Poster X6 - Layer-by-layer nanoparticles for controlled release of therapeutic cytokines in ovarian cancer minimal residual disease

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

MB

Margaret Billingsley, PhD

Post Doctoral Associate
Massachusetts Institute of Technology
Hockessin, Delaware, United States

Co-Author(s)

MP

Mae Pryor

Graduate Student
MIT, United States

Last Author(s)

PH

Paula Hammond

Institute Professor and Vice Provost for Faculty
MIT, United States

Introduction: Over 20,000 cases of ovarian cancer are diagnosed in the United States each year with 70% of these diagnoses occurring at late stages in disease progression. Though aggressive interventions such as debulking surgeries and combination chemotherapies may lead to lower tumor burden and an apparent cure, 85% of these patients will relapse with incurable disease. This is due, in part, to minimal residual disease (MRD)—the persistence of small, undetectable cancer cell populations that survive initial interventions and expand into an incurable relapse. Here, to address this pivotal point in disease progression, we explore cytokine immunotherapies to eliminate the MRD in ovarian cancer patients. Specifically, we utilize layer-by-layer nanoparticles (LbL-NPs) with a poly-L-glutamate (PLE) surface chemistry that drives specific accumulation on ovarian cancer cell surfaces, allowing for the extracellular presentation of cytokine cargo. In this work, we explore the delivery of XCL-1 and IFN-alpha—cytokines responsible for recruiting and activating potent anti-tumor dendritic cells (DCs), respectively. Further, to optimize the delivery of these cytokines, this work explores the use of conjugation chemistries with varied stabilities including Nickel-His tag, Cobalt-His tag, and maleimide-cysteine. By characterizing the in vivo pharmacokinetics of cytokines conjugated via these chemistries, we establish a LbL-NP delivery platform with tunable release of its therapeutic cytokine cargo. Thus, by rationally designing each LbL-NP platform to release its cytokine at the therapeutically optimal rate, we can create more effective, tunable immunotherapies to improve patient outcomes in ovarian cancer.

Materials and

Methods: To generate the cytokine-conjugated LbL-NPs, liposomal cores were first synthesized by generating lipid films to then hydrate and extrude to produce ~100nm liposomes. These liposome formulations contained functional conjugation components including either maleimide-PE (mal-NPs), Ni-NTA DGS (Ni-NPs), or Cobalt-porphyrin (Co-NPs) to allow for protein loading onto their surface in an overnight incubation. Following conjugation, the liposomes were layered with polymers of alternating charges to allow for either a PLD or PLE surface as these polymers are associated with ovarian cancer targeting via glutamine receptors. The LbL-NPs were characterized by size, PDI, zeta potential, and weight percent protein loading throughout this process. To then evaluate cell association the LbL-NPs, Cy5 labeled NPs were incubated with the BPPNM and CPAK ovarian cancer cell lines used to model MRD, and LbL-NP signal in the cell populations were measured over multiple timepoints using flow cytometry. To confirm this localization in vivo in an MRD context, CPAK cells were used to inoculate mice and NPs were administered IP to observe organ-level biodistribution at 1 hour via IVIS. To assess the varied conjugation chemistries, ICG-tagged IL-12 was conjugated to the surface of the liposomal cores before layering, allowing for IL-12 release to be measured with fluorescent signal. The release of IL-12 from the Ni-NPs and Co-NPs was assessed in both serum containing media and cell free murine ascites. Further, the CPAK model was used to assess the pharmacokinetics of IP injected IL-12 as free cytokine as well as for IL-12 conjugated to mal-, Ni-, and Co-NPs.

Results, Conclusions, and Discussions: The cytokine-loaded LbL-NPs were synthesized incorporating one of three conjugation chemistries and then coated in polymer layers to facilitate ovarian cancer targeting (Figure 1A). After characterization to ensure sizes < 150 nm and negative surface charges, the LbL-NPs were evaluated for delivery. LbL-NPs with the PLE and PLD outer layer demonstrated enhanced association with the MRD cell lines, validating that these ovarian cancer targeting layers extend their specificity to MRD (Figure 1B). These associations were further demonstrated in vivo via biodistribution measurements confirming colocalization with MRD nodules. To characterize the release kinetics of the conjugation strategies, the Co-NPs, Ni-NPs, and mal-NPs were loaded with IL-12, and its release from each NP was measured over 24 hours in either serum containing media or ascites fluid collected from mice with ovarian cancer. In these studies, Co-NPs demonstrated more stable conjugation with slower release of the conjugated cytokine. This was further demonstrated in vivo in the CPAK model with Co-NPs and mal-NPs achieving prolonged IP retention of IL-12 over 120 hours while Ni-NPs demonstrated rapid clearance (Figure 1C). Thus, for cytokines requiring more rapid release, Ni-NPs may be the optimal platform whereas prolonged release or localization of cytokine is better accomplished by Co-NPs and mal-NPs. These results demonstrate the ability of LbL-NPs to deliver therapeutic cytokine cargos with tunable release kinetics in murine models of ovarian cancer MRD. Further, the in vivo results establish cytokine conjugation chemistry as an important consideration in the design of NP-based immunotherapies.

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