Sonodelivery in Skeletal Muscle: Current Approaches and Future Potential

There are currently multiple approaches to facilitate gene therapy via intramuscular gene delivery, such as electroporation, viral delivery, or direct DNA injection with or without polymeric carriers. Each of these methods has benefits, but each method also has shortcomings preventing it from being established as the ideal technique. A promising method, ultrasound-mediated gene delivery (or sonodelivery) is inexpensive, widely available, reusable, minimally invasive, and safe. Hurdles to utilizing sonodelivery include choosing from a large variety of conditions, which are often dependent on the equipment and/or research group, and moderate transfection efficiencies when compared to some other gene delivery methods. In this review, we provide a comprehensive look at the breadth of sonodelivery techniques for intramuscular gene delivery and suggest future directions for this continuously evolving field

Effect of Oligopeptide Orientation on Polymer-based DNA Delivery

Non-viral synthetic gene therapy reagents offer excellent structural and chemical versatility within non-immunogenic delivery systems, coupled with high therapeutic gene carrying capacity and long shelf life, making them attractive alternatives to viral systems. The success of non-viral transfection using polymers hinges on efficient nuclear uptake of nucleic acid cargo and overcoming intra- and extracellular barriers. This poster will describe the integration of the PKKKRKV heptapeptide (the Simian virus SV40 large T-antigen nuclear localization sequence, NLS) onto a polymer backbone, and the resultant high reporter gene expression in mice when administered by intramuscular ultrasound-mediated delivery. These novel polymers afforded protein expression higher than JetPEI™ in vivo, and in cell culture outperformed commercial reagents JetPEI™ and Lipofectamine 2000™, the latter being notorious for coupling high transfection efficiency with cytotoxicity. The orientation of the NLS peptide grafts relative to the polymer backbone markedly affected transfection performance both in vitro and in vivo. Quantitative polymerase chain reaction (qPCR) studies on transfected cells showed that polymers having the NLS attached at the valine residue afforded higher nuclear translocation, and subsequently higher protein expression, relative to those having the NLS groups attached in the opposite orientation. Besides nuclear uptake, the superior binding characteristics of these comb polymers compared to linear polylysine, as judged by atomistic and coarse grain simulations as well as polymer-DNA binding experiments, contributes to their enhanced transfection performance. Polyplexes formed from these comb polymer structures exhibit low cytotoxicity and high transfection efficiency both in vitro and in vivo, demonstrating the therapeutic promise of these novel gene therapy reagents

A Novel Approach to Targeted Oncologic Therapy - Co-culture Viability of Polymer Prodrug Conjugation to Mesenchymal Stem Cells

Background/Purpose: Conjugation of polymer prodrugs to tumor homing cells, such as Mesenchymal Stem Cells (MSCs), could provide a vehicle for actively targeted delivery of polymer prodrugs. Methods: Human Bone Marrow MSCs were conjugated to either a doxorubicin polymer prodrug or free doxorubicin and were co-cultured with T-cells. Viability was assessed through the use of a Vi-cell counter. In Vivo Migration Analysis was performed NOD SCID mice implanted with subcutaneous MDA MB-231 breast cancer xenografts. Following tumor establishment, mice were injected via lateral tail vein injection with either saline or polymer loaded MSCs. Five days following stem cell injection, mice were euthanized, tumors were harvested and sections were analyzed using fluorescent microscopy and immuno-histochemical staining for cd105. Results: T-cell viability was reduced when co-cultured with MSCs conjugated to free doxorubicin although cells co-cultured with MSCs conjugated to doxorubicin polymer did not exhibit reduced viability. Polymer loaded MSCs displayed intact tumor homing migratory ability in vivo (Figure 1). Conclusion: MSCs conjugated to doxorubicin released the drug, resulting in reduced neighboring T-cells viability. MSCs loaded with polymer maintained their migratory capacity were able to migrate to tumors in vivo. MSCs therefore represent a potential vehicle for targeted drug delivery. Future work will focus on developing methods for releasing the drug upon successful delivery to the target in vivo

In Vivo Evaluation of a Biomimetic Polymer-Doxorubicin Conjugate for Cancer Therapy

This poster will describe a novel polymer pro-drug platform designed for conjugation and delivery of chemotherapeutics. Specifically, polymer pro-drugs were prepared from functional polymer zwitterions and doxorubicin (DOX), and evaluated in vivo to assess toxicological, pharmacokinetic and therapeutic properties. The biocompatible polymer scaffold (PolyMPC) consists of zwitterionic phosphorylcholine pendent groups, which mimic the natural hydrophilic moieties of phospholipids in cell membranes, and hydrazone linkages that allow for pH-triggered release of DOX. PolyMPC-DOX pro-drugs were isolated as dry solids using a facile strategy that allows for precise control of molecular weight and DOX incorporation. In vivo toxicity of PolyMPC and PolyMPC-DOX was assessed in a murine model. The maximum tolerated dose of the pro-drug was five times greater than that of free DOX, while PolyMPC alone exhibited no toxicity even at a dose of 800 mg/kg. A pharmacokinetic study in tumor-bearing mice demonstrated a significant increase in circulation half-life of conjugated DOX (t1/2=2 hours) compared to free DOX (t1/2=15 minutes), with conjugated DOX detectable in blood serum for longer than 24 hours. This pronounced enhancement in circulation time was attributed to the macromolecular scaffold, which precludes rapid renal clearance compared to native DOX. Examination of mice given PolyMPC-DOX five days after injection in the PK study showed a three-fold increase of drug accumulated in tumor tissue compared to that of mice treated with free DOX and drug accumulation in off-target organs was reduced for mice given DOX conjugate. The therapeutic efficacy of the PolyMPC-DOX conjugates was then assessed in an orthotopic murine breast cancer model. The treatment group given PolyMPC-DOX exhibited a two-fold increase in overall survival and a significant reduction in average tumor volume compared to the free DOX and saline control groups. A study evaluating the therapeutic efficacy of PolyMPC-DOX in a human ovarian xenograft tumor model is ongoing

Reengineering Tumor Microenvironment with Sequential Interleukin Delivery

Some cytokines can reengineer anti-tumor immunity to modify the tumor micro-environment. Interleukin-27 (IL-27) can partially reduce tumor growth in several animal models, including prostate cancer. We hypothesized that addition of IL-18, which can induce the proliferation of several immune effector cells through inducing IFNγ could synergize with IL-27 to enhance tumor growth control. We describe our findings on the effects of IL-27 gene delivery on prostate cancer cells and how sequential therapy with IL-18 enhanced the efficacy of IL-27. The combination of IL-27 followed by IL-18 (27→18) successfully reduced cancer cell viability, with significant effects in cell culture and in an immunocompetent mouse model. We also examined a novel chimeric cytokine, comprising an IL-27 targeted at the C-terminus with a short peptide, LSLITRL (27pepL). This novel cytokine targets a receptor upregulated in tumor cells (IL-6Rα) via the pepL ligand. Interestingly, when we compared the 27→18 combination with the single 27pepL therapy, we observed a similar efficacy for both. This efficacy was further enhanced when 27pepL was sequenced with IL-18 (27pepL→18). The observed reduction in tumor growth and significantly enriched canonical pathways and upstream regulators, as well as specific immune effector signatures (as determined by bioinformatics analyses in the tumor microenvironment) supported the therapeutic design, whereby IL-27 or 27pepL can be more effective when delivered with IL-18. This cytokine sequencing approach allows flexible incorporation of both gene delivery and recombinant cytokines as tools to augment IL-27’s bioactivity and reengineer efficacy against prostate tumors and may prove applicable in other therapeutic settings

Zwitterionic PEG-PC hydrogels modulate the foreign body response in a modulus-dependent manner

Reducing the foreign body response (FBR) to implanted biomaterials will enhance their performance in tissue engineering. Poly(ethylene glycol) (PEG) hydrogels are increasingly popular for this application due to their low cost, ease of use, and the ability to tune their compliance via molecular weight and crosslinking densities. PEG hydrogels can elicit chronic inflammation in vivo, but recent evidence has suggested that extremely hydrophilic, zwitterionic materials and particles can evade the immune system. To combine the advantages of PEG-based hydrogels with the hydrophilicity of zwitterions, we synthesized hydrogels with co-monomers PEG and the zwitterion phosphorylcholine (PC). Recent evidence suggests that stiff hydrogels elicit increased immune cell adhesion to hydrogels, which we attempted to reduce by increasing hydrogel hydrophilicity. Surprisingly, hydrogels with the highest amount of zwitterionic co-monomer elicited the highest FBR we observed. Lowering the hydrogel modulus (165 kPa to 3 kPa), or PC content (20 wt% to 0 wt%), mitigated this effect. A high density of macrophages was found at the surface of implants associated with a high FBR, and mass spectrometry analysis of the proteins adsorbed to these gels implicated extracellular matrix, immune response, and cell adhesion protein categories as drivers of macrophage recruitment to these hydrogels. Overall, we show that modulus regulates macrophage adhesion to zwitterionic-PEG hydrogels, and demonstrate that chemical modifications to hydrogels should be studied in parallel with their physical properties to optimize implant design

Effect of Polymer Chemistry on the Linear Viscoelasticity of Complex Coacervates

Complex coacervates can form through the electrostatic complexation of oppositely charged polymers. The material properties of the resulting coacervates can change based on the polymer chemistry and the complex interplay between electrostatic interactions and water structure, controlled by salt. We examined the effect of varying the polymer backbone chemistry using methacryloyl- and acryloyl-based complex coacervates over a range of polymer chain lengths and salt conditions. We simultaneously quantified the coacervate phase behavior and the linear viscoelasticity of the resulting coacervates to understand the interplay between polymer chain length, backbone chemistry, polymer concentration, and salt concentration. Time-salt superposition analysis was used to facilitate a broader characterization and comparison of the stress relaxation behavior between different coacervate samples. Samples with mismatched polymer chain lengths highlighted the ways in which the shortest polymer chain can dominate the resulting coacervate properties. A comparison between coacervates formed from methacryloyl vs acryloyl polymers demonstrated that the presence of a backbone methyl group affects the phase behavior, and thus the rheology in such a way that coacervates formed from methacryloyl polymers have a similar phase behavior to those of acryloyl polymers with ∼10× longer polymer chains

Quantum dots coordinated with conjugated organic ligands: new nanomaterials with novel photophysics

CdSe quantum dots functionalized with oligo-(phenylene vinylene) (OPV) ligands (CdSe-OPV nanostructures) represent a new class of composite nanomaterials with significantly modified photophysics relative to bulk blends or isolated components. Single-molecule spectroscopy on these species have revealed novel photophysics such as enhanced energy transfer, spectral stability, and strongly modified excited state lifetimes and blinking statistics. Here, we review the role of ligands in quantum dot applications and summarize some of our recent efforts probing energy and charge transfer in hybrid CdSe-OPV composite nanostructures

The use of 4-substituted pyridines to afford amphiphilic, pegylated cadmium selenide nanoparticles

Amphiphilic cadmium selenide (CdSe) nanoparticles were prepared by surface functionalization with novel ligands 1 and 2, composed of pyridine moieties substituted in the 4-position with polyethylene glycol (PEG) chains

PEGylated polymers for medicine: from conjugation to self-assembled systems

Synthetic polymers have transformed society in many areas of science and technology, including recent breakthroughs in medicine. Synthetic polymers now offer unique and versatile platforms for drug delivery, as they can be “bio-tailored” for applications as implants, medical devices, and injectable polymer-drug conjugates. However, while several currently used therapeutic proteins and small molecule drugs have benefited from synthetic polymers, the full potential of polymer-based drug delivery platforms has not yet been realized. This review examines both general advantages and specific cases of synthetic polymers in drug delivery, focusing on PEGylation in the context of polymer architecture, self-assembly, and conjugation techniques that show considerable effectiveness and/or potential in therapeutics