Chronic safety assessment of hemostatic self-expanding foam: 90-day survival study and intramuscular biocompatibility.

BACKGROUND: Noncompressible hemorrhage is a significant cause of preventable death in trauma, with no effective presurgical treatments. We previously described the efficacy and 28-day safety of a self-expanding hemostatic foam in swine models. We hypothesized that the 28-day results would be confirmed at a second site and that results would be consistent over 90 days. Finally, we hypothesized that the foam material would be biocompatible following intramuscular implantation. METHODS: Foam treatment was administered in swine following a closed-cavity splenic injury. The material was explanted after 3 hours, and the animals were monitored to 28 days (n = 6) or 90 days (n = 4). Results were compared with a control group with injury alone (n = 6 at 28 days, n = 3 at 90 days). In a separate study, foam samples were implanted in rabbit paravertebral muscle and assessed at 28 days and 90 days relative to a Food and Drug Administration-approved polyurethane mesh (n = 3 per group). RESULTS: All animals survived the acute phase of the study, and the foam animals required enterorrhaphy. One animal developed postoperative ileus and was euthanized; all other animals survived to the 28-day or 90-day end point without clinically significant complications. Histologic evaluation demonstrated that remnant particles were associated with a fibrotic capsule and mild inflammation. The foam was considered biocompatible in 28-day and 90-day intramuscular implant studies. CONCLUSION: Foam treatment was not associated with significant evidence of end-organ dysfunction or toxicity at 28 days or 90 days. Remnant foam particles were well tolerated. These results support the long-term safety of this intervention for severely bleeding patients.

High-throughput methods for screening polymeric transfection reagents.

The clinical success of gene therapy requires the development of a safe and efficient delivery system for DNA. Cationic polymers are nonviral vectors that can associate electrostatically with plasmid DNA to form nanocomplexes. In some cases, this is sufficient for cellular uptake and transfection, although the precise mechanisms by which polymers facilitate gene delivery remain unclear. A robust and reliable method to screen for efficacy is essential for the development of effective polycationic transfection reagents. Numerous parameters must be controlled and optimized, such as polymer structure, polymer-DNA-binding conditions (mixing method, pH, ionic strength, incubation time, concentrations, ratios), transfection media (type, serum content), DNA dose and incubation time with the cells, cell specificity, and assay conditions. In this protocol, we describe a high-throughput method for assessing polymer-mediated transfection. The technique uses 96-well plates, which allows many transfection parameters to be varied and optimized in parallel. Hundreds of polymers can be tested in quadruplicate in a single day and the technique can easily be automated to efficiently and reproducibly test large material libraries. One limitation is that many plate types, solutions, and equipment must be stocked and sterilized. Moreover, because all polymers are processed simultaneously in very small volumes, it is difficult to validate each step for each polymer to ensure solution uniformity and adequate polymer-DNA complexation. Despite these drawbacks, this high-throughput screening method has already been used successfully in the development of efficient polycation vectors.

Nanoparticle-delivered multimeric soluble CD40L DNA combined with Toll-Like Receptor agonists as a treatment for melanoma.

Stimulation of CD40 or Toll-Like Receptors (TLR) has potential for tumor immunotherapy. Combinations of CD40 and TLR stimulation can be synergistic, resulting in even stronger dendritic cell (DC) and CD8+ T cell responses. To evaluate such combinations, established B16F10 melanoma tumors were injected every other day X 5 with plasmid DNA encoding a multimeric, soluble form of CD40L (pSP-D-CD40L) either alone or combined with an agonist for TLR1/2 (Pam(3)CSK(4) ), TLR2/6 (FSL-1 and MALP2), TLR3 (polyinosinic-polycytidylic acid, poly(I:C)), TLR4 ( monophosphoryl lipid A, MPL), TLR7 (imiquimod), or TLR9 (Class B CpG phosphorothioate oligodeoxynucleotide, CpG). When used by itself, pSP-D-CD40L slowed tumor growth and prolonged survival, but did not lead to cure. Of the TLR agonists, CpG and poly(I:C) also slowed tumor growth, and the combination of these two TLR agonists was more effective than either agent alone. The triple combination of intratumoral pSP-D-CD40L + CpG + poly(I:C) markedly slowed tumor growth and prolonged survival. This treatment was associated with a reduction in intratumoral CD11c+ dendritic cells and an influx of CD8+ T cells. Since intratumoral injection of plasmid DNA does not lead to efficient transgene expression, pSP-D-CD40L was also tested with cationic polymers that form DNA-containing nanoparticles which lead to enhanced intratumoral gene expression. Intratumoral injections of pSP-D-CD40L-containing nanoparticles formed from polyethylenimine (PEI) or C32 (a novel biodegradable poly(B-amino esters) polymer) in combination with CpG + poly(I:C) had dramatic antitumor effects and frequently cured mice of B16F10 tumors. These data confirm and extend previous reports that CD40 and TLR agonists are synergistic and demonstrate that this combination of immunostimulants can significantly suppress tumor growth in mice. In addition, the enhanced effectiveness of nanoparticle formulations of DNA encoding immunostimulatory molecules such as multimeric, soluble CD40L supports the further study of this technology for tumor immunotherapy.

Nanoparticle-delivered suicide gene therapy effectively reduces ovarian tumor burden in mice.

There is currently no effective therapy for patients with advanced ovarian cancer. To address the need for a more effective treatment for this deadly disease, we conducted preclinical tests in ovarian tumor-bearing mice to evaluate the therapeutic efficacy of using a cationic biodegradable poly(beta-amino ester) polymer as a vector for nanoparticulate delivery of DNA encoding a diphtheria toxin suicide protein (DT-A). The promoter sequences of two genes that are highly active in ovarian tumor cells, MSLN and HE4, were used to target DT-A expression to tumor cells. Administration of DT-A nanoparticles directly to s.c. xenograft tumors and to the peritoneal cavity of mice bearing primary and metastatic ovarian tumors resulted in a significant reduction in tumor mass and a prolonged life span compared to control mice. Minimal nonspecific tissue and blood chemistry toxicity was observed following extended treatment with nanoparticles. DT-A nanoparticle therapy suppressed tumor growth more effectively than treatment with clinically relevant doses of cisplatin and paclitaxel. Our findings suggest that i.p. administration of polymeric nanoparticles to deliver DT-A encoding DNA, combined with transcriptional regulation to target gene expression to ovarian tumor cells, holds promise as an effective therapy for advanced-stage ovarian cancer.

Poly(beta-amino esters): procedures for synthesis and gene delivery.

Non-viral gene delivery systems are promising as they avoid many problems of viral gene therapy by having increased design flexibility, high safety, large DNA cargo capacity, and ease of manufacture. Here, we describe the use of polymeric vectors, in particular biodegradable poly(beta-amino esters) (PBAEs), for non-viral gene delivery. These polymers are able to self-assemble with DNA and form positively charged gene delivery nanoparticles. Methods for synthesis of these polymers, particle self-assembly, and transfection using these particles are delineated. A standard protocol is presented as well as a high-throughput screening technique that can be used to more quickly optimize transfection parameters for efficient delivery.

Intracellular delivery of core-shell fluorescent silica nanoparticles.

Highly fluorescent core-shell silica nanoparticles made by the modified Stöber process (C dots) are promising as tools for sensing and imaging subcellular agents and structures but will only be useful if they can be easily delivered to the cytoplasm of the subject cells. This work shows that C dots can be electrostatically coated with cationic polymers, changing their surface charge and enabling them to escape from endosomes and enter the cytoplasm and nucleus. As an example of cellular delivery, we demonstrate that these particles can also be complexed with DNA and mediate and trace DNA delivery and gene expression.

Gene delivery properties of end-modified poly(beta-amino ester)s.

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.

Rapid optimization of gene delivery by parallel end-modification of poly(beta-amino ester)s.

Poly(beta-amino ester)s are cationic degradable polymers that have significant potential as gene delivery vectors. Here we present a generalized method to modify poly(beta-amino ester)s at the chain ends to improve their delivery performance. End-chain coupling reactions were developed so that polymers could be synthesized and tested in a high-throughput manner, without the need for purification. In this way, many structural variations at the polymer terminus could be rapidly evaluated. End-modification of the terminal amine structure of a previously optimized poly(beta-amino ester), C32, significantly enhanced its in vitro transfection efficiency. In vivo, intraperitoneal (IP) gene delivery using end-modified C32 polymers resulted in expression levels over one order of magnitude higher than unmodified C32 and jet-polyethylenimine (jet-PEI) levels in several abdominal organs. The rapid end-modification strategy presented here has led to the discovery of many effective polymers for gene delivery and may be a useful method to develop and optimize cationic polymers for gene therapy.

Rapid Optimization of Gene Delivery by Parallel End-modification of Poly(ß-amino ester)s.

Poly(ß-amino ester)s are cationic degradable polymers that have significant potential as gene delivery vectors. Here we present a generalized method to modify poly(ß-amino ester)s at the chain ends to improve their delivery performance. End-chain coupling reactions were developed so that polymers could be synthesized and tested in a high-throughput manner, without the need for purification. In this way, many structural variations at the polymer terminus could be rapidly evaluated. End-modification of the terminal amine structure of a previously optimized poly(ß-amino ester), C32, significantly enhanced its in vitro transfection efficiency. In vivo, intraperitoneal (IP) gene delivery using end-modified C32 polymers resulted in expression levels over one order of magnitude higher than unmodified C32 and jet-polyethylenimine (jet-PEI) levels in several abdominal organs. The rapid end-modification strategy presented here has led to the discovery of many effective polymers for gene delivery and may be a useful method to develop and optimize cationic polymers for gene therapy.

Synthesis of poly(beta-amino ester)s with thiol-reactive side chains for DNA delivery.

The safe and efficient delivery of DNA remains the major barrier to the clinical application of non-viral gene therapy. Here, we present novel, biodegradable polymers for gene delivery that are capable of simple graft modification and demonstrate the ability to respond to intracellular conditions. We synthesized poly(beta-amino ester)s using a new amine monomer, 2-(pyridyldithio)-ethylamine (PDA). These cationic, degradable polymers contain pyridyldithio functionalities in the side chains that react with high specificity toward thiol ligands. This reactivity is demonstrated using both mercaptoethylamine (MEA) and the thiol peptide RGDC, a ligand that binds with high affinity to certain integrin receptors. These two polymer derivatives displayed strong DNA binding as determined using electrophoresis and dye exclusion assays. In addition, the MEA-based polymer and plasmid DNA were shown to self-assemble into cationic complexes with effective diameters as low as 100 nm. Furthermore, this DNA binding ability was substantially reduced in response to intracellular glutathione concentrations, which may aid in DNA unpackaging inside the cell. These complexes also displayed low cellular toxicity and were able to mediate transfection at levels comparable to PEI in human hepatocellular carcinoma cells. These results suggest that PDA-based poly(beta-amino ester)s may serve as a modular platform for polymer-mediated gene delivery.