Nanotopography-based lymphatic delivery for improved anti-tumor responses to checkpoint blockade immunotherapy.

Rationale: Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is a co-inhibitory checkpoint receptor that is expressed by naïve T-cells in lymph nodes (LNs) to inhibit activation against "self" antigens (Ags). In cancer, anti-CTLA-4 blocks inhibitory action, enabling robust activation of T-cells against tumor Ags presented in tumor draining LNs (TDLNs). However, anti-CTLA-4 is administered intravenously with limited exposure within TDLNs and immune related adverse events (irAEs) are associated with over-stimulation of the immune system. Methods: Herein, we first deliver anti-CTLA-4 in an orthotopic mammary carcinoma murine model using a nanotopographical microneedle-array device to compare its anti-tumor response to that from systemic administration. Additionally, to demonstrate the feasibility of lymphatic delivery in humans using the device, we use near-infrared fluorescence imaging to image delivery of ICG to LNs. Results: Our data show that lymphatic infusion results in more effective tumor growth inhibition, arrest of metastases, increased tumor infiltrating lymphocytes and complete responses when compared to conventional systemic administration. In clinical studies, we demonstrate for the first time that nanotopographic infusion can deliver ICG through the lymphatics directly to the axilla and inguinal LNs of healthy human volunteers. Conclusion: Taken together, these results suggest that regional delivery using a nanotopography-based microneedle array could revolutionize checkpoint blockade immunotherapy by reducing systemic drug exposure and maximizing drug delivery to TDLNs where tumor Ags present. Future work is needed to determine whether lymphatic delivery of anti-CTLA-4 can alleviate irAEs that occur with systemic dosing.

Lymphatic delivery of etanercept via nanotopography improves response to collagen-induced arthritis.

BACKGROUND: Evidence suggests lymphatic function mediates local rheumatoid arthritis (RA) flares. Yet biologics that target the immune system are dosed systemically via the subcutaneous (SC) administration route, thereby inefficiently reaching local lymphatic compartments. Nanotopography has previously been shown to disrupt tight cellular junctions, potentially enhancing local lymphatic delivery and potentially improving overall therapeutic efficacy. METHOD: We first characterized nanotopography (SOFUSA™) delivery of an anti-TNF drug, etanercept, by comparing pharmacokinetic profiles to those obtained by conventional SC, intravenous (IV), and intradermal (ID) routes of administration, and assessed uptake of radiolabeled etanercept in draining lymph nodes (LNs) in single dosing studies. We then compared etanercept efficacy in a progressive rat model of collagen-induced arthritis (CIA), administered systemically via SC route of administration; via the regional lymphatics through ID delivery; or through a nanotopography (SOFUSA™) device at 10, 12, and 14 days post CIA induction. Measurements of hind limb swelling and near-infrared fluorescence (NIRF) imaging of afferent lymph pumping function and reflux were conducted on days 11, 13, and 18 post CIA induction and compared to untreated CIA animals. Univariate and multivariate analysis of variance were used to compare the group differences for percentage swelling and lymphatic contractile activity. RESULTS: Even though all three modes of administration delivered an equal amount of etanercept, SOFUSA™ delivery resulted in increased lymphatic pumping and significantly reduced swelling as compared to untreated, ID, and SC groups. Pharmacokinetic profiles in serum and LN uptake studies showed that using the nanotopography device resulted in the greatest uptake and retention in draining LNs. CONCLUSIONS: Locoregional lymphatic delivery of biologics that target the immune system may have more favorable pharmacodynamics than SC or IV administration. Nanotopography may provide a more efficient method for delivery of anti-TNF drugs to reverse impairment of lymphatic function and reduce swelling associated with RA flares.

Calibrated flux measurements reveal a nanostructure-stimulated transcytotic pathway.

Transport of therapeutic agents across epithelial barriers is an important element in drug delivery. Transepithelial flux is widely used as a measure of transit across an epithelium, however it is most typically employed as a relative as opposed to absolute measure of molecular movement. Here, we have used the calcium switch approach to measure the maximum rate of paracellular flux through unencumbered intercellular junctions as a method to calibrate the flux rates for a series of tracers ranging in 0.6-900kDa in size across barriers composed of human colon epithelial (Caco-2) cells. We then examined the effects of nanostructured films (NSFs) on transepithelial transport. Two different NSF patterns were used, Defined Nanostructure (DN) 2 imprinted on polypropylene (PP) and DN3 imprinted on polyether ether ketone (PEEK). NSFs made direct contact with cells and decreased their barrier function, as measured by transepithelial resistance (TER), however cell viability was not affected. When NSF-induced transepithelial transport of Fab fragment (55kDa) and IgG (160kDa) was measured, it was unexpectedly found to be significantly greater than the maximum paracellular rate as predicted using cells cultured in low calcium. These data suggested that NSFs stimulate an active transport pathway, most likely transcytosis, in addition to increasing paracellular flux. Transport of IgG via transcytosis was confirmed by immunofluorescence confocal microscopy, since NSFs induced a significant level of IgG endocytosis by Caco-2 cells. Thus, NSF-induced IgG flux was attributable to both transcytosis and the paracellular route. These data provide the first demonstration that transcytosis can be stimulated by NSFs and that this was concurrent with increased paracellular permeability. Moreover, NSFs with distinct architecture paired with specific substrates have the potential to provide an effective means to regulate transepithelial transport in order to optimize drug delivery.

The effect of nanotopography on modulating protein adsorption and the fibrotic response.

Understanding and modulating the cellular response to implanted biomaterials is crucial for the field of tissue engineering and regenerative medicine. Since cells typically reside in an extracellular matrix containing nanoscale architecture, identifying synthetic nanostructures that induce desirable cellular behaviors could greatly impact the field. Using nanoimprint lithography, nanostructured patterns were generated on thin film polymeric materials. The ability of these surfaces to influence protein adsorption, fibroblast proliferation and morphology, and fibrotic markers was investigated. Nanostructured features with aspect ratios greater than five allowed for less protein adsorption, resulting in decreased fibroblast proliferation and rounded cellular morphology. These nanofeatures also induced significantly lower gene expression of collagen 1α2, collagen 3α1, and growth factors such as connective tissue growth factor, integrin linked kinase, transforming growth factor ß1 (TGF-ß1), and epidermal growth factor, key factors associated with a fibrotic response. The results demonstrate that select nanostructured surfaces could be used to modulate the fibrotic behavior in cells and have the potential to be used as antifibrotic architecture for medical implants or tissue engineering scaffolds.

Nanostructure-mediated transport of biologics across epithelial tissue: enhancing permeability via nanotopography.

Herein, we demonstrate that nanotopographical cues can be utilized to enable biologics >66 kDa to be transported across epithelial monolayers. When placed in contact with epithelial monolayers, nanostructured thin films loosen the epithelial barrier and allow for significantly increased transport of FITC-albumin, FITC-IgG, and a model therapeutic, etanercept. Our work highlights the potential to use drug delivery systems which incorporate nanotopography to increase the transport of biologics across epithelial tissue.