The reduced-charge melittin analogue MelP5 improves the transfection of non-viral DNA nanoparticles.

Melittin is a 26-amino acid amphiphilic alpha-helical peptide derived from honeybee venom. Prior studies have incorporated melittin into non-viral delivery systems to effect endosomal escape of DNA nanoparticles and improve transfection efficiency. Recent advances have led to the development of two newer melittin analogues, MelP5 and Macrolittin 70, with improved pore formation in lipid bilayers while possessing fewer positive charges relative to natural melittin. Consequently, MelP5 and Macrolittin 70 were conjugated through a disulfide bond to a DNA binding polyacridine peptide. The resulting peptide conjugates were used to prepare DNA nanoparticles to compare their relative endosomolytic potency by transfection of HepG2 cells. Melittin and MelP5 conjugates were equally potent at mediating in vitro gene transfer, whereas PEGylation of DNA nanoparticles revealed improved transfection with MelP5 relative to melittin. The results demonstrate the ability to substitute a potent, reduced-charge analogue of melittin to improve overall DNA nanoparticle biocompatibility needed for in vivo testing.

Gene transfection of primary mouse hepatocytes in 384-well plates.

We report the development of an improved in vitro transfection assay to test the efficiency of non-viral vector DNA nanoparticle transfection of primary hepatocytes. The protocol describes the isolation of viable hepatocytes from a mouse by collagenous perfusion. Primary mouse hepatocytes are plated in 384-well plates and cultured for 24 h prior to transfection with polyethylenimine (PEI) or peptide DNA nanoparticles. Luciferase expression is measured after 24 h following the addition of ONE-Glo substrate. The gene transfer assay for primary hepatocytes was optimized for cell plating number, DNA dose, and PEI to DNA ratio. The assay was applied to compare the expression mediated by mRNA relative to two plasmids possessing different promoters. The reported assay provides reliable in vitro expression results that allow direct comparison of the efficiency of different non-viral gene delivery vectors.

Fluorescent labeling of plasmid DNA for gene delivery: Implications of dye hydrophobicity on labeling efficiencies and nanoparticle size.

Covalent fluorescent labels are important tools for monitoring the in vitro and in vivo localization of plasmid DNA nanoparticles, but must meet several criteria including high DNA labeling efficiencies and minimal impact on nanoparticle size. We developed a novel fluorescent labeling strategy utilizing an aryl azide photolabel conjugated to a short cationic peptide to label plasmid DNA with Cyanine 5 and sulfo-Cyanine 5. Using a simple camera flash apparatus, photolabel-peptide-dyes can be conjugated to DNA in minutes with preservation of DNA structure and minimal dye photobleaching. The addition of two anionic sulfonates to the Cyanine 5 core greatly improved labeling efficiencies from ~13 to ~53% and mitigated PEGylated polyacridine peptide-DNA nanoparticle size increases over a range of labeling densities. Comparison of our sulfo-Cyanine 5 peptide label to the Mirus Bio Label IT-Cy5 kit revealed that while both did not affect nanoparticle sizes appreciably, labeling efficiencies with our conjugate were higher, possibly due to the higher positive charge density on the peptide linker. The results from this work provide important considerations for choosing fluorophore tags to track DNA nanoparticles.

Heat-shrinking DNA nanoparticles for in vivo gene delivery.

The particle size of a PEG-peptide DNA nanoparticle is a key determinant of biodistribution following i.v. dosing. DNA nanoparticles of <100 nm in diameter are sufficiently small to cross through fenestrated endothelial cells to target hepatocytes in the liver. In addition, DNA nanoparticles must be close to charge-neutral to avoid recognition and binding to scavenger receptors found on Kupffer cells and endothelial cells in the liver. In the present study, we demonstrate an approach to heat shrink DNA nanoparticles to reduce their size to <100 nm to target hepatocytes. An optimized protocol heated plasmid DNA at 100 °C for 10 min resulting in partial denaturation. The immediate addition of a polyacridine PEG-peptide followed by cooling to room temperature resulted in heat-shrunken DNA nanoparticles that were ~70 nm in diameter compared with 170 nm when heating was omitted. Heat shrinking resulted in the conversion of supercoiled DNA into open circular to remove strain during compaction. Heat-shrunken DNA nanoparticles were stable to freeze-drying and reconstitution in saline. Hydrodynamic dosing established that 70 nm heat-shrunken DNA nanoparticles efficiently expressed luciferase in mouse liver. Biodistribution studies revealed that 70 nm DNA nanoparticles are rapidly and transiently taken up by liver whereas 170 nm DNA nanoparticles avoid liver uptake due to their larger size. The results provide a new approach to decrease the size of polyacridine PEG-peptide DNA nanoparticles to allow penetration of the fenestrated endothelium of the liver for the purpose of transfecting hepatocytes in vivo.

Metabolically stabilized double-stranded mRNA polyplexes.

The metabolic instability of mRNA currently limits its utility for gene therapy. Compared to plasmid DNA, mRNA is significantly more susceptible to digestion by RNase in the circulation following systemic dosing. To increase mRNA metabolic stability, we hybridized a complementary reverse mRNA with forward mRNA to generate double-stranded mRNA (dsmRNA). RNase A digestion of dsmRNA established a 3000-fold improved metabolic stability compared to single-stranded mRNA (ssmRNA). Formulation of a dsmRNA polyplex using a PEG-peptide further improved the stability by 3000-fold. Hydrodynamic dosing and quantitative bioluminescence imaging of luciferase expression in the liver of mice established the potent transfection efficiency of dsmRNA and dsmRNA polyplexes. However, hybridization of the reverse mRNA against the 5' and 3' UTR of forward mRNA resulted in UTR denaturation and a tenfold loss in expression. Repeat dosing of dsmRNA polyplexes produced an equivalent transient expression, suggesting the lack of an immune response in mice. Co-administration of excess uncapped dsmRNA with a dsmRNA polyplex failed to knock down expression, suggesting that dsmRNA is not a Dicer substrate. Maximal circulatory stability was achieved using a fully complementary dsmRNA polyplex. The results established dsmRNA as a novel metabolically stable and transfection-competent form of mRNA.

PEG-Peptide Inhibition of Scavenger Receptor Uptake of Nanoparticles by the Liver.

PEGylated polylysine peptides represent a new class of scavenger receptor inhibitors that may find utility at inhibiting DNA nanoparticle uptake by Kupffer cells in the liver. PEG-peptides inhibit scavenger receptors in the liver by a novel mechanism involving in situ formation of albumin nanoparticles. The present study developed a new in vivo assay used to explore the structure-activity-relationships of PEG-peptides to find potent scavenger receptor inhibitors. Radio-iodinated PEG-peptides were dosed i.v. in mice and shown to saturate liver uptake in a dose-dependent fashion. The inhibition potency (IC50) was dependent on both the length of a polylysine repeat and PEG molecular weight. PEG30kda-Cys-Tyr-Lys25 was confirmed to be a high molecular weight (33.5 kDa) scavenger receptor inhibitor with an IC50 of 18 µM. Incorporation of multiple Leu residues improved potency, allowing a decrease in PEG MW and Lys repeat, resulting in PEG5kda-Cys-Tyr-Lys-(Leu-Lys4)3-Leu-Lys that inhibited scavenger receptors with an IC50 = 20 µM. A further decrease in PEG MW to 2 kDa increased potency further, resulting in a low molecular weight (4403 g/mol) PEG-peptide with an IC50 of 3 µM. Optimized low molecular weight PEG-peptides also demonstrated potency when inhibiting the uptake of radio-iodinated DNA nanoparticles by the liver. This study demonstrates an approach to discover low molecular weight PEG-peptides that serve as potent scavenger receptor inhibitors to block nanoparticle uptake by the liver.

Structure-Activity Relationship of PEGylated Polylysine Peptides as Scavenger Receptor Inhibitors for Non-Viral Gene Delivery.

PEGylated polylysine peptides of the general structure PEG30 kDa-Cys-Trp-LysN (N = 10 to 30) were used to form fully condensed plasmid DNA (pGL3) polyplexes at a ratio of 1 nmol of peptide per µg of DNA (ranging from N:P 3:1 to 10:1 depending on Lys repeat). Co-administration of 5 to 80 nmols of excess PEG-peptide with fully formed polyplexes inhibited the liver uptake of (125)I-pGL3-polyplexes. The percent inhibition was dependent on the PEG-peptide dose and was saturable, consistent with inhibition of scavenger receptors. The scavenger receptor inhibition potency of PEG-peptides was dependent on the length of the Lys repeat, which increased 10-fold when comparing PEG30 kDa-Cys-Trp-Lys10 (IC50 of 20.2 µM) with PEG30 kDa-Cys-Trp-Lys25 (IC50 of 2.1 µM). We hypothesize that PEG-peptides inhibit scavenger receptors by spontaneously forming small 40 to 60 nm albumin nanoparticles that bind to and saturate the receptor. Scavenger receptor inhibition delayed the metabolism of pGL3-polyplexes, resulting in efficient gene expression in liver hepatocytes following delayed hydrodynamic dosing. PEG-peptides represent a new class of scavenger inhibitors that will likely have broad utility in blocking unwanted liver uptake and metabolism of a variety of nanoparticles.

"Evolving nanoparticle gene delivery vectors for the liver: What has been learned in 30 years".

Nonviral gene delivery to the liver has been under evolution for nearly 30years. Early demonstrations established relatively simple nonviral vectors could mediate gene expression in HepG2 cells which understandably led to speculation that these same vectors would be immediately successful at transfecting primary hepatocytes in vivo. However, it was soon recognized that the properties of a nonviral vector resulting in efficient transfection in vitro were uncorrelated with those needed to achieve efficient nonviral transfection in vivo. The discovery of major barriers to liver gene transfer has set the field on a course to design biocompatible vectors that demonstrate increased DNA stability in the circulation with correlating expression in liver. The improved understanding of what limits nonviral vector gene transfer efficiency in vivo has resulted in more sophisticated, low molecular weight vectors that allow systematic optimization of nanoparticle size, charge and ligand presentation. While the field has evolved DNA nanoparticles that are stable in the circulation, target hepatocytes, and deliver DNA to the cytosol, breaching the nucleus remains the last major barrier to a fully successful nonviral gene transfer system for the liver. The lessons learned along the way are fundamentally important to the design of all systemically delivered nanoparticle nonviral gene delivery systems.

Miniaturization of gene transfection assays in 384- and 1536-well microplates.

The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)-DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35µl and in 1536-well plates in a total assay volume of 8µl. A luciferase assay performed in 384-well plates produced a Z' score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250cellsperwell in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.

Nanoparticle ligand presentation for targeting solid tumors.

Among the many scientific advances to come from the study of nanoscience, the development of ligand-targeted nanoparticles to eliminate solid tumors is predicted to have a major impact on human health. There are many reports describing novel designs and testing of targeted nanoparticles to treat cancer. While the principles of the technology are well demonstrated in controlled lab experiments, there are still many hurdles to overcome for the science to mature into truly efficacious targeted nanoparticles that join the arsenal of agents currently used to treat cancer in humans. One of these hurdles is overcoming unwanted biodistribution to the liver while maximizing delivery to the tumor. This almost certainly requires advances in both nanoparticle stealth technology and targeting. Currently, it continues to be a challenge to control the loading of ligands onto polyethylene glycol (PEG) to achieve maximal targeting. Nanoparticle cellular uptake and subcellular targeting of genes and siRNA also remain a challenge. This review examines the types of ligands that have been most often used to target nanoparticles to solid tumors. As the science matures over the coming decade, careful control over ligand presentation on nanoparticles of precise size, shape, and charge will likely play a major role in achieving success.

Iterative Reducible Ligation to form Homogeneous Penicillamine Cross-linked Polypeptides.

The syntheses of homogeneous penicillamine disulfide cross-linked polypeptides are reported. Dodecapeptides containing N-terminal, C-terminal, or N- and C-terminal Pen were serially ligated into 36 amino acid polypeptides linked through Cys-Pen, Pen-Cys or Pen-Pen disulfide bonds. Critical to the syntheses was the incorporation of thiazolidine masked Cys and Pen as the N-terminal residues and selective hydrolysis with silver trifluoromethanesulfonate in acidic aqueous conditions to generate a free thiol for subsequent ligation. This approach allows the synthesis of homogeneous disulfide cross-linked polypeptides that have different reductive stabilities and have application in gene delivery by undergoing a tempered reductive triggered release of DNA.

PEG length and chemical linkage controls polyacridine peptide DNA polyplex pharmacokinetics, biodistribution, metabolic stability and in vivo gene expression.

The pharmacokinetics (PK), biodistribution and metabolism of non-viral gene delivery systems administered systemically are directly related to in vivo efficacy. The magnitude of luciferase expression in the liver of mice following a tail vein dose of a polyplex, composed of 1 µg of pGL3 in complex with a polyethylene glycol (PEG) polyacridine peptide, followed by a delayed hydrodynamic (HD) stimulation (1-9 h), depends on the HD stimulation delay time and the structure of the polyacridine peptide. As demonstrated in the present study, the PEG length and the type of chemical linkage joining PEG to the polyacridine peptide dramatically influence the in vivo gene transfer efficiency. To understand how PEG length, linkage and location influence gene transfer efficiency, detailed PK, biodistribution and HD-stimulated gene expression experiments were performed on polyplexes prepared with an optimized polyacridine peptide modified through a single terminal Cys or Pen (penicillamine) with a PEG chain of average length of 2, 5, 10, 20, or 30 kDa. The chemical linkage was examined by attaching PEG(5 kDa) to the polyacridine peptide through a thiol-thiol (SS), thiol-maleimide (SM), thiol-vinylsulfone (SV), thiol-acetamide (SA), penicillamine-thiol-maleimide (PM) or penicillamine-thiol-thiol (PS). The influence of PEG location was analyzed by attaching PEG(5 kDa) to the polyacridine peptide through a C-terminal, N-terminal, or a middle Cys residue. The results established rapid metabolism of polyplexes containing SV and SA chemical linkages that leads to a decreased polyplex PK half-life and a complete loss of HD-stimulated gene expression at delay times of 5 h. Conversely, polyplexes containing PM, PS, and SM chemical linkages were metabolically stable, allowing robust HD-stimulated expression at delay times up to 5h post-polyplex administration. The location of PEG(5 kDa) within the polyacridine peptide exerted only a minor influence on the gene transfer of polyplexes. However, varying the PEG length from 2, 5, 10, 20, or 30 kDa dramatically altered polyplex biodistribution, with a 30 kDa PEG maximally blocking liver uptake to 13% of dose, while maintaining the ability to mediate HD-stimulated gene expression. The combination of results establishes important relationships between PEGylated polyacridine peptide structure, physical properties, in vivo metabolism, PK and biodistribution resulting in an optimal PEG length and linkage that leads to a robust HD-stimulated gene expression in mice.

A Convergent Synthesis of Homogeneous Reducible Polypeptides.

The convergent syntheses of homogeneous disulfide cross-linked polypeptides are reported. Reducible polypeptides were synthesized containing four and eight dodecapeptides in two and three linear conjugation steps. Critical for the convergent methodology was the use of orthogonally protected cysteines as either acetamidomethyl (Acm) or Fmoc-thiazolidine (Thz). Both groups could be selectively deprotected with silver trifluoromethanesulfonate in the presence of internal disulfide bonds using TFA and aqueous conditions, respectively. This approach allows for large, reducible polypeptides to be synthesized in efficient yields and minimizes the number of conjugation steps, allowing the development and optimization of gene delivery polypeptides containing multiple peptide components necessary to overcome the numerous in vivo barriers for efficacious gene delivery.

Synthesis of homogenous disulfide cross-linked polypeptides by iterative reducible ligation.

A new method of directed solution phase synthesis of polypeptides linked through iterative formation of disulfide bonds is reported. Four dodecapeptides were successfully ligated into a single 48 amino acid polypeptide using an N-terminal Fmoc-thiazolidine and a novel acidic silver trifluoromethanesulfonate thiazolidine hydrolysis to achieve efficient ligation in the presence of internal disulfide bonds. The approach allows the synthesis of homogeneous disulfide cross-linked polypeptides that have application in gene delivery by undergoing a reductively triggered release of DNA.

N-glycan targeted gene delivery to the dendritic cell SIGN receptor.

A novel nonviral gene delivery vector composed of a high-mannose N-glycan conjugated to a polyacridine peptide was prepared. The glycopeptide was designed to bind to plasmid DNA by a combination of polyintercalation and ionic binding, and to the DC-SIGN (dendritic cell-specific intracellular adhesion molecule-3 grabbing nonintegrin) receptor expressed on CHO cells by recognition of the high-mannose N-glycan. The glycopeptide conjugate was prepared by purification of a high-mannose N-glycan from affinity fractionated soybean agglutinin (SBA). The SBA was proteolyzed to release the N-glycan which was then modified on its N-terminus with Tyr and a propionate maleimide. A DNA binding polyacridine peptide, Cys-(Acr-Lys)(4), was prepared by solid-phase peptide synthesis using Fmoc-Lys(Acr), then conjugated to the maleimide on the N-glycan to produce a glycopeptide. The glycopeptide bound to DNA with high affinity as determined by fluorophore displacement assay and DNA band shift on agarose gel. When bound to Cy5 labeled DNA, the glycopeptide mediated specific uptake in DC-SIGN CHO (+) cells as determined by FACS analysis. In vitro gene transfer studies established that the glycopeptide increased the specificity of gene transfer in DC-SIGN CHO (+) cells 100-fold relative to CHO (-) cells. These studies suggest that a high-mannose N-glycan conjugated to a polyacridine peptide may also facilitate receptor mediated gene delivery in dendritic cells and thereby find utility in the delivery of DNA vaccines.

In silico prediction of the 3D structure of trimeric asialoglycoprotein receptor bound to triantennary oligosaccharide.

In this study, we present a general-purpose methodology for deriving the three-dimensional (3D) arrangement of multivalent transmembrane complexes in the presence of their ligands. Specifically, we predict the most likely families of structures of the experimentally intractable trimeric asialoglycoprotein receptor (ASGP-R), which consists of human hepatic subunits (two subunits of H1 and one subunit of H2), bound to a triantennary oligosaccharide (TA). Because of the complex nature of this multivalent type-II transmembrane hetero-oligomeric receptor, structural studies have to date been unable to provide the 3D arrangement of these subunits. Our approach is based on using the three-pronged ligand of ASGP-R as a computational probe to derive the 3D conformation of the complex and then using this information to predict the relative arrangement of the protein subunits on the cell surface. Because of interprotein subunit clashes, only a few families of TA conformers are compatible with the trimeric structure of ASGP-R. We find that TA displays significant flexibility, matching that detected previously in FRET experiments, and that the predicted complexes derived from the viable TA structures are asymmetric. Significant variation exists with respect to TA presentation to the receptor complex. In summary, this study provides detailed information about TA-ASGP-R interactions and the symmetry of the complex.

Discovery of metabolically stabilized electronegative polyacridine-PEG peptide DNA open polyplexes.

Cationic condensing peptides and polymers bind electrostatically to DNA to form cationic polyplexes. While many cationic polyplexes are able to achieve in vitro transfection mediated through electrostatic interactions, few have been able to mediate gene transfer in vivo. The present study describes the development and testing of polyacridine PEG-peptides that bind to plasmid DNA by intercalation resulting in electronegative open polyplex DNA. Polyacridine PEG-peptides were prepared by chemically conjugating 6-(9-acridinylamino) hexanoic acid onto side chains of Lys in PEG-Cys-Trp-(Lys)(3, 4, or 5). The resulting PEG-Cys-Trp-(Lys-(Acr))(3, 4, or 5) peptides bound tightly to DNA by polyintercalation, rather than electrostatic binding. Unlike polycationic polyplexes, polyacridine PEG-peptide polyplexes were anionic and open coiled, as revealed by zeta potential and atomic force microscopy. PEG-Cys-Trp-(Lys-(Acr))(5) showed the highest DNA binding affinity and the greatest ability to protect DNA from metabolism by DNase. Polyacridine PEG-peptide DNA open polyplexes were dosed intramuscularly and electroporated in mice to demonstrate their functional activity in gene transfer. These results establish polyacridine PEG-peptide DNA open polyplexes as a novel gene delivery method for in vivo use.

Synthesis and in vitro testing of new potent polyacridine-melittin gene delivery peptides.

The combination of a polyacridine peptide modified with a melittin fusogenic peptide results in a potent gene transfer agent. Polyacridine peptides of the general formula (Acr-X)(n)-Cys were prepared by solid-phase peptide synthesis, where Acr is Lys modified on its epsilon-amine with acridine, X is Arg, Leu, or Lys and n is 2, 3, or 4 repeats. The Cys residue was modified by either a maleimide-melittin or a thiolpyridine-Cys-melittin fusogenic peptide resulting in reducible or non-reducible polyacridine-melittin peptides. Hemolysis assays established that polyacridine-melittin peptides retained their membrane lytic potency relative to melittin at pH 7.4 and 5. When combined with plasmid DNA, the membrane lytic potency of polyacridine-melittin peptides was neutralized. Gene transfer experiments in multiple cell lines established that polyacridine-melittin peptides mediate expression as efficiently as PEI. The expression was very dependent upon a disulfide bond linking polyacridine to melittin. The gene transfer was most efficient when X is Arg and n is 3 or 4 repeats. These studies establish polyacridine peptides as a novel DNA binding anchor peptide.

Quantitative in vivo imaging of non-viral-mediated gene expression and RNAi-mediated knockdown.

Bioluminescent imaging (BLI) coupled with hydrodynamic (HD) dosing of luciferase-expressing plasmid DNA (pDNA) has proven to be a powerful method for quantitatively benchmarking non-viral gene expression in the liver. The expression of luciferase or knockdown of luciferase by RNA interference (RNAi) in the liver is quantifiable over five-orders of magnitude in living mice. The photon emission data derived from BLI can be converted to the absolute amount of luciferase expression by comparison with a standard curve developed using luciferase as a primary standard. Quantitative BLI is also applicable to luciferase expression in other tissues, such as skeletal muscle, following intramuscular (IM) dosing and electroporation (EP) of pDNA. The primary advantages of using quantitative BLI in mouse liver and muscle are the sensitivity of the assay, the speed and ease of making measurements, the precision and linearity of the dose-response curves, and the ability to conduct serial sampling of gene expression over many days or months while eliminating the need to euthanize animals.

Engineered nanoscaled polyplex gene delivery systems.

Improving the transfection efficiencies of nonviral gene delivery requires properly engineered nanoscaled delivery carriers that can overcome the multiple barriers associated with the delivery of oligonucleotides from the site of administration to the nucleus or cytoplasm of the target cell. This article reviews the current advantages and limitation of polyplex nonviral delivery systems, including the apparent barriers that limit gene expression efficiency compared to physical methods such as hydrodynamic dosing and electroporation. An emphasis is placed on engineered nanoscaled polyplexes (NSPs) of modular design that both self-assemble and systematically disassemble at the desired stage of delivery. It is suggested that NSPs of increasingly sophisticated designs are necessary to improve the efficiency of the rate limiting steps in gene delivery.