UAN polymer nanoparticles for targeted delivery of anti-inflammatory drugs

Immune cell adhesion has been implicated in both cancers and autoimmune diseases to emerge as a target for nanoparticle imaging and therapy. Prolonged inflammation at the tumor site contributes to angiogenesis and activation of leukocyte integrins. Amphiphilic urethane acrylate nonionomer (UAN) polymer nanoparticles have been equipped with activation-specific antibodies against leukocyte integrins to deliver a suite of diagnostic and therapeutic agents. The molecular signature of integrin activation and subsequent signaling in the tumor micro-environment reveals lymphocyte function and macrophage efflux to the lymphatics. UAN nanoparticles encapsulating dye (FITC), super paramagnetic iron oxide (SPIO), and anti-inflammatory drugs (diclofenac and celastrol) were delivered to leukemic monocyte, leukemic T lymphocyte, and neutrophil cells and evaluated for specific targeting with immunofluorescence flow cytometry. Inflammatory signaling was studied using qPCR upon stimulation with inflammatory agents (LPS, PMA, and FMLP). Nanoparticle uptake analyzed by Perl’s Prussian Blue staining, histological sectioning, and FACS showed tumor specific targeting. Diclofenac and celastrol inhibited LPS, PMA, and FMLP-induced inflammatory responses in vitro by reducing the level of ICAM-1 and the release of inflammatory cytokines TNF-alpha and IFN-gamma. Nanoparticles with activation-specific antibodies encapsulating anti-inflammatory drugs may effectively suppress leukocyte recruitment to inflamed sites for the prevention and treatment of autoimmune diseases.

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Figure 1. TEM micrographs of UAN-NTA-SPIO-Ni Nanoparticle in 1% UA stain.

INTRODUCTION

The integrin lymphocyte function-associated antigen-1 (LFA-1) is expressed on the surface of leukocytes, and in the activated state promotes heterotypic adhesion to intercellular adhesion molecules on other immune cells. Stimulation of LFA-1 in the presence of Mg2+ and EGTA (or activating antibody CBRLFA-1/2) yields a conformational change, thereby creating a high affinity (HA) form of LFA-1 (1,9).

The activation epitope of LFA-1 has been localized to the inserted (I) domain of the α subunit, which suggests that this domain may assist in regulating the interconversion between the high and low affinity binding conformations of LFA-1 (6). The I domain has also been mapped to a larger subset of integrin α subunits signifying an essential role for the I domain in controlling integrin activity and ligand binding (2,3).

In particular, the ICAM family has been uniquely presented to be closely tied with leukocyte integrin immune function. ICAM-1, -2, and -3 are confirmed to bind with an identical docking mechanism onto the I domain and are similarly expressed on leukocyte surfaces as well as endothelium (5). These molecular interactions mark the activation of lymphocytes by engagement of the T Cell Receptor (TCR) or chemokine receptors by eliciting intracellular signaling cascades that lead to transient up-regulation of HA LFA-1. Cyclins and other cell-cycle regulating proteins could represent potential therapeutic targets for regulating autoimmune diseases and limiting immune over-activation (1).

scFv-protamine fusion proteins have been constructed from antibodies to readily associate with activation-dependent conformational changes in LFA-1 (7, 10). Selectively targeting activated leukocytes can be used to down-regulate gene expression and cell proliferation. Engineered antibodies have been developed including AL-57, which preferentially binds to HA LFA-1 and not to low-affinity LFA-1, and TS1/22, which binds to LFA-1 in both the active and inactive conformation. This nonviral delivery approach using targeted fusion proteins could even be used to induce RNAi in primary leukocytes (11).

The conformational change of LFA-1 on activated immune cells serves as an attractive and specific target for therapeutic intervention. While blocking LFA-1 by itself has not been sufficient to suppress inflammation in certain disease models, siRNA delivery in conjunction with blocking LFA-1 antibodies may transduce only activated subsets of immune cells (4). A powerful therapeutic approach to study in vivo transfer of siRNA in combination with LFA-1 blocking antibodies could implement nanoparticles modified with a membrane translocation peptide for intracellular delivery. Nanoparticles that interact with activated leukocytes can be monitored to evaluate the accumulation of the siRNA and contrast agent with simultaneous magnetic resonance imaging (MRI).

This investigation of tumor-specific immune responses will help identify tumor-associated antigens and propose avenues to manipulate the immunoregulatory environment of the tumor. By constructing and comparing various platforms for drug delivery the efficacy of immune-mediated cancer therapies can be increased (12).

In this study, the Jin Lab proposes the UAN-DPA drug delivery platform equipped with engineered antibodies to selectively target integrin structures expressed on the surface of leukocytes. AL-57 and TS1/22 have been demonstrated as site-specific targeting antibodies. Single chain variable fragments (scFv) #1 and #17 contain the I domain sequence and have also been selected for conformation-specific activity. These antibodies containing a 6x His tag interact with the UAN-DPA nanoparticles by Ni-NTA chelation. Delivery and binding of UAN-DPA with and without antibody will be examined in yeast, leukemic T lymphocyte, and leukemic monocyte cell lines. The experiment’s cells, antibodies, and conditions are detailed in 45 trials (Table 1).

OBJECTIVE

The UAN-DPA nanoparticle platform has been engineered for targeted delivery to organs involved in acute and chronic inflammation, which includes the thymus, bone marrow, spleen, or lymph nodes. Examining conformation-specific activity of integrins will provide a method to probe endothelial interactions during lymphocyte migration in order to detect a trigger for inflammation. The UAN-DPA platform with antibody will be demonstrated as a drug delivery platform to suppress an inflammatory response by competitively binding with integrin ligands, such as ICAM-1. The nanoparticles conjugated with activation-specific antibodies can help elucidate mechanisms of autoimmune diseases (diabetes, multiple sclerosis (MS), rheumatoid arthritis (RA), and Crohn’s Disease), blood cancers (leukemia), sepsis, as well as cardiovascular diseases (atherosclerosis). By examining and interfering with the interactions of LFA-1 and Mac-1 with ICAM-1, the role of lymphocyte function and macrophage efflux to the lymphatics can be studied by controlling the activation of endothelial-leukocyte adhesion.

REFERENCES

1. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1

2. Effects of I Domain Deletion on the Function of the β2 Integrin Lymphocyte Function-associated Antigen-1

3. A Novel LFA-1 Activation Epitope Maps to the I Domain


Alexander Veach ajv25@cornell.edu