate immune cells was monitored by bleeding mice at pre-determined timepoints and staining blood for FACs analysis.

Results and Discussion: As the pre-conditioning regimen for HSCT depletes all immune cell lineages, we sought to compare the effect of depleting one or more lineages of immune cells on the degradation kinetics of HA cryogels. Fluorescently labeled HA cryogels were injected into the subcutaneous space under the dorsal flank and the degradation kinetics were compared between of untreated mice and mice depleted of T-cells, B-cells, macrophages, and in NSG mice. HA cryogel degradation rate was unchanged in cohorts of mice depleted of T-cells and macrophages. The initial degradation rate of HA cryogels in the B-cell depleted mice was reduced relative to untreated mice, but full degradation was achieved in 6 weeks, similar to untreated cohort. In contrast, NSG mice failed to clear HA cryogels over 100 days. We analyzed cellular infiltrates in HA cryogels at one- and ten-days post- injection and characterized reduced myeloid lineage infiltration in the B-cell depleted mice. We measured the degradation of HA cryogels in a syngeneic HSCT model of transient innate and adaptive immunodeficiency. HA cryogels were administered to mice concurrent to HSCT following lethal radiation. We HA cryogel degradation was delayed for ~3 weeks coinciding with full reconstitution of peripheral innate immunity and innate immune cell infiltration into the HA cryogel. As the degradation of the HA cryogel was substantially delayed in post-HSCT mice, we developed the HA cryogel as a depot to mediate sustained delivery of G-CSF to accelerate peripheral blood neutrophil reconstitution. A single post-HSCT administration of G-CSF encapsulated HA cryogels was able to mediate its sustained release as determined by accelerated neutrophil reconstitution at days 7 and 12 post-HSCT as compared to mice that did not receive G-CSF. The accelerated rate of neutrophil reconstitution accelerated HA cryogel degradation, as determined by differences in fluorescence signal intensity at days 12 and days 18 post-injection.

Conclusions: Immune deficiency substantially limits the applicability of potentially curative hematopoietic stem cell transplantation in patients, and is associated with opportunistic infections. Here, we report the development and in vivo evaluation of an injectable, biodegradable hyaluronic acid (HA)-based scaffold, termed HA cryogel, with neutrophil responsive degradation behavior. In mouse models of innate and adaptive immune deficiency, we show that the infiltration of functional myeloid-lineage cells is essential to mediate HA cryogel degradation. In HSCT mouse models, we harnessed the delay in trafficking of myeloid cells to sustain the release of granulocyte colony stimulating factor (G-CSF) from HA cryogels. In comparison to a single dose of G-CSF, sustained release from HA cryogels accelerated post-HSCT neutrophil recovery neutrophil by 4-fold, with full recovery achieved within 14 days post-HSCT and accelerated the rate of degradation of HA cryogels. The HA-cryogel provides an off-the-shelf approach for enhancing neutrophil recovery in settings of immune deficiency and reducing the risk of immune-deficiency related complications.