Validation of a small animal model for soft tissue filler characterization.

BACKGROUND: Rigorous preclinical testing of soft tissue fillers has been lacking. No animal model has emerged as an accepted standard to evaluate tissue filler longevity. OBJECTIVE: To validate a small animal model to compare soft tissue filler degradation and tissue reaction. METHODS: Preliminary experiments compared caliper with magnetic resonance imaging volumetric analysis. Next, four hyaluronic acid (HA) fillers were injected into the dermis of Sprague-Dawley rats. The three dimensions of the implants were measured at day 0, day 1, and monthly for 1 year or complete resorption of the filler. Volumetric, histologic, and statistical analyses were performed. RESULTS: Magnetic resonance imaging results validated caliper-based volumetric measurements. Histology demonstrated injections in the subcutaneous space just deep to the dermis and panniculus carnosus. High- and very high-concentration HA fillers maintained significantly greater volumes and volume ratios than low-concentration HA fillers throughout the duration of the study. CONCLUSIONS: The rat subcutis model demonstrated the ability to differentiate between HA fillers with different residence times. The caliper-based rat-subcutis method demonstrated consistent volumetric analysis and correlated with human residence times of HA fillers. These quantitative results validate the rat subcutis model as an expedited preclinical model for HA fillers.

Photoactivated composite biomaterial for soft tissue restoration in rodents and in humans.

Soft tissue reconstruction often requires multiple surgical procedures that can result in scars and disfiguration. Facial soft tissue reconstruction represents a clinical challenge because even subtle deformities can severely affect an individual's social and psychological function. We therefore developed a biosynthetic soft tissue replacement composed of poly(ethylene glycol) (PEG) and hyaluronic acid (HA) that can be injected and photocrosslinked in situ with transdermal light exposure. Modulating the ratio of synthetic to biological polymer allowed us to tune implant elasticity and volume persistence. In a small-animal model, implanted photocrosslinked PEG-HA showed a dose-dependent relationship between increasing PEG concentration and enhanced implant volume persistence. In direct comparison with commercial HA injections, the PEG-HA implants maintained significantly greater average volumes and heights. Reversibility of the implant volume was achieved with hyaluronidase injection. Pilot clinical testing in human patients confirmed the feasibility of the transdermal photocrosslinking approach for implantation in abdomen soft tissue, although an inflammatory response was observed surrounding some of the materials.

Doxorubicin and beta-lapachone release and interaction with micellar core materials: experiment and modeling.

Polymer micelles with two different core-forming blocks, poly(d,l -lactide) (PLA) and poly(epsilon-caprolactone) (PCL), but the same coronal material, poly(ethylene glycol) (PEG), were investigated in this study as nanoscopic drug carriers. The release of two different drugs, doxorubicin (DOX) and beta-lapachone (beta-lap), from PEG(5k)-b-PCL(5k) and PEG(5k)-b-PLA(5k) micelles was studied at pH 5.0 and 7.4. Mathematical solutions of both Higuchi's model and Fickian diffusion equations were utilized to elucidate the differences between the micelle core materials for the two drugs. The neutral and smaller of the two drugs tested, beta-lap, demonstrated faster, pH-independent release, suggesting that no substantial changes occurred in either micelle core at lower pH. In contrast, the release rate of DOX was found to noticeably increase at lower pH with a larger cumulative amount of drug released. Different core materials were shown to have considerable influence on the release kinetics of both drugs: in both cases, the more hydrophobic PCL core showed slower drug release rates compared with the less hydrophobic PLA core.

Beta-lapachone-containing PEG-PLA polymer micelles as novel nanotherapeutics against NQO1-overexpressing tumor cells.

Beta-lapachone (beta-lap) is a novel anticancer agent that is bioactivated by NADP(H): quinone oxidoreductase 1 (NQO1), an enzyme overexpressed in a variety of tumors. Despite its therapeutic promise, the poor aqueous solubility of beta-lap hinders its preclinical evaluation and clinical translation. Our objective was to develop beta-lap-containing poly(ethylene glycol)-block-poly(D,L-lactide) (PEG-PLA) polymer micelles for the treatment of NQO1-overexpressing tumors. Several micelle fabrication strategies were examined to maximize drug loading. A film sonication method yielded beta-lap micelles with relatively high loading density (4.7+/-1.0% to 6.5+/-1.0%) and optimal size (29.6+/-1.5 nm). Release studies in phosphate-buffered saline (pH 7.4) showed the time (t(1/2)) for 50% of drug release at 18 h. In vitro cytotoxicity assays were performed in NQO1-overexpressing (NQO1+) and NQO1-null (NQO1-) H596 lung, DU-145 prostate, and MDA-MB-231 breast cancer cells. Cytotoxicity data showed that after a 2 h incubation with beta-lap micelles, a marked increase in toxicity was shown in NQO1+ cells over NQO1- cells, resembling free drug both in efficacy and mechanism of cell death. In summary, these data demonstrate the potential of beta-lap micelles as an effective therapeutic strategy against NQO1-overexpressing tumor cells.

Functionalized micellar systems for cancer targeted drug delivery.

Polymer micelles are rapidly becoming a powerful nanomedicine platform for cancer therapeutic applications due to their small size (10-100 nm), in vivo stability, ability to solubilize water insoluble anticancer drugs, and prolonged blood circulation times. Recent data from clinical trials with three micelle formulations have highlighted these and other pharmacokinetic advantages with reduced systemic toxicity and patient morbidity compared to conventional drug formulation. While the initial anti-tumor efficacy of these systems seems promising, a strong research impetus has been placed on micelle functionalization in order to achieve tumor targeting and site-specific drug release, with the hope of reaching a more pronounced tumor response. Hence, the purpose of this review is to draw attention to the new developments of multi-functional polymer micelles for cancer therapy with special focus on tumor targeting and controlled drug release strategies.

Efficient suppression of secretory clusterin levels by polymer-siRNA nanocomplexes enhances ionizing radiation lethality in human MCF-7 breast cancer cells in vitro.

Small interfering RNA molecules (siRNA) hold great promise to specifically target cytoprotective factors to enhance cancer therapy. Like antisense RNA strategies, however, the use of siRNA is limited because of in vivo instability. As a first step to overcome delivery issues, a series of graft copolymers of polyethylene glycol and polyethylenimine (PEI-g-PEG) were synthesized and investigated as nontoxic carriers for delivery of siRNA targeting the signaling peptide of secretory clusterin (sCLU), a prosurvival factor that protects cells from ionizing radiation (IR) injury, as well as chemotherapeutic agents. Three copolymers with different PEG grafting densities were tested for their abilities to bind and form nanocomplexes with siRNA. A copolymer composed of 10 PEG grafts (2 kDa each) per PEI polymer (2k10 copolymer) gave the highest binding affinity to siRNA by ethidium bromide exclusion assays, and had the smallest nanocomplex size (115 +/- 13 nm diameter). In human breast cancer MCF-7 cells, 2k10-siRNA-sCLU nanocomplexes suppressed both basal as well as IR-induced sCLU protein expression, which led to an over 3-fold increase in IR-induced lethality over 2k10-siRNA scrambled controls. In summary, this study demonstrates the proof-of-principle in using nanoparticle-mediated delivery of specific siRNAs to enhance the lethality of IR exposure in vitro, opening the door for siRNA-mediated knockdown of specific cytoprotective factors, such as DNA repair, anti-apoptotic, free radical scavenging, and many other proteins.

Development of beta-lapachone prodrugs for therapy against human cancer cells with elevated NAD(P)H:quinone oxidoreductase 1 levels.

beta-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 reduces beta-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1 rendered cells resistant to beta-lapachone. Thus, beta-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non-small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of beta-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to beta-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to beta-lapachone. Once converted, beta-lapachone derivatives caused NQO1-dependent, mu-calpain-mediated cell death in human cancer cells identical to that caused by beta-lapachone. Interestingly, coadministration of N-acetyl-l-cysteine, prevented derivative-induced cytotoxicity but did not affect beta-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of beta-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-l-cysteine, preventing their conversion to beta-lapachone. The use of beta-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.

Combined modeling and experimental approach for the development of dual-release polymer millirods.

This paper describes a combined modeling and experimental approach for the design and development of a polymer device to provide local drug therapy to thermally ablated solid tumors. The polymer device, in the shape of cylindrical millirod, will be implanted via image-guided procedures into the center of the ablated tumor. Drug released from the millirod aims to eliminate residual cancer cells at the boundary of the normal and ablated tissue following thermal ablation to provide an effective treatment of the total tumor volume. The design of the millirod release kinetics is based on a mathematical model of drug transport in the ablated tumor and the surrounding normal tissue. The optimal release kinetics consists of a dual-release process-a burst release followed by sustained release-to provide the most optimal drug pharmacokinetics at the ablation boundary. Model analysis leads to a quantitative correlation of burst dose and release rates to the ablation size and the drug concentration at the ablation boundary. A three-layer polymer millirod is produced by a dip-coating method, and in vitro study demonstrates the dual-release kinetics in which a burst release occurs within 2 h followed by a sustained release over 7 -10 days. Independent control of the burst and sustained release rates is achieved by varying the structural composition of the outer and middle layers of the millirods, respectively. Results from this study provide the rational basis and experimental feasibility of dual-release millirods for further efficacy studies in solid tumors.