Abdulgader Baoum

ICAM-1 Targeting of Doxorubicin-Loaded PLGA Nanoparticles to Lung Epithelial Cells

Interaction of leukocyte function associated antigen-1 (LFA-1) on T-lymphocytes and intercellular adhesion molecule-1 (ICAM-1) on epithelial cells controls leukocyte adhesion, spreading, and extravasation. This process plays an important role in leukocyte recruitment to a specific site of inflammation and has been identified as a biomarker for certain types of carcinomas. Cyclo-(1,12)-PenITDGEATDSGC (cLABL) has been shown to inhibit LFA-1 and ICAM-1 interaction via binding to ICAM-1. In addition, cLABL has been shown to internalize after binding ICAM-1. The possibility of using cLABL conjugated nanoparticles (cLABL-NP) as a targeted and controlled release drug delivery system has been investigated in this study. The cLABL peptide was conjugated to a modified Pluronic surfactant on poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles. The cLABL-NP showed more rapid cellular uptake by A549 lung epithelial cells compared to nanoparticles without peptide. The specificity of ICAM-1-mediated internalization was confirmed by blocking the uptake of cLABL-NP to ICAM-1 using free cLABL peptide to block the binding of cLABL-NP to ICAM-1 on the cell surface. Cell studies suggested that cLABL-NPs targeted encapsulated doxorubicin to ICAM-1 expressing cells. Cytotoxicity assay confirmed the activity of the drug incorporated in nanoparticles. Sustained release of doxorubicin afforded by PLGA nanoparticles may enable cLABL-NP as a targeted, controlled release drug delivery system.

Controlling ligand surface density optimizes nanoparticle binding to ICAM-1

During infection, pathogens utilize surface receptors to gain entry into intracellular compartments. Multiple receptor-ligand interactions that lead to pathogen internalization have been identified and the importance of multivalent ligand binding as a means to facilitate internalization has emerged. The effect of ligand density, however, is less well known. In this study, ligand density was examined using poly(DL-lactic-co-glycolic acid) nanoparticles (PLGA NPs). A cyclic peptide, cLABL, was used as a targeting moiety, as it is a known ligand for intercellular cell adhesion molecule-1 (ICAM-1). To modulate the number of reactive sites on the surface of PLGA NPs, modified Pluronic with carboxyl groups and Pluronic with hydroxyl groups were combined in different ratios and the particle properties were examined. Utilizing a surfactant mixture directly affected the particle charge and the number of reactive sites for cLABL conjugation. The surface density of cLABL peptide increased as the relative amount of reactive Pluronic was increased. Studies using carcinomic human alveolar basal epithelial cells (A549) showed that cLABL density might be optimized to improve cellular uptake. These results complement other studies, suggesting that surface density of the targeting moiety on the NP surface should be considered to enhance the effect of ligands used for cell targeting.

Intratracheal Administration of a Nanoparticle-Based Therapy with the Angiotensin II Type 2 Receptor Gene Attenuates Lung Cancer Growth

Targeted gene delivery, transfection efficiency, and toxicity concerns remain a challenge for effective gene therapy. In this study, we dimerized the HIV-1 TAT peptide and formulated a nanoparticle vector (dTAT NP) to leverage the efficiency of this cell-penetrating strategy for tumor-targeted gene delivery in the setting of intratracheal administration. Expression efficiency for dTAT NP-encapsulated luciferase or angiotensin II type 2 receptor (AT2R) plasmid DNA (pDNA) was evaluated in Lewis lung carcinoma (LLC) cells cultured in vitro or in vivo in orthotopic tumor grafts in syngeneic mice. In cell culture, dTAT NP was an effective pDNA transfection vector with negligible cytotoxicity. Transfection efficiency was further increased by addition of calcium and glucose to dTAT/pDNA NP. In orthotopic tumor grafts, immunohistochemical analysis confirmed that dTAT NP successfully delivered pDNA to the tumor, where it was expressed primarily in tumor cells along with the bronchial epithelium. Notably, gene expression in tumor tissues persisted at least 14 days after intratracheal administration. Moreover, bolus administration of dTAT NP-encapsulated AT2R or TNF-related apoptosis-inducing ligand (TRAIL) pDNA markedly attenuated tumor growth. Taken together, our findings offer a preclinical proof-of-concept for a novel gene delivery system that offers an effective intratracheal strategy for administering lung cancer gene therapy.

Calcium condensed LABL-TAT complexes effectively target gene delivery to ICAM-1 expressing cells.

Targeted gene delivery using nonviral vectors is a highly touted scheme to reduce the potential for toxic or immunological side effects by reducing dose. In previous reports, TAT polyplexes with DNA have shown relatively poor gene delivery. The transfection efficiency has been enhanced by condensing TAT/DNA complexes to a small particle size using calcium. To explore the targetability of these condensed TAT complexes, LABL peptide targeting intercellular cell-adhesion molecule-1 (ICAM-1) was conjugated to TAT peptide using a polyethylene glycol (PEG) spacer. PEGylation reduced the transfection efficiency of TAT, but TAT complexes targeting ICAM-1 expressing cells regained much of the lost transfection efficiency. Targeted block peptides properly formulated with calcium offer promise for gene delivery to ICAM-1 expressing cells at sites of injury or inflammation.

Cationic surface modification of PLG nanoparticles offers sustained gene delivery to pulmonary epithelial cells

Biodegradable polymeric nanoparticles are currently being explored as a nonviral gene delivery system; however, many obstacles impede the translation of these nanomaterials. For example, nanoparticles delivered systemically are inherently prone to adsorbing serum proteins and agglomerating as a result of their large surface/volume ratio. What is desired is a simple procedure to prepare nanoparticles that may be delivered locally and exhibit minimal toxicity while improving entry into cells for effectively delivering DNA. The objective of this study was to optimize the formulation of poly(D,L-lactide-co-glycolide) (PLG) nanoparticles for gene delivery performance to a model of the pulmonary epithelium. Using a simple solvent diffusion technique, the chemistry of the particle surface was varied by using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (approximately 200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for 2 weeks. In A549 alveolar lung epithelial cells, high levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least 2 weeks. In contrast, PEI gene expression ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium.

Calcium Condensation of DNA Complexed with Cell-Penetrating Peptides Offers Efficient, Noncytotoxic Gene Delivery

Drug delivery strategies using cell-penetrating peptides (CPPs) have been widely explored to improve the intracellular delivery of a large number of cargo molecules. Electrostatic complexation of plasmid DNA using CPPs has been less explored due to the relatively large complexes formed and the low levels of gene expression achieved when using these low-molecular-weight polycations as DNA condensing agents. Here, condensing nascent CPP polyplexes using CaCl(2) produced small and stable nanoparticles leading to gene expression levels higher than observed for control polyethylenimine gene vectors. This simple formulation approach showed negligible cytotoxicity in A549 lung epithelial cells and maintained particle size and transfection efficiency even in the presence of serum.