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1.
Chem Sci ; 14(37): 10318-10328, 2023 Sep 27.
Article in English | MEDLINE | ID: mdl-37772096

ABSTRACT

Early stages of life likely employed catalytic RNAs (ribozymes) in many functions that are today filled by proteins. However, the earliest life forms must have emerged from heterogenous chemical mixtures, which included amino acids, short peptides, and many other compounds. Here we explored whether the presence of short peptides can help the emergence of catalytic RNAs. To do this, we conducted an in vitro selection for catalytic RNAs from randomized sequence in the presence of ten different peptides with a prebiotically plausible length of eight amino acids. This in vitro selection generated dozens of ribozymes, one of them with ∼900-fold higher activity in the presence of one specific peptide. Unexpectedly, the beneficial peptide had retained its N-terminal Fmoc protection group, and this group was required to benefit ribozyme activity. The same, or higher benefit resulted from peptide conjugates with prebiotically plausible polyaromatic hydrocarbons (PAHs) such as fluorene and naphthalene. This shows that PAH-peptide conjugates can act as potent cofactors to enhance ribozyme activity. The results are discussed in the context of the origin of life.

2.
J Control Release ; 224: 103-111, 2016 Feb 28.
Article in English | MEDLINE | ID: mdl-26772876

ABSTRACT

The tumor microenvironment in the majority of cancers is known to favor polarization of tumor-associated macrophages (TAMs) to alternatively activated M2 phenotype, promoting disease progression and reducing patient survival. Effective therapy targeting this M2 macrophage population is thus a promising adjuvant to approved cancer therapies. One of the challenges in targeting M2-like TAMs is a lack of high affinity targeting ligand with good selectivity over anti-tumor M1-like TAMs. We have previously identified an M2 macrophage-targeting peptide (M2pep) that binds preferentially to murine M2 macrophages and M2-like TAMs. A fusion peptide of M2pep with pro-apoptotic peptide KLA (M2pepKLA) was further used to reduce TAM population in vivo but high concentrations and frequent dosing were required due to low binding affinity of M2pep for M2 macrophage. The goal of this study was to develop more potent TAM depletion constructs by increasing the valency of both the M2pep targeting and KLA drug domains. Divalent and tetravalent displays of M2pep ([M2pep]2-Biotin and [M2pep]4-Biotin) were synthesized and evaluated for improvement in binding avidity to the murine macrophages. High avidity and selective binding of [M2pep]2-Biotin to M2 macrophages were achieved with at least 10-fold lower concentration than required for monovalent M2pep activity. Increasing M2pep valency to four, however, resulted in a reduction in both binding activity and selectivity. Surprisingly, both divalent and tetravalent M2pep, without conjugation of any cytotoxic drug cargo, exhibited M2 macrophage-selective toxicity not observed in monovalent M2pep treatment. We next synthesized divalent M2pep with monovalent and divalent KLA ([M2pep]2-[KLA] and [M2pep]2-[KLA]2) to evaluate its enhanced potency compared to M2pepKLA. While both constructs were significantly more toxic than M2pepKLA to primary, bone marrow-derived M2 macrophage, desired selectivity was retained only with [M2pep]2-[KLA]. Finally, we evaluated all multivalent M2pep and M2pepKLA analogs using a syngeneic CT-26 tumor cell suspension. In this setting, [M2pep]4-Biotin and [M2pep]2-[KLA]2 exhibited selective toxicity to both M2-like TAMs and malignant cells but not to M1-like TAMs. Therefore, these constructs are promising anti-cancer constructs with dual-modality mechanisms: malignant cell killing and TAM-based immunomodulation.


Subject(s)
Macrophages/drug effects , Peptides/chemical synthesis , Peptides/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Biotin/chemistry , Bone Marrow Cells/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Drug Delivery Systems , Humans , Mice , Mice, Inbred C57BL , Tumor Microenvironment
4.
Biomater Sci ; 3(1): 112-20, 2015 Jan.
Article in English | MEDLINE | ID: mdl-26214195

ABSTRACT

Polycations have been successfully used as gene transfer vehicles both in vitro and in vivo; however, their cytotoxicity has been associated with increasing molecular weight. Polymers that can be rapidly degraded after internalization are typically better tolerated by mammalian cells compared to their non-degradable counterparts. Here, we report the use of a dibromomaleimide-alkyne (DBM-alkyne) linking agent to reversibly bridge cationic polymer segments for gene delivery and to provide site-specific functionalization by azide-alkyne cycloaddition chemistry. A panel of reducible and non-reducible, statistical copolymers of (2-dimethylamino)ethyl methacrylate (DMAEMA) and oligo(ethylene glycol)methyl ether methacrylate (OEGMA) were synthesized and evaluated. When complexed with plasmid DNA, the reducible and non-reducible polymers had comparable DNA condensation properties, sizes, and transfection efficiencies. When comparing cytotoxicity, the DBM-linked, reducible polymers were significantly less toxic than the non-reducible polymers. To demonstrate polymer functionalization by click chemistry, the DBM-linked polymers were tagged with an azide-fluorophore and were used to monitor cellular uptake. Overall, this polymer system introduces the use of a reversible linker, DBM-alkyne, to the area of gene delivery and allows for facile, orthogonal, and site-specific functionalization of gene delivery vehicles.


Subject(s)
Alkynes/chemistry , Azides/chemistry , Cations/chemistry , Drug Carriers/chemistry , Maleimides/chemistry , Maleimides/toxicity , Methacrylates/chemistry , Polymers/chemistry , Animals , Gene Transfer Techniques , Genetic Therapy , Kinetics , Molecular Weight , Transfection
5.
ACS Nano ; 7(12): 10612-20, 2013 Dec 23.
Article in English | MEDLINE | ID: mdl-24195594

ABSTRACT

Nanoparticle morphology has been shown to affect cellular uptake, but there are few studies investigating the impact of particle shape on biologic drug delivery. Recently, our group synthesized a series of N-(2-hydroxypropyl) methacrylamide (HPMA)-oligolysine brush polymers for nucleic acid delivery that varied in oligolysine peptide length and polymer molecular weight. Interestingly, a 50% longer peptide (K15) transfected very poorly compared to the optimized polymer comprised of K10 peptide despite similar chemical composition and molecular weight. We hypothesized that differences in particle morphology contributed to the differences in plasmid DNA delivery. We found that particles formed with plasmid DNA and a polymer with the longer oligolysine peptide (pHK15) had larger aspect ratios than particles formed with optimized polymer (pHK10). Even though both formulations showed similar percentages of cellular association, particles of a higher aspect ratio were internalized to a lesser extent. Furthermore, the rod-like particles accumulated more in endosomal/lysosomal compartments, leading to delayed nuclear delivery. Other parameters, such as particle surface charge, unpackaging ability, uptake mechanism, intracellular trafficking, and the presence of heparan sulfate proteoglycans did not significantly differ between the two polymer formulations. These results indicate that, for this system, polyplex morphology primarily impacts nucleic acid delivery efficiency through differences in cellular internalization rates.


Subject(s)
Drug Delivery Systems , Nanoparticles/chemistry , Transgenes , Animals , CHO Cells , Cricetinae , Cricetulus , DNA/genetics , Endocytosis , Endosomes/metabolism , Gene Transfer Techniques , HeLa Cells , Heparan Sulfate Proteoglycans/chemistry , Humans , Lysine/chemistry , Lysosomes/metabolism , Microscopy, Electron, Transmission , Molecular Weight , Nanomedicine , Nucleic Acids/administration & dosage , Nucleic Acids/chemistry , Peptides/chemistry , Plasmids/metabolism , Polymers/chemistry , Transfection , Water/chemistry
6.
J Biol Eng ; 7(1): 25, 2013 Oct 24.
Article in English | MEDLINE | ID: mdl-24156736

ABSTRACT

The complex nature of in vivo gene transfer establishes the need for multifunctional delivery vectors capable of meeting these challenges. An additional consideration for clinical translation of synthetic delivery formulations is reproducibility and scale-up of materials. In this review, we summarize our work over the last five years in developing a modular approach for synthesizing peptide-based polymers. In these materials, bioactive peptides that address various barriers to gene delivery are copolymerized with a hydrophilic backbone of N-(2-hydroxypropyl)methacrylamide (HPMA) using reversible-addition fragmentation chain-transfer (RAFT) polymerization. We demonstrate that this synthetic approach results in well-defined, narrowly-disperse polymers with controllable composition and molecular weight. To date, we have investigated the effectiveness of various bioactive peptides for DNA condensation, endosomal escape, cell targeting, and degradability on gene transfer, as well as the impact of multivalency and polymer architecture on peptide bioactivity.

7.
ACS Macro Lett ; 2(8): 725-730, 2013 Aug 20.
Article in English | MEDLINE | ID: mdl-24044103

ABSTRACT

Endosomal release peptides have been incorporated in synthetic gene delivery formulations to increase transfection efficiencies. In this work, cationic copolymers containing sHGP, a membrane-lytic peptide derived from HIV gp41, were synthesized and evaluated. Diblock, with sHGP displayed on one block, and statistical, with sHGP randomly displayed, copolymers were prepared via RAFT polymerization. While the statistical copolymer existed as unimers in solution, amphiphilic diblock copolymers self-assembled into cationic micelles in aqueous solution as evidenced by TEM and dynamic light scattering analyses. This self-assembly sequestered the lytic domain and significantly reduced the cytotoxicity of the materials. However, when complexed with plasmid DNA, both the diblock and statistical copolymers of sHGP showed higher gene delivery efficacy compared to the copolymers without the membrane lytic motif. The ability of amphiphilic, diblock copolymers containing endosomal release motifs to self-assemble and sequester lytic domains is a promising feature for the nucleic acid delivery.

8.
Biomaterials ; 34(37): 9632-7, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24041424

ABSTRACT

Targeted gene delivery vectors can enhance cellular specificity and transfection efficiency. We demonstrated previously that conjugation of Tet1, a peptide that binds to the GT1b ganglioside, to polyethylenimine results in preferential transfection of neural progenitor cells in vivo. In this work, we investigate the effect of Tet1 ligand density on gene delivery to neuron-like, differentiated PC-12 cells. A series of statistical, cationic peptide-based polymers containing various amounts (1-5 mol%) of Tet1 were synthesized via one-pot reversible addition-fragmentation chain transfer (RAFT) polymerization by copolymerization of Tet1 and oligo-l-lysine macromonomers with N-(2-hydroxypropyl)methacrylamide (HPMA). When complexed with plasmid DNA, the resulting panel of Tet1-functionalized polymers formed particles with similar particle size as particles formed with untargeted HPMA-oligolysine copolymers. The highest cellular uptake in neuron-like differentiated PC-12 cells was observed using polymers with intermediate Tet1 peptide incorporation. Compared to untargeted polymers, polymers with optimal incorporation of Tet1 increased gene delivery to neuron-like PC-12 cells by over an order of magnitude but had no effect compared to control polymers in transfecting NIH/3T3 control cells.


Subject(s)
DNA/administration & dosage , Lysine/analogs & derivatives , Methacrylates/chemistry , Neurons/metabolism , Peptides/chemistry , Plasmids/administration & dosage , Transfection , Amino Acid Sequence , Animals , Mice , Molecular Sequence Data , NIH 3T3 Cells , PC12 Cells , Rats
9.
Biomater Sci ; 1(7): 736-744, 2013 Jul 01.
Article in English | MEDLINE | ID: mdl-23750319

ABSTRACT

Polyethylenimine (PEI), one of the most frequently used polycations for non-viral nucleic acid delivery, exhibits good transfection efficiency to cultured cells but generally has to be used in restricted concentration ranges due to high cytotoxicity. We recently reported a family of HPMA-co-oligolysine brush copolymers that show nucleic acid delivery efficiencies approaching that of PEI. Guanidine-containing polymers have been reported in some systems to be more effective at cellular delivery of cargo than their primary-amine analogs. The goal of this work is to investigate the effect of guanidinylation on gene transfer ability of HPMA-co-oligolysine copolymers. Several parameters were evaluated: arginine versus homoarginine monomers, oligopeptide length, and charge density within the peptide. Using reversible addition-fragmentation chain transfer (RAFT) polymerization, a series of six copolymers were synthesized containing the cationic peptides K10, R10, K5, and (GK)5. Lysine-containing copolymers were functionalized with guanidine by reaction with O-methylisourea to generate an additional five homoarginine-based copolymers. All eleven copolymers readily condensed DNA into small, < 150 nm polyplexes and remained stable in physiological salt conditions. The best performing copolymers provided more efficient gene transfection with less associated cytotoxicity than PEI. Reducing the number of charge centers (from 10 to 5) further reduced toxicity while retaining comparable transfection efficiency to PEI.

10.
Biomacromolecules ; 14(6): 1961-70, 2013 Jun 10.
Article in English | MEDLINE | ID: mdl-23641942

ABSTRACT

One of the major intracellular barriers to nonviral gene delivery is efficient endosomal escape. The incorporation of histidine residues into polymeric constructs has been found to increase endosomal escape via the proton sponge effect. Statistical and diblock copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA), oligolysine, and oligohistidine were synthesized via reversible-addition fragmentation chain transfer (RAFT) polymerization and tested for in vitro transfection efficiency, buffering ability, and polyplex uptake mechanism via the use of chemical endocytic inhibitors. Interestingly, histidine-containing statistical and diblock polymers exhibited increased buffer capacity in different endosomal pH ranges. Statistical copolymers transfected better than block copolymers that contained similar amounts of histidine. In addition, only the polymer containing the highest incorporation of oligohistidine residues led to increases in transfection efficiency over the HPMA-oligolysine base polymer. Thus, for these polymer architectures, high histidine incorporation may be required for efficient endosomal escape. Furthermore, inhibitor studies indicate that nonacidified caveolae-mediated endocytosis may be the primary route of transfection for these copolymers, suggesting that alternative approaches for increasing endosomal escape may be beneficial for enhancing transfection efficiency with these HPMA-oligolysine copolymers.


Subject(s)
Buffers , Histidine/chemistry , Lysine/chemistry , Methacrylates/chemistry , Polymers/chemistry , Transfection , Chloroquine/pharmacology , Macrolides/pharmacology , Microscopy, Electron, Transmission , Plasmids
11.
Biomaterials ; 34(9): 2318-26, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23261217

ABSTRACT

Non-viral gene delivery systems capable of transfecting cells in the brain are critical in realizing the potential impact of nucleic acid therapeutics for diseases of the central nervous system. In this study, the membrane-lytic peptide melittin was incorporated into block copolymers synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The first block, designed for melittin conjugation, was composed of N-(2-hydroxypropyl)methacrylamide (HPMA) and pyridyl disulfide methacrylamide (PDSMA) and the second block, designed for DNA binding, was composed of oligo-l-lysine (K10) and HPMA. Melittin modified with cysteine at the C-terminus was conjugated to the polymers through the pyridyl disulfide pendent groups via disulfide exchange. The resulting pHgMelbHK10 copolymers are more membrane-lytic than melittin-free control polymers, and efficiently condensed plasmid DNA into salt-stable particles (~100-200 nm). The melittin-modified polymers transfected both HeLa and neuron-like PC-12 cells more efficiently than melittin-free polymers although toxicity associated with the melittin peptide was observed. Optimized formulations containing the luciferase reporter gene were delivered to mouse brain by intraventricular brain injections. Melittin-containing polyplexes produced about 35-fold higher luciferase activity in the brain compared to polyplexes without melittin. Thus, the melittin-containing block copolymers described in this work are promising materials for gene delivery to the brain.


Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Melitten/chemistry , Methacrylates/chemistry , Polymers/chemistry , Acrylamides/chemistry , Animals , Brain/drug effects , Brain/metabolism , DNA-Binding Proteins/chemistry , Female , Genes, Reporter , HeLa Cells , Humans , Luciferases/metabolism , Lysine/analysis , Lysine/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , PC12 Cells , Polymerization , Rats , Transfection
12.
J Am Chem Soc ; 134(40): 16554-7, 2012 Oct 10.
Article in English | MEDLINE | ID: mdl-23013485

ABSTRACT

Adaptation of in vitro optimized polymeric gene delivery systems for in vivo use remains a significant challenge. Most in vivo applications require particles that are sterically stabilized, which significantly compromises transfection efficiency of materials shown to be effective in vitro. We present a multifunctional well-defined block copolymer that forms particles useful for cell targeting, reversible shielding, endosomal release, and DNA condensation. We show that targeted and stabilized particles retain transfection efficiencies comparable to the nonstabilized formulations. A novel, double-head agent that combines a reversible addition-fragmentation chain transfer agent and an atom transfer radical polymerization initiator through a disulfide linkage is used to synthesize a well-defined cationic block copolymer containing a hydrophilic oligoethyleneglycol and a tetraethylenepentamine-grafted polycation. This material effectively condenses plasmid DNA into salt-stable particles that deshield under intracellular reducing conditions. In vitro transfection studies show that the reversibly shielded polyplexes afford up to 10-fold higher transfection efficiencies than the analogous stably shielded polymer in four different mammalian cell lines. To compensate for reduced cell uptake caused by the hydrophilic particle shell, a neuron-targeting peptide is further conjugated to the terminus of the block copolymer. Transfection of neuron-like, differentiated PC-12 cells demonstrates that combining both targeting and deshielding in stabilized particles yields formulations that are suitable for in vivo delivery without compromising in vitro transfection efficiency and are thus promising carriers for in vivo gene delivery applications.


Subject(s)
DNA/administration & dosage , Ethylenediamines/chemistry , Neurons/metabolism , Plasmids/administration & dosage , Polyethylene Glycols/chemistry , Transfection , Animals , Cell Line , DNA/pharmacokinetics , Endocytosis , Humans , Neurons/cytology , Peptides/chemistry , Peptides/metabolism , Plasmids/pharmacokinetics , Polyamines/chemistry , Polyelectrolytes , Polymerization , Rats
13.
Acc Chem Res ; 45(7): 1089-99, 2012 Jul 17.
Article in English | MEDLINE | ID: mdl-22242774

ABSTRACT

Therapeutic gene delivery can alter protein function either through the replacement of nonfunctional genes to restore cellular health or through RNA interference (RNAi) to mask mutated and harmful genes. Researchers have investigated a range of nucleic acid-based therapeutics as potential treatments for hereditary, acquired, and infectious diseases. Candidate drugs include plasmids that induce gene expression and small, interfering RNAs (siRNAs) that silence target genes. Because of their self-assembly with nucleic acids into virus-sized nanoparticles and high transfection efficiency in vitro, cationic polymers have been extensively studied for nucleic acid delivery applications, but toxicity and particle stability have limited the clinical applications of these systems. The advent of living free radical polymerization has improved the quality, control, and reproducibility of these synthesized materials. This process yields well-defined, narrowly disperse materials with designed architectures and molecular weights. As a result, researchers can study the effects of polymer architecture and molecular weight on transfection efficiency and cytotoxicity, which will improve the design of next-generation vectors. In this Account, we review findings from structure-function studies that have elucidated key design motifs necessary for the development of effective nucleic acid vectors. Researchers have used robust methods such as atom transfer radical polymerization (ATRP), reverse addition-fragmentation chain transfer polymerization (RAFT), and ring-opening metastasis polymerization (ROMP) to engineer materials that enhance extracellular stability and cellular specificity and decrease toxicity. In addition, we discuss polymers that are biodegradable, form supramolecular structures, target specific cells, or facilitate endosomal release. Finally, we describe promising materials with a range of in vivo applications from pulmonary gene delivery to DNA vaccines.


Subject(s)
Free Radicals/chemistry , Nucleic Acids/metabolism , Polymers/chemistry , Animals , Mice , Nucleic Acids/genetics , Plasmids/genetics , Plasmids/metabolism , Polymerization , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transfection
14.
Int J Pharm ; 427(1): 113-22, 2012 May 01.
Article in English | MEDLINE | ID: mdl-21893178

ABSTRACT

Biodegradability can be incorporated into cationic polymers via use of disulfide linkages that are degraded in the reducing environment of the cell cytosol. In this work, N-(2-hydroxypropyl)methacrylamide (HPMA) and methacrylamido-functionalized oligo-l-lysine peptide monomers with either a non-reducible 6-aminohexanoic acid (AHX) linker or a reducible 3-[(2-aminoethyl)dithiol] propionic acid (AEDP) linker were copolymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Both of the copolymers and a 1:1 (w/w) mixture of copolymers with reducible and non-reducible peptides were complexed with DNA to form polyplexes. The polyplexes were tested for salt stability, transfection efficiency, and cytotoxicity. The HPMA-oligolysine copolymer containing the reducible AEDP linkers was less efficient at transfection than the non-reducible polymer and was prone to flocculation in saline and serum-containing conditions, but was also not cytotoxic at charge ratios tested. Optimal transfection efficiency and toxicity were attained with mixed formulation of copolymers. Flow cytometry uptake studies indicated that blocking extracellular thiols did not restore transfection efficiency and that the decreased transfection of the reducible polyplex is therefore not primarily caused by extracellular polymer reduction by free thiols. The decrease in transfection efficiency of the reducible polymers could be partially mitigated by the addition of low concentrations of EDTA to prevent metal-catalyzed oxidation of reduced polymers.


Subject(s)
Acrylamides/chemistry , DNA/administration & dosage , Drug Carriers/chemistry , Oligopeptides/chemistry , Polymers/chemical synthesis , Animals , CHO Cells , Cell Survival/drug effects , Cricetinae , DNA/chemistry , Drug Carriers/toxicity , HeLa Cells , Humans , Lysine/chemistry , Mice , NIH 3T3 Cells , Oligopeptides/administration & dosage , Polymerization , Polymers/administration & dosage , Transfection/methods
15.
Org Biomol Chem ; 10(8): 1521-6, 2012 Feb 28.
Article in English | MEDLINE | ID: mdl-22132412

ABSTRACT

Multivalent single chain variable fragments (scFv) show increased affinity to tumor-associated antigens compared to monovalent scFv and intact monoclonal antibodies (mAb). Multivalent constructs can be derived from self-associating or covalent scFv with covalent constructs offering improved in vivo and in vitro stability. Covalent attachment of scFv can be achieved using genetically engineered expression vectors that afford scFv with site specific cysteine functionality. Expression vectors for di-scFv-C wherein the cysteine is located in the center of two scFv have also been developed for attaching chemically reactive linkers. In the example illustrated here, the di-scFv-C is derived from a mAb directed against the MUC1 epitope, which is presented on cancer cells. To achieve multivalency, a chemical crosslinking strategy utilizing various azide and multi-alkyne functionalized polyethylene glycol (PEG) linkers was implemented. Conjugation was achieved by attachment of these linkers to the scFv thiol functionality. Chemoselective ligation was employed to covalently link different protein conjugates via copper(I) catalyzed azide alkyne 1,3-dipolar cycloaddition reaction (CuAAC) chemistry. Ligations were achieved in >70% yield using a specific set of linkers as determined by SDS-PAGE and densitometry. ELISA showed increased tumor binding of a tetravalent scFv providing a versatile chemical crosslinking strategy for construction of multivalent and bi-specific immunoconjugates that retain biological activity and have potential application in pre-targeted radioimmunotherapy and imaging.


Subject(s)
Single-Chain Antibodies/chemistry , Azides/chemistry , Cyclization , Molecular Structure
16.
Biomaterials ; 32(35): 9536-45, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21959008

ABSTRACT

Adenoviral (AdV) gene vectors offer efficient nucleic acid transfer into both dividing and non-dividing cells. However issues such as vector immunogenicity, toxicity and restricted transduction to receptor-expressing cells have prevented broad clinical translation of these constructs. To address this issue, engineered AdV have been prepared by both genetic and chemical manipulation. In this work, a polymer-coated Ad5 formulation is optimized by evaluating a series of N-(2-hydroxypropyl) methacrylamide (HPMA)-co-oligolysine copolymers synthesized by living polymerization techniques. This synthesis approach was used to generate highly controlled and well-defined polymers with varying peptide length (K(5), K(10) and K(15)), polymer molecular weight, and degradability to coat the viral capsid. The optimal formulation was not affected by the presence of serum during transduction and significantly increased Ad5 transduction of several cell types that lack the Coxsackie and Adenovirus Receptor (CAR) by up to 6-fold compared to unmodified AdV. Polymer-coated Ad5 also retained high transduction capability in the presence of Ad5 neutralizing antibodies. The critical role of heparan sulfate proteoglycans (HSPGs) in mediating cell binding and internalization of polymer-coated AdV was also demonstrated by evaluating transduction in HSPG-defective recombinant CHO cells. The formulations developed here are attractive vectors for ex vivo gene transfer in applications such as cell therapy. In addition, this platform for adenoviral modification allows for facile introduction of alternative targeting ligands.


Subject(s)
Acrylamides/chemistry , Adenoviridae/metabolism , Antibodies, Neutralizing/pharmacology , Cytoprotection/drug effects , Polylysine/analogs & derivatives , Receptors, Virus/metabolism , Transduction, Genetic/methods , Adenoviridae/drug effects , Animals , CHO Cells , Coxsackie and Adenovirus Receptor-Like Membrane Protein , Cricetinae , Cricetulus , HeLa Cells , Heparan Sulfate Proteoglycans/genetics , Humans , Mice , Microscopy, Electron, Transmission , Mutation/genetics , Polylysine/chemistry , Polymerization/drug effects , Virus Internalization/drug effects
17.
J Control Release ; 155(2): 303-11, 2011 Oct 30.
Article in English | MEDLINE | ID: mdl-21782863

ABSTRACT

Polycations are one of the most frequently used classes of materials for non-viral gene transfer in vivo. Several studies have demonstrated a sensitive relationship between polymer structure and delivery activity. In this work, we used reverse addition-fragmentation chain transfer (RAFT) polymerization to build a panel of N-(2-hydroxypropyl)methacrylamide (HPMA)-oligolysine copolymers with varying peptide length and polymer molecular weight. The panel was screened for optimal DNA-binding, colloidal stability in salt, high transfection efficiency, and low cytotoxicity. Increasing polyplex stability in PBS correlated with increasing polymer molecular weight and decreasing peptide length. Copolymers containing K(5) and K(10) oligocations transfected cultured cells with significantly higher efficiencies than copolymers of K(15). Four HPMA-oligolysine copolymers were identified that met the desired criteria. Polyplexes formed with these copolymers demonstrated both salt stability and transfection efficiencies on-par with poly(ethylenimine) PEI in cultured cells.


Subject(s)
Drug Carriers/chemistry , Gene Transfer Techniques , Oligopeptides/chemistry , Polylysine/chemistry , Polymethacrylic Acids/chemistry , Cell Survival/drug effects , Chromatography, Gel , DNA/administration & dosage , DNA/genetics , Drug Carriers/chemical synthesis , Drug Carriers/toxicity , Drug Stability , HeLa Cells , Humans , Light , Molecular Structure , Molecular Weight , Oligopeptides/chemical synthesis , Oligopeptides/toxicity , Polylysine/chemical synthesis , Polylysine/toxicity , Polymethacrylic Acids/chemical synthesis , Polymethacrylic Acids/toxicity , Protein Conformation , Scattering, Radiation
18.
J Am Chem Soc ; 133(10): 3230-3, 2011 Mar 16.
Article in English | MEDLINE | ID: mdl-21341746

ABSTRACT

A fully synthetic trivalent mimotope of gp120 conjugated to pan allelic HLA DR binding epitope was prepared using solid-phase peptide synthesis and optimized copper-catalyzed azide-alkyne cycloaddition. The methodology efficiently provides chemically uniform heteromultimeric peptide constructs with enhanced binding, avidity, and specificity toward an established HIV-neutralizing human antibody, MAb b12. The versatile synthetic strategy serves as a powerful platform for the development of synthetic peptides as potential HIV-1 vaccine candidates.


Subject(s)
Epitopes, T-Lymphocyte/chemistry , HIV Envelope Protein gp120/chemical synthesis , HLA-DR Antigens/chemistry , Immunodominant Epitopes/chemistry , Peptides/chemical synthesis , Amino Acid Sequence , Epitopes, T-Lymphocyte/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , HLA-DR Antigens/immunology , Immunodominant Epitopes/immunology , Malaria Vaccines/chemistry , Molecular Sequence Data , Peptides/chemistry , Peptides/immunology , T-Lymphocytes, Helper-Inducer/immunology
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