Haoyan Zhou

Noninvasive Characterization of the Effect of Varying PLGA Molecular Weight Blends on In Situ Forming Implant Behavior Using Ultrasound Imaging

In situ forming implants (ISFIs) have shown promise in drug delivery applications due to their simple manufacturing and minimally invasive administration. Precise, reproducible control of drug release from ISFIs is essential to their successful clinical application. This study investigated the effect of varying the molar ratio of different molecular weight (Mw) poly(D,L-lactic-co-glycolic acid) (PLGA) polymers within a single implant on the release of a small Mw mock drug (sodium fluorescein) both in vitro and in vivo. Implants were formulated by dissolving three different PLGA Mw (15, 29, and 53kDa), as well as three 1:1 molar ratio combinations of each PLGA Mw in 1-methyl-2-pyrrolidinone (NMP) with the mock drug fluorescein. Since implant morphology and microstructure during ISFI formation and degradation is a crucial determinant of implant performance, and the rate of phase inversion has been shown to have an effect on the implant microstructure, diagnostic ultrasound was used to noninvasively quantify the extent of phase inversion and swelling behavior in both environments. Implant erosion, degradation, as well as the in vitro and in vivo release profiles were also measured using standard techniques. A non-linear mathematical model was used to correlate the drug release behavior with polymer phase inversion, with all formulations yielding an R2 value greater than 0.95. Ultrasound was also used to create a 3D image reconstruction of an implant over a 12 day span. In this study, swelling and phase inversion were shown to be inversely related to the polymer Mw with 53kDa polymer implants increasing at an average rate of 9.4%/day compared with 18.6%/day in the case of the 15 kDa PLGA. Additionally the onset of erosion, complete phase inversion, and degradation facilitated release required 9 d for 53 kDa implants, while these same processes began 3 d after injection into PBS with the 15 kDa implants. It was also observed that PLGA blends generally had intermediate properties when compared to pure polymer formulations. However, release profiles from the blend formulations were governed by a more complex set of processes and were not simply averages of release profiles from the pure polymers preparations. This study demonstrated that implant properties such as phase inversion, swelling and drug release could be tailored to by altering the molar ratio of the polymers used in the depot formulation.

Ultrasound Imaging Beyond the Vasculature with New Generation Contrast Agents

Current commercially available ultrasound contrast agents are gas-filled, lipid- or protein-stabilized microbubbles larger than 1 µm in diameter. Because the signal generated by these agents is highly dependent on their size, small yet highly echogenic particles have been historically difficult to produce. This has limited the molecular imaging applications of ultrasound to the blood pool. In the area of cancer imaging, microbubble applications have been constrained to imaging molecular signatures of tumor vasculature and drug delivery enabled by ultrasound-modulated bubble destruction. Recently, with the rise of sophisticated advancements in nanomedicine, ultrasound contrast agents, which are an order of magnitude smaller (100-500 nm) than their currently utilized counterparts, have been undergoing rapid development. These agents are poised to greatly expand the capabilities of ultrasound in the field of targeted cancer detection and therapy by taking advantage of the enhanced permeability and retention phenomenon of many tumors and can extravasate beyond the leaky tumor vasculature. Agent extravasation facilitates highly sensitive detection of cell surface or microenvironment biomarkers, which could advance early cancer detection. Likewise, when combined with appropriate therapeutic agents and ultrasound-mediated deployment on demand, directly at the tumor site, these nanoparticles have been shown to contribute to improved therapeutic outcomes. Ultrasounds safety profile, broad accessibility and relatively low cost make it an ideal modality for the changing face of healthcare today. Aided by the multifaceted nano-sized contrast agents and targeted theranostic moieties described herein, ultrasound can considerably broaden its reach in future applications focused on the diagnosis and staging of cancer.

Effect of cargo properties on in situ forming implant behavior determined by noninvasive ultrasound imaging

Diagnostic ultrasound has been shown to be an effective method for the noninvasive characterization of in situ forming implant behavior both in vivo and in vitro through the evaluation of the echogenic signal that forms as a consequence of the polymer phase transition from liquid to solid. The kinetics of this phase transition have a direct effect on drug release and can be altered through factors that change the mass transfer events of the solvent and aqueous environment, including properties of the entrapped active agent. This study examined the effect of payload properties on implant phase inversion, swelling, drug release, and polymer degradation. Poly(dl-lactide-co-gylcolide) implants were loaded with either: sodium fluorescein, bovine serum albumin (BSA), doxorubicin (Dox), or 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). Fluorescein and Dox were released at near equivalent rates throughout the diffusion phase of release but due to differing drug–matrix interactions, Dox-loaded implants released a lower mass of drug during the degradation phase of release. DiI was not readily released, and due to increased depot hydrophobicity, resulted in significantly lower swelling than the other formulations. The initial echogenicity was higher in Dox-loaded implants than those loaded with fluorescein, but after the initial precipitation, phase inversion and drug release occurred at near equivalent rates for both Dox and fluorescein-loaded implants. Nonlinear mathematical fitting was used to correlate drug release and phase inversion, providing a noninvasive method for evaluating implant release (R2 > 0.97 for Dox, BSA, and fluorescein; DiI had a correlation coefficient of 0.56).

Ultrasound molecular imaging of ovarian cancer with CA-125 targeted nanobubble contrast agents

Ultrasound is frequently utilized in diagnosis of gynecologic malignancies such as ovarian cancer. Because epithelial ovarian cancer (EOC) is often characterized by overexpression of cancer antigen 125 (CA-125), ultrasound contrast agents able to target this molecular signature could be a promising complementary strategy. In this work, we demonstrate application of CA-125-targeted echogenic lipid and surfactant-stabilized nanobubbles imaged with standard clinical contrast harmonic ultrasound for imaging of CA-125 positive OVCAR-3 tumors in mice. Surface functionalization of the nanobubbles with a CA-125 antibody achieved rapid significantly (P < 0.05) enhanced tumor accumulation, higher peak ultrasound signal intensity and slower wash out rates in OVCAR-3 tumors compared to CA-125 negative SKOV-3 tumors. Targeted nanobubbles also exhibited increased tumor retention and prolonged echogenicity compared to untargeted nanobubbles. Data suggest that ultrasound molecular imaging using CA-125 antibody-conjugated nanobubbles may contribute to improved diagnosis of EOC.

Macroporous acrylamide phantoms improve prediction of in vivo performance of in situ forming implants.

In situ forming implants (ISFIs) have shown promise as a sustained, local drug delivery system for therapeutics in a variety of applications. However, development of ISFIs has been hindered by poor correlation between in vitro study results and in vivo performance. In contrast to oral dosage forms, there is currently no clear consensus on a standard for in vitro drug dissolution studies for parenteral formulations. Recent studies have suggested that the disparity between in vivo and in vitro behavior of phase-inverting ISFIs may be, in part, due to differences in injection site stiffness. Accordingly, this study aimed to create acrylamide-based hydrogel phantoms of varying porosity and stiffness, which we hypothesized would better predict in vivo performance. Implant microstructure and shape were found to be dependent on the stiffness of the phantoms, while drug release was found to be dependent on both phantom porosity and stiffness. Specifically, SEM analysis revealed that implant porosity and interconnectivity decreased with increasing phantom stiffness and better mimicked the microstructure seen in vivo. Burst release of drug increased from 31% to 43% when in standard acrylamide phantoms vs macroporous phantoms (10kPa), improving the correlation to the burst release seen in vivo. Implants in 30kPa macroporous phantoms had the best correlation with in vivo burst release, significantly improving (p<0.05) the burst release relative to in vivo from 64%, using a standard PBS dissolution method, to 92%. These findings confirm that implant behavior is affected by injection site stiffness. Importantly, with appropriate optimization and validation, hydrogel phantoms such as the one investigated here could be used to improve the in vitro-in vivo correlation of in situ forming implant formulations and potentially augment their advancement to clinical use.

Induction of HEXIM1 activities by HMBA derivative 4a1: Functional consequences and mechanism

We have been studying the role of Hexamethylene bisacetamide (HMBA) Induced Protein 1 (HEXIM1) as a tumor suppressor whose expression is decreased in tamoxifen resistant and metastatic breast cancer. HMBA was considered the most potent and specific inducer for HMBA inducible protein 1 (HEXIM1) prior to our studies. Moreover, the ability of HMBA to induce differentiation is advantageous for its therapeutic use when compared to cytotoxic agents. However, HMBA induced HEXIM1 expression required at mM concentrations and induced dose limiting toxicity, thrombocytopenia. Thus we structurally optimized HMBA and identified a more potent inducer of HEXIM1 expression, 4a1. The studies reported herein tested the ability of 4a1 to induce HEXIM1 activities using a combination of biochemical, cell phenotypic, and in vivo assays. 4a1 induced breast cell differentiation, including the stem cell fraction in triple negative breast cancer cells. Clinically relevant HEXIM1 activities that are also induced by 4a1 include enhancement of the inhibitory effects of tamoxifen and inhibition of breast tumor metastasis. We also provide mechanistic basis for the phenotypic effects of 4a1. Our results support the potential of an unsymmetrical HMBA derivative, such as 4a1, as lead compound for further drug development.