Poster T9 - Hybrid membrane-coated nanoparticles for multi-cellular targeting in the tumor microenvironment

Introduction: The tumor microenvironment consists of various cell types that perform multitudes of functions to propagate disease and hinder the efficacy of administered therapeutics including nanoparticles (NPs). Beyond cancer cells, cancer-associated fibroblasts (CAFs) are a predominant population of cells in the tumor stroma that create a protective physical barrier that impedes NP delivery throughout the tumor volume. CAFs also secrete a variety of growth factors and cytokines that increase tumor initiation, angiogenesis, progression, metastasis, and therapy resistance. Accordingly, both CAFs and cancer cells must be targeted and eliminated by therapies to ensure disease eradiation. Previous research has shown that NPs coated with cancer cell-derived biological membranes can precisely target and deliver cargo to cancer cells because cell adhesion proteins, such as Tf-antigen, EpCAM, CD44, and others, embedded in the membrane facilitate binding to homotypic cancer cells. To enable targeting of both cancer cells and CAFs within triple-negative breast cancer (TNBC) tumors, we have developed poly(lactic-co-glycolic acid) (PLGA) NPs that are coated with a hybrid combination of membranes derived from both TNBC cells and activated fibroblasts (AFs). Here, we show these Hybrid NPs can efficiently enter both TNBC cells and fibroblasts in vitro. Additionally, the Hybrid NPs exhibit ~2X greater accumulation in orthotopic mammary fat pad TNBC tumors in female Balb/c mice than NPs coated with only cancer cell membranes (CCNPs), and similar accumulation in the tumors as NPs coated with only AF membranes (AFNPs). These exciting results warrant further investigation into the use of Hybrid NPs as cancer therapeutics.

Materials and

Methods: PLGA NPs loaded with Cy5-tagged siRNA for tracking were made by double emulsion synthesis. To create activated fibroblasts (AFs), 3T3 murine fibroblast cells were treated with TGFβ-1 in serum-free media for two days before membrane extraction. Then, 4T1 murine TNBC cells and AFs were lysed, mechanically homogenized, and differentially centrifuged to remove intracellular components and isolate membranes vesicles (MVs). The MVs and unwrapped NPs were co-extruded at a 2:1 ratio to form cancer cell membrane-wrapped NPs (CCNPs), AF membrane-wrapped NPs (AFNPs), or Hybrid NPs (Figure 1). For Hybrid NPs, 4T1 and AF MVs were used at a 1:1 ratio. The size, zeta potential, and concentration of Bare NPs, MVs, and wrapped NPs was measured by nanoparticle tracking analysis and dynamic light scattering. NPs were also visualized with transmission electron microscopy (TEM). NP uptake by 4T1 and AF cells in the absence or presence of endocytosis inhibitors was measured using flow cytometry and confocal microscopy. To evaluate tumor targeting in vivo, 4T1-luciferase expressing cells were injected in the mammary fat pad of immune-competent female Balb/c mice to form primary tumors. Once tumors were ~50-150 mm^3 in volume per caliper measurements, mice received IV injections of saline, CCNPs, AFNPs, or Hybrid NPs. After ~18 hours, the primary tumors and major organs were excised and imaged ex vivo to assess the NPs’ relative biodistribution. Additionally, tumors were enzymatically digested into single cell suspensions and analyzed by flow cytometry to determine the percent of live tumor cells that were positive for Cy5.

Results, Conclusions, and Discussions: Characterization studies showed that Hybrid NPs, CCNPs, and AFNPs were ~135 nm diameter and had a charge near that of MVs (-15 mV) (Figure 2A). The presence of a membrane coating around the PLGA core was also clearly identifiable in TEM images (Figure 2B). Regarding cellular uptake, Hybrid NPs entered both 4T1 and 3T3 cells, and 4T1 cells internalized more Hybrid NPs, CCNPs, and AFNPs than Bare (unwrapped) NPs (Figure 2C,D). Inhibitor assays revealed the NPs enter cells primarily via lipid-raft and dynamin-dependent endocytosis (Figure 2D). In vivo, we performed IVIS imaging of tissues excised ~18 hours post systemic administration of NPs to mice bearing orthotopic 4T1 tumors. This analysis showed that Hybrid NPs and AFNPs exhibited 1.94X and 1.90X, respectively, greater radiant efficiency per tumor mass (milligram) than CCNPs (Figure 3A-C). These results support the inclusion of AF membranes in the Hybrid NP design to enhance tumor delivery. Finally, flow cytometry of excised tumors showed that ~5.6% of the total live cells within tumors were positive for Hybrid NPs compared to ~5.3% for AFNPs and ~2.2% for CCNPs (Figure 3D). This approximate 2.5X increase for Hybrid NPs and AFNPs compared to CCNPs agrees with the differences in tumor accumulation observed by IVIS. Moving forward, future biodistribution studies will examine the NPs’ interactions with specific cells in tumors (namely, cancer cells, CAFs, and tumor-associated macrophages) to determine whether the precise membrane coating drives delivery to different cells within tumors. We expect that CCNPs will primarily bind cancer cells, AFNPs will primarily bind CAFs, and Hybrid NPs will have balanced interactions. We also plan to analyze accumulation in small (~200 mm^3) tumors as compared to large (~600 mm^3) tumors because both the vascular density and cellular composition of tumors changes as disease progresses, which may influence NP accumulation in and transport throughout tumors to reach specific cells. Collectively, the data we have gathered provide critical insight to the mechanisms of tumor delivery of membrane-wrapped NPs and warrant further investigation of Hybrid NPs for multi-cellular targeting in the tumor microenvironment.

Acknowledgements (Optional): The authors acknowledge funding support from the Delaware Bioscience Center for Advanced Technology, the National Science Foundation (DMR1752009), and the National Institutes of Health (R01CA211925).