pH-Sensitive, N-ethoxybenzylimidazole (NEBI) bifunctional crosslinkers enable triggered release of therapeutics from drug delivery carriers.

This paper presents a pH-sensitive bifunctional crosslinker that enables facile conjugation of small molecule therapeutics to macromolecular carriers for use in drug delivery systems. This N-ethoxybenzylimidazole (NEBI) bifunctional crosslinker was designed to exploit mildly acidic, subcellular environments to trigger the release of therapeutics upon internalization in cells. We demonstrate that an analog of doxorubicin (a representative example of an anticancer therapeutic) conjugated to human serum albumin (HSA, a representative example of a macromolecular carrier) via this NEBI crosslinker can internalize and localize into acidic lysosomes of ovarian cancer cells. Fluorescence imaging and cell viability studies demonstrate that the HSA-NEBI-doxorubicin conjugate exhibited improved uptake and cytotoxic activity compared to the unconjugated doxorubicin analog. The pH-sensitive NEBI group was also shown to be relatively stable to biologically-relevant metal Lewis acids and to serum proteins, supporting that these bifunctional crosslinkers may be useful for constructing drug delivery systems that will be stable in biological fluids such as blood.

Development of a Translatable Ultrasound Molecular Imaging Agent for Inflammation.

This study details the development, characterization and non-clinical efficacy of an ultrasound molecular imaging agent intended for molecular imaging of P-selectin in humans. A targeting ligand based on a recently discovered human selectin ligand was manufactured as fusion protein, and activity for human and mouse P- and E-selectin was evaluated by functional immunoassay. The targeting ligand was covalently conjugated to a lipophilic anchor inserted into a phospholipid microbubble shell. Three lots of the targeted microbubble drug product, TS-07-009, were produced, and assays for size distribution, zeta potential and morphology were established. The suitability of TS-07-009 as a molecular imaging agent was evaluated in vitro in a flow-based adhesion assay and in vivo using a canine model of transient myocardial ischemia. Selectivity for P-selectin over E-selectin was observed in both the human and murine systems. Contrast agent adhesion increased with P-selectin concentration in a dynamic adhesion assay. Significant contrast enhancement was observed on ultrasound imaging with TS-07-009 in post-ischemic canine myocardium at 30 or 90 min of re-perfusion. Negligible enhancement was observed in resting (no prior ischemia) hearts or with a control microbubble 90 min after ischemia. The microbubble contrast agent described here exhibits physiochemical properties and in vivo behavior suitable for development as a clinical imaging agent.

Experimental observations and numerical modeling of lipid-shell microbubbles with calcium-adhering moieties for minimally-invasive treatment of urinary stones.

A novel treatment modality incorporating calcium-adhering microbubbles has recently entered human clinical trials as a new minimally-invasive approach to treat urinary stones. In this treatment method, lipid-shell gas-core microbubbles can be introduced into the urinary tract through a catheter. Lipid moities with calcium-adherance properties incorporated into the lipid shell facilitate binding to stones. The microbubbles can be excited by an extracorporeal source of quasi-collimated ultrasound. Alternatively, the microbubbles can be excited by an intraluminal source, such as a fiber-optic laser. With either excitation technique, calcium-adhering microbubbles can significantly increase rates of erosion, pitting, and fragmentation of stones. We report here on new experiments using high-speed photography to characterize microbubble expansion and collapse. The bubble geometry observed in the experiments was used as one of the initial shapes for the numerical modeling. The modeling showed that the bubble dynamics strongly depends on bubble shape and stand-off distance. For the experimentally observed shape of microbubbles, the numerical modeling showed that the collapse of the microbubbles was associated with pressure increases of some two-to-three orders of magnitude compared to the excitation source pressures. This in-vitro study provides key insights into the use of microbubbles with calcium-adhering moieties in treatment of urinary stones.

Short Hairpin RNA Knockdown of Connective Tissue Growth Factor by Ultrasound-Targeted Microbubble Destruction Improves Renal Fibrosis.

The purpose of this study was to evaluate whether ultrasound-targeted microbubble destruction transfer of interfering RNA against connective tissue growth factor (CTGF) in the kidney would ameliorate renal fibrosis in vivo. A short hairpin RNA (shRNA) targeting CTGF was cloned into a tool plasmid and loaded onto the surface of a cationic microbubble product. A unilateral ureteral obstruction (UUO) model in mice was used to evaluate the effect of CTGF knockdown. Mice were administered the plasmid-carrying microbubble intravenously, and ultrasound was applied locally to the obstructed kidney. Mice undergoing a sham UUO surgery and untreated UUO mice were used as disease controls, and mice administered plasmid alone, plasmid with ultrasound treatment and microbubbles and plasmid without ultrasound were used as treatment controls. Mice were treated once and then evaluated at day 14. CTGF in the kidney was measured by quantitative reverse transcription polymerase chain reaction and Western blot. Expression of CTGF, transforming growth factor ß1, α smooth muscle actin and type I collagen in the obstructed kidney was evaluated by immunohistochemistry. The cohort treated with plasmid-carrying microbubbles and ultrasound exhibited reduced mRNA and protein expression of CTGF (p < 0.01). Furthermore, CTGF gene silencing decreased the interstitial deposition of transforming growth factor ß1, α smooth muscle actin and type I collagen as assessed in immunohistochemistry, as well as reduced renal fibrosis in pathologic alterations (p < 0.01). No significant changes in target mRNA, protein expression or disease pathology were observed in the control cohorts. A single treatment of ultrasound-targeted microbubble destruction is able to deliver sufficient shRNA to inhibit the expression of CTGF and provide a meaningful reduction in disease severity. This technique may be a potential therapy for treatment of renal fibrosis.

Acidic hydrolysis of N-Ethoxybenzylimidazoles (NEBIs): potential applications as pH-sensitive linkers for drug delivery.

This paper describes the development of a new class of N-linked imidazoles as potential pH-sensitive, cleavable linkers for use in cancer drug delivery systems. Kinetic analysis of eight derivatives of N-ethoxybenzylimidazoles (NEBIs) showed that their rates of hydrolysis are accelerated in mild aqueous acidic solutions compared to in solutions at normal, physiological pH. Incorporation of electron donating or electron withdrawing substituents on the phenyl ring of the NEBI resulted in the ability to tune the rates of hydrolysis under mild acidic conditions with half-lives ranging from minutes to months. A derivative of NEBI carrying doxorubicin, a widely used anticancer agent, also showed an increased rate of hydrolysis under mild acid compared to that at normal physiological pH. The doxorubicin analogue resulting from hydrolysis from the NEBI exhibited good cytotoxic activity when exposed to human ovarian cancer cells. These results demonstrate a potentially useful, general strategy for conjugating a wide range of drugs to imidazole-containing delivery vessels via NEBI functionalities for controlled release of therapeutics for drug delivery applications.