Tyrosine-derived polymeric surfactant nanospheres insert cholesterol in cell membranes.

HYPOTHESIS: The design of biodegradable tyrosine-derived polymeric surfactants (TyPS) through the use of calculated thermodynamic parameters could lead to phospholipid membrane surface modifiers capable of controlling cellular properties such as viability. Delivery of cholesterol by TyPS nanospheres into membrane phospholipid domains could provide further controlled modulation of membrane physical and biological properties. EXPERIMENT: Calculated Hansen solubility parameters (∂T) and hydrophile:lipophile balances (HLB) were applied to design and synthesize a small family of diblock and triblock TyPS with different hydrophobic blocks and PEG hydrophilic blocks. Self-assembled TyPS/cholesterol nanospheres were prepared in aqueous media via co-precipitation. Cholesterol loading and Langmuir film balance surface pressures of phospholipid monolayers were obtained. TyPS and TyPS/cholesterol nanosphere effects on human dermal cell viability were evaluated by cell culture using poly(ethylene glycol) (PEG) and Poloxamer 188 as controls. FINDINGS: Stable TyPS nanospheres incorporated between 1% and 5% cholesterol. Triblock TyPS formed nanosphere with dimensions significantly smaller than diblock TyPS nanospheres. In accord calculated thermodynamic parameters, cholesterol binding increased with increasing TyPS hydrophobicity. TyPS inserted into phospholipid monolayer films in a manner consistent with their thermodynamic properties and TyPS/cholesterol nanospheres delivered cholesterol into the films. Triblock TyPS/cholesterol nanospheres increased human dermal cell viability, which was indicative of potentially beneficial TyPS effects on cell membrane surface properties.

Nanosphere size control by varying the ratio of poly(ester amide) block copolymer blends.

HYPOTHESIS: Blending amphiphilic triblock (A-B-A) and diblock (A-B) copolymers comprised of the same hydrophobic tyrosine-derived oligomeric B-block and hydrophilic poly(ethylene glycol) methyl ether (mPEG) A-block can provide highly tunable self-assembled nanosphere particle sizes suitable for biomedical applications. EXPERIMENT: Triblock and diblock copolymers were synthesized via carbodiimide chemistry and were characterized by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The amount of free PEG present in the purified copolymers was determined using a standard addition calibration curve and GPC peak deconvolution methods. Nanospheres were prepared by co-precipitation of each copolymer and of copolymer blends over a range of mole ratios. Nanospheres were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and % polymer recovery post-preparation. FINDING: Precise synthesis control produced triblock and diblock copolymers with narrow molecular weight distributions and minimal residual reactants. Self-assembled nanosphere particle sizes were 33 nm for the triblock and 129 nm for the diblock, and the size of their blends increased continuously as a function of mole ratio within that biomedically relevant range. Addition of unreacted PEG had minimal impact on either triblock or diblock nanosphere particle sizes whereas addition of unreacted oligomeric B-block increased nanosphere sizes.

Delivery of antisense oligonucleotides using poly(alkylene oxide)-poly(propylacrylic acid) graft copolymers in conjunction with cationic liposomes.

The clinical application of gene silencing is hindered by poor stability and low delivery efficiency of naked oligonucleotides. Here, we present the in vitro and in vivo behaviors of a rationally designed, ternary, self-assembled nanoparticle complex, consisting of an anionic copolymer, cationic DOTAP liposome, and antisense oligonucleotide (AON). The multifunctional copolymers are based on backbone poly(propylacrylic acid) (PPAA), a pH-sensitive hydrophobic polymer, with grafted poly(alkylene oxides) (PAOs) varying in extent of grafting and PAO chemistry. The nanoparticle complexes with PPAA-g-PAO copolymers enhance antisense gene silencing effects in A2780 human ovarian cancer cells. A greater amount of AON is delivered to ovarian tumor xenografts using the ternary copolymer-stabilized delivery system, compared to a binary DOTAP/AON complex, following intraperitoneal injection in mice. Further, intratumoral injection of the nanoparticle complexes containing 1 mol% grafted PAO reduced tumoral bcl-2 expression by up to 60%. The data for complexes across the set of PAO polymers support a strong role for the hydrophilic-lipophilic balance of the graft copolymer in achieving serum stability and cellular uptake. Based upon these results, we anticipate that this novel nanoparticle delivery system can be extended to the delivery of plasmid DNA, siRNA, or aptamers for preclinical and clinical development.

Graft copolymer polyelectrolyte complexes for delivery of cationic antimicrobial peptides.

Peptides have enormous potential as therapeutic agents for the treatment of infection, in immunomodulation and for other medical applications, but their hydrolytic degradation in biological fluids is a serious limitation to their in vivo performance. Here we demonstrate the potential utility of polyelectrolyte nanoparticle complexes of novel self-assembling anionic graft copolymers for protecting peptides from degradation in human plasma. The anionic graft copolymers are synthesized by covalently attaching pendent polyetheramine chains to poly(alkylacrylic acid) backbones by carbodiimide coupling. The peptide:copolymer nanocomplexes' particle size, zeta-potential, peptide binding, and controlled release of the peptide are shown to be dependent upon the pendent chain graft density, polymer backbone alkyl groups (propyl vs. methyl), and the nanocomplexes' electrostatic charge ratio. The nanocomplexes can provide substantial protection to the bound peptides from degradation in human plasma for at least 24 h and, in standard microbiological assays are shown to retain some or all of the peptide's antimicrobial activity against a clinically relevant strain of Staphylococcus aureus.

Delivery of siRNA silencing Runx2 using a multifunctional polymer-lipid nanoparticle inhibits osteogenesis in a cell culture model of heterotopic ossification.

Heterotopic ossification (HO) associated with traumatic neurological or musculoskeletal injuries remains a major clinical challenge. One approach to understanding better and potentially treating this condition is to silence one or more genes believed to be responsible for osteogenesis by small interfering RNA (siRNA) post-injury. Improved methods of delivering siRNA to myoprogenitor cells as well as relevant cell culture models of HO are needed to advance this approach. We utilize a model of HO featuring C2C12 myoprogenitor cells stimulated to the osteogenic phenotype by addition of BMP-2. For siRNA delivery, we utilize a nanocomposite consisting of DOTAP-based cationic liposomes coated with a graft copolymer of poly(propylacrylic acid) grafted with polyetheramine (Jeffamine), as this system has been shown previously to deliver antisense oligonucleotides safely into cells and out of endosomes for gene silencing in vitro and in vivo. Delivery of siRNA targeting Runx2, a transcription factor downstream of BMP-2, to stimulated C2C12 cells produced greater than 60% down-regulation of the Runx2 gene. This level of gene silencing was sufficient to inhibit alkaline phosphatase activity over the course of several days and calcium phosphate deposition over the course of 2 weeks. These results show the utility of the BMP-2/C2C12 model for capturing the cellular cell-fate decision in HO. Further, they suggest DOTAP/PPAA-g-Jeffamine as a promising delivery system for siRNA-based therapy for HO.

Poly(alkylene oxide) copolymers for nucleic acid delivery.

The advancement of gene-based therapeutics to the clinic is limited by the ability to deliver physiologically relevant doses of nucleic acids to target tissues safely and effectively. Over the last couple of decades, researchers have successfully employed polymer and lipid based nanoassemblies to deliver nucleic acids for the treatment of a variety of diseases. Results of phase I/II clinical studies to evaluate the efficacy and biosafety of these gene delivery vehicles have been encouraging, which has promoted the design of more efficient and biocompatible systems. Research has focused on designing carriers to achieve biocompatibility, stability in the circulatory system, biodistribution to target the disease site, and intracellular delivery, all of which enhance the resulting therapeutic effect. The family of poly(alkylene oxide) (PAO) polymers includes random, block, and branched structures, among which the ABA type triblocks copolymers of ethylene oxide (EO) and propylene oxide (PO) (commercially known as Pluronic) have received the greatest consideration. In this Account, we highlight examples of polycation-PAO conjugates, liposome-PAO formulations, and PAO micelles for nucleic acid delivery. Among the various polymer design considerations, which include molecular weight of polymer, molecular weight of blocks, and length of blocks, the overall hydrophobic-lipophilic balance (HLB) is a critical parameter in defining the behavior of the polymer conjugates for gene delivery. We discuss the effects of varying this parameter in the context of improving gene delivery processes, such as serum stability and association with cell membranes. Other innovative macromolecular modifications discussed in this category include our work to enhance the serum stability and efficiency of lipoplexes using PAO graft copolymers, the development of a PAO gel-based carrier for sustained and stimuli responsive delivery, and the development of biodegradable PAO-based amphiphilic block copolymers.

Polymer-xerogel composites for controlled release wound dressings.

Many polymers and composites have been used to prepare active wound dressings. These materials have typically exhibited potentially toxic burst release of the drugs within the first few hours followed by a much slower, potentially ineffective drug release rate thereafter. Many of these materials also degraded to produce inflammatory and cytotoxic products. To overcome these limitations, composite active wound dressings were prepared here from two fully biodegradable and tissue compatible components, silicon oxide sol-gel (xerogel) microparticles that were embedded in tyrosine-poly(ethylene glycol)-derived poly(ether carbonate) copolymer matrices. Sustained, controlled release of drugs from these composites was demonstrated in vitro using bupivacaine and mepivacaine, two water-soluble local anesthetics commonly used in clinical applications. By systematically varying independent compositional parameters of the composites, including the hydrophilic:hydrophobic balance of the tyrosine-derived monomers and poly(ethylene glycol) in the copolymers and the porosity, weight ratio and drug content of the xerogels, drug release kinetics approaching zero-order were obtained. Composites with xerogel mass fractions up to 75% and drug payloads as high as 13% by weight in the final material were fabricated without compromising the physical integrity or the controlled release kinetics. The copolymer-xerogel composites thus provided a unique solution for the sustained delivery of therapeutic agents from tissue compatible wound dressings.

Novel graft copolymers enhance in vitro delivery of antisense oligonucleotides in the presence of serum.

Antisense technology holds tremendous potential in the research and clinical settings. However, successful delivery of antisense oligodeoxynucleotides (ODNs) to the intracellular site of action requires the passage of many barriers, including survival against extracellular serum nucleases and escape from endolysosomal degradation. Previous work has shown that the effectiveness of antisense delivery by the cationic liposome, dioleoyl-3-trimethylammonium-propane (DOTAP), is enhanced substantially by the incorporation of a pH-sensitive polymer, poly (propylacrylic acid) (PPAA), in serum-free media. To improve this system for application in serum-containing media conditions, PPAA was modified in this work by grafting onto it either poly(ethylene oxide) (PEO) or a more hydrophobic analog, poly (oxyalkylene amine), known as Jeffamine. The ternary formulation of DOTAP/ODN/PPAA-g-Jeffamine resulted in 8-fold increased uptake of fluorescently-labeled ODNs compared to DOTAP/ODN/PPAA and ~80% silencing of green fluorescent protein (GFP) expression in CHO-d1EGFP cells treated in the presence of 10% FBS-containing media. In contrast, the carrier systems that contained PPAA or PPAA-g-PEO failed to display any significant antisense activity in the presence of serum, even though all of the delivery systems displayed moderate to high levels of antisense activity in serum-free conditions. The results reveal that the carrier system with the Jeffamine graft copolymer effectively mediates specific gene silencing in the presence of serum, while the system with the PEO graft copolymer fails to do so. While the pH-dependent lytic functionality of PPAA was found to be lost upon grafting with PEO or Jeffamine, the hydrophobicity of the latter was sufficient to mediate cellular internalization and endosomal escape. Thus, the PPAA-g-Jeffamine copolymers hold substantial promise as agents for controlled therapeutic delivery of antisense oligonucleotides.