Huafeng Fang

Shape effects of nanoparticles conjugated with cell-penetrating peptides (HIV Tat PTD) on CHO cell uptake

In order to probe the nanoparticle shape/size effect on cellular uptake, a spherical and two cylindrical nanoparticles, whose lengths were distinctively varied, were constructed by the selective cross-linking of amphiphilic block copolymer micelles. Herein, we demonstrate that, when the nanoparticles were functionalized with the protein transduction domain of human immunodeficiency virus type 1 Tat protein (HIV Tat PTD), the smaller, spherical nanoparticles had a higher rate of cell entry into Chinese hamster ovary (CHO) cells than did the larger, cylindrical nanoparticles. It was also found that nanoparticles were released after internalization and that the rate of cell exit was dependent on both the nanoparticle shape and the amount of surface-bound PTD.

Cationic shell-crosslinked knedel-like nanoparticles for highly efficient gene and oligonucleotide transfection of mammalian cells.

In this work, a robust synthetic nanostructure was designed for the effective packaging of DNA and it was shown to be an efficient agent for cell transfection. An amphiphilic block copolymer, poly(acrylamidoethylamine)(128)-b-polystyrene(40) (PAEA(128)-b-PS(40)), was synthesized, micellized in water and shell-crosslinked using a diacid-derivatized crosslinker, to give cationic shell-crosslinked nanoparticles (cSCKs) with a mean hydrodynamic diameter of 14 +/- 2 nm. A series of discrete complexes of the cSCKs with plasmid DNA (pDNA) was able to be formed over a broad range of polymer amine:pDNA phosphate ratios (N/P ratio), 2:1-20:1. The sizes of the complexes and their ability to fully bind the pDNA were dependent upon the N/P ratio, as characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and gel retardation assay. A luciferase activity assay and EGFP expression were used to evaluate intracellular delivery of a splice-correcting phosphorothioate and genetic material, respectively, by the cSCKs, which indicated that an N/P ratio of 6:1 gave the highest transfection. It was shown by both luciferase activity assay (48 h) and EGFP transfection data that high transfection efficiencies were achieved for HeLa cells transfected by cSCK/CCUCUUACCUCAGUUACA and cSCK/pEGFP-N1 plasmid, respectively. The cSCK/pEGFP-N1 plasmid transfection efficiency of 27% far exceeded the performance of Polyfect (PAMAM dendrimers), which achieved only 12% transfection efficiency, under the same conditions. Cytotoxicities for the cSCKs were evaluated for HeLa and CHO cells.

Cationic shell-cross-linked knedel-like (cSCK) nanoparticles for highly efficient PNA delivery.

Peptide nucleic acids have a number of features that make them an ideal platform for the development of in vitro biological probes and tools. Unfortunately, their inability to pass through membranes has limited their in vivo application as diagnostic and therapeutic agents. Herein, we describe the development of cationic shell-cross-linked knedel-like (cSCK) nanoparticles as highly efficient vehicles for the delivery of PNAs into cells, either through electrostatic complexation with a PNA * ODN hybrid, or through a bioreductively cleavable disulfide linkage to a PNA. These delivery systems are better than the standard Lipofectamine/ODN-mediated method and much better than the Arg(g)-mediated method for PNA delivery in HeLa cells, showing lower toxicity and higher bioactivity. The cSCKs were also found to facilitate both endocytosis and endosomal release of the PNAs, while themselves remaining trapped in the endosomes.

Benzaldehyde-functionalized polymer vesicles.

Polymer vesicles with diameters of ca. 100-600 nm and bearing benzaldehyde functionalities within the vesicular walls were constructed through self-assembly of an amphiphilic block copolymer PEO(45)-b-PVBA(26) in water. The reactivity of the benzaldehyde functionalities was verified by cross-linking the polymersomes and also by a one-pot cross-linking and functionalization approach to further render the vesicles fluorescent, each via reductive amination. In vitro studies found these labeled nanostructures to undergo cell association.

Photocrosslinking of human telomeric G-quadruplex loops by anti cyclobutane thymine dimer formation

The unusual structural forms of telomere DNA, which protect the ends of chromosomes during replication, may render it vulnerable to unprecedented photodamage, possibly involving nonadjacent bases that are made proximate by folding. The G-quadruplex for the human telomere sequence consisting of a repeating d(TTAGGG) is one unusual form. Tel22, d[AGGG(TTAGGG)3], forms a basket structure in the presence of Na+ and may form multiple equilibrating structures in the presence of K+ with hybrid-type structures predominating. UVB irradiation of d[AGGG(TTAGGG)3] in the presence of Na+ results in a cis,syn thymine dimer between two adjacent Ts in a TTA loop and a mixture of nonadjacent anti thymine dimers between various loops. Irradiation in the presence of K+, however, produces, in addition to these same products, a large amount of specific anti thymine dimers formed between either T in loop 1 and the central T in loop 3. These latter species were not observed in the presence of Na+. Interloop-specific anti thymine dimers are incompatible with hybrid-type structures, but could arise from a chair or basket-type structure or from triplex intermediates involved in interconverting these structures. If these unique nonadjacent anti thymine dimer photoproducts also form in vivo, they would constitute a previously unrecognized type of DNA photodamage that may interfere with telomere replication and present a unique challenge to DNA repair. Furthermore, these unusual anti photoproducts may be used to establish the presence of G-quadruplex or quadruplex-like structures in vivo.

Well-defined Cationic Shell Crosslinked Nanoparticles for Efficient Delivery of DNA or Peptide Nucleic Acids

This mini-review highlights developments that have been made over the past year to advance the construction of well-defined nanoscale objects to serve as devices for cell transfection. Design of the nanoscale objects originated from biomimicry concepts, using histones as the model, to afford cationic shell crosslinked knedel-like (cSCK) nanoparticles. Packaging and delivery of plasmid DNA, oligonucleotides, and peptide nucleic acids were studied by dynamic light scattering, transmission electron microscopy, gel electrophoresis, biological activity assays, RT-PCR measurements, flow cytometry, and confocal fluorescence microscopy. With the demonstration of more efficient cell transfection in vitro than that achieved using commercially-available transfection agents, together with the other features offered by the robust nanostructural framework, work continues toward the application of these cSCKs for in vivo molecular recognition of genetic material, for imaging and therapy targeted specifically to pulmonary injury and disease.

Phospholipid Conjugate for Intracellular Delivery of Peptide Nucleic Acids

Peptide nucleic acids (PNAs) have a number of attractive features that have made them an ideal choice for antisense and antigene-based tools, probes, and drugs, but their poor membrane permeability has limited their application as therapeutic or diagnostic agents. Herein, we report a general method for the synthesis of phospholipid-PNAs (LP-PNAs) and compare the effect of noncleavable lipids and bioreductively cleavable lipids (L and LSS) and phospholipid (LP) on the splice-correcting bioactivity of a PNA bearing the cell penetrating Arg9 group (PNA-R9). While the three constructs show similar and increasing bioactivity at 1-3 microM, the activity of LP-PNA-R9 continues to increase from 4-6 microM, while the activity of L-PNA-R9 remains constant and that of LSS-PNA-R9 decreases rapidly in parallel with their relative cytotoxicity. The activity of both LP-PNA-R9 and L-PNA-R9 dramatically increased in the presence of chloroquine, as expected for an endocytotic entry mechanism. The constructs were also found to have CMC values of 1.0 and 4.5 microM, respectively, in 150 mM NaCl, pH 7 water, suggesting that micelle formation may play a hitherto unrecognized role in modulating toxicity and/or facilitating endocytosis.

Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells

We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase random oligonucleotide library (RT-ROL) method that we have improved, and by a serial analysis of antisense binding sites (SAABS) method that we have developed which is similar to another recently described method. The relative binding affinities of oligodeoxynucleotides (ODNs) complementary to the antisense binding sites were then qualitatively ranked by a new Dynabead-based dot blot assay. Dissociation constants for a subset of the ODNs were determined by a new Dynabead-based solution assay and were found to be 300 pM for the best binders in 1 M salt. PNAs corresponding to the ODNs with the highest affinities were synthesized with an N-terminal CysTyr and C-terminal Lys4 sequence. Dissociation constants of these hybrid PNAs were determined by the Dynabead-based solution assay to be about 10 pM for the highest affinity binders.

Native mRNA antisense-accessible sites library for the selection of antisense oligonucleotides, PNAs, and siRNAs.

A procedure for rapidly generating a library of antisense-accessible sites on native mRNAs (mRNA antisense-accessible sites library [MASL]) is described that involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific polymerase chain reaction (PCR). Antisense phosphorothioate oligodeoxynucleotides (ODNs), peptide nucleic acids (PNAs), and small interfering RNAs (siRNAs) can then be identified by screening against the antisense-accessible sites. The utility of this methodology is demonstrated for the identification of more effective inhibitors of inducible nitric oxide synthase (iNOS) induction than have previously been reported. This method may also be useful for constraining folding calculations of native mRNAs and for designing mRNA imaging probes.

Composite soft-matter nanoscale objects: Nanocylinder-templated assembly of nanospheres

We extend the surface layer-by-layer self-assembly strategy to the preparation of block copolymer-based composite nanoconstructs utilizing nanoscale building blocks. These composite materials are well-defined, hierarchically-organized nanoscale objects of a cylindrical form and core–shell morphology, having an outer coating of cationic shell-crosslinked knedel-like nanospheres. In addition to standard characterization techniques, a fluorescence resonance energy transfer experiment is designed and used to study the coating of anionic cylinders by cationic spheres. Expedited HeLa cell uptake is found for the composite nanostructures compared with the non-coated cylinders, indicating that this simple assembly strategy is a facile method to allow the secondary structure to inherit properties from its individual building blocks.