Nonviral Vectors for Gene Delivery

The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,L-lactide-co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry 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 (~200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) 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. On the other hand, a more simple method to synthesize 50-200 nm complexes capable of high transfection efficiency or high gene knockdown was also explored. Positively charged CPPs were complexed with pDNA or siRNA, which resulted in `loose' (~1 micron) particles. These were then condensed into small nanoparticles by using calcium, which formed "soft" crosslinks by interacting with both phosphates on nucleic acids and amines on CPPs. An optimal amount of CaCl2 produced stable, ~100 nm complexes that exhibited higher transfection efficiency and gene silencing than PEI polyplexes. CPPs also displayed negligible cytotoxicity up to 5 mg/mL. Biophysical studies of the pDNA structure within complexes suggested that pDNA within CPP complexes (condensed with calcium) had similar structure, but enhanced thermal stability compared to PEI complexes. Thus, CPP complexes emerged as simple, attractive candidates for future studies on nonviral gene delivery in vivo

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 pDNA 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 CaCl2 produced small and stable nanoparticles leading to gene expression levels higher than observed for control PEI 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

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 (~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

“Soft” Calcium Crosslinks Enable Highly Efficient Gene Transfection Using TAT Peptide

The original publication is available at www.springerlink.comPurpose Typically, low molecular weight cationic peptides or polymers exhibit poor transfection efficiency due to an inability to condense plasmid DNA into small nanoparticles. Here, efficient gene delivery was attained using TAT/pDNA complexes containing calcium crosslinks. Methods Electrostatic complexes of pDNA with TAT or PEI were studied with increasing calcium concentration. Gel electrophoresis was used to determine DNA condensation. The morphology of the complexes was probed by transmission electron microscopy. Transfection efficiency was assessed using a luciferase reporter plasmid. The accessibility of phosphate and amine groups within complexes was evaluated to determine the effect of calcium on structure. Results TAT/pDNA complexes were condensed into small, 50–100 nm particles by optimizing the concentration of calcium. Complexes optimized for small size also exhibited higher transfection efficiency than PEI polyplexes in A549 cells. TAT and TAT complexes displayed negligible cytotoxicity up to 5 mg/mL, while PEI exhibited high cytotoxicity, as expected. Probing the TAT-Ca/pDNA structure suggested that calcium interacted with both phosphate and amine groups to compact the complexes; however, these “soft” crosslinks could be competitively disrupted to facilitate DNA release. Conclusion Small and stable TAT-Ca/pDNA complexes were obtained via “soft” calcium crosslinks leading to sustained gene expression levels higher than observed for control PEI gene vectors. TAT-Ca/pDNA complexes were stable, maintaining particle size and transfection efficiency even in the presence of 10% of FBS. TAT-Ca complexes offer an effective vehicle offering potential for translatable gene delivery

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

Targeted gene delivery using non-viral 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

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 at 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 may be optimized to improve cellular uptake. These results compliment other studies suggesting surface density of the targeting moiety on the NP surface should be considered to enhance the effect of ligands employed for cell targeting

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

Interaction of leukocyte function associated antigen-1 (LFA-1) on T-lymphoctytes 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 indentified 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

Synthesis of novel conjugates of a saccharide, amino acids, nucleobase and the evaluation of their cell compatibility

This article reports the synthesis of a novel type of conjugate of three fundamental biological build blocks (i.e., saccharide, amino acids, and nucleobase) and their cell compatibility. The facile synthesis starts with the synthesis of nucleobase and saccharide derivatives, then uses solid-phase peptide synthesis (SPPS) to build the peptide segment (Phe-Arg-Gly-Asp or naphthAla-Phe-Arg-Gly-Asp with fully protected groups), and later, an amidation reaction in liquid phase connects these three parts together. The overall yield of these multiple step synthesis is about 34%. Besides exhibiting excellent solubility, these conjugates of saccharide–amino acids–nucleobase (SAN), like the previously reported conjugates of nucleobase–amino acids–saccharide (NAS) and nucleobase–saccharide–amino acids (NSA), are mammalian cell compatible

Synthesis and evaluation of the biostability and cell compatibility of novel conjugates of nucleobase, peptidic epitope, and saccharide

This article reports the synthesis of a new class of conjugates containing a nucleobase, a peptidic epitope, and a saccharide and the evalution of their gelation, biostability, and cell compatibility. We demonstrate a facile synthetic process, based on solid-phase peptide synthesis of nucleopeptides, to connect a saccharide with the nucleopeptides for producing the target conjugates. All the conjugates themselves (1–8) display excellent solubility in water without forming hydrogels. However, a mixture of 5 and 8 self-assembles to form nanofibers and results in a supramolecular hydrogel. The proteolytic stabilities of the conjugates depend on the functional peptidic epitopes. We found that TTPV is proteolytic resistant and LGFNI is susceptible to proteolysis. In addition, all the conjugates are compatible to the mammalian cells tested