Chemical and mechanical modulation of polymeric micelle assembly.

Recently, polymeric micelles self-assembled from amphiphilic polymers have been studied for various industrial and biomedical applications. This nanoparticle self-assembly typically occurs in a solvent-exchange process. In this process, the quality of the resulting particles is uncontrollably mediated by polymeric solubility and mixing conditions. Here, we hypothesized that improving the solubility of an amphiphilic polymer in an organic solvent via chemical modification while controlling the mixing rate of organic and aqueous phases would enhance control over particle morphology and size. We examined this hypothesis by synthesizing a poly(2-hydroxyethyl)aspartamide (PHEA) grafted with controlled numbers of octadecyl (C18) chains and oligovaline groups (termed "oligovaline-PHEA-C18"). The mixing rate of DMF and water was controlled either by microfluidic mixing of laminar DMF and water flows or through turbulent bulk mixing. Interestingly, oligovaline-PHEA-C18 exhibited an increased solubility in DMF compared with PHEA-C18, as demonstrated by an increase of mixing energy. In addition, increasing the mixing rate between water and DMF using the microfluidic mixer resulted in a decrease of the diameter of the resulting polymeric micelles, as compared with the particles formed from a bulk mixing process. Overall, these findings will expand the parameter space available to control particle self-assembly while also serving to improve existing nanoparticle processing techniques.

"Click Chip" Conjugation of Bifunctional Chelators to Biomolecules.

There is a growing demand for diagnostic procedures including in vivo tumor imaging. Radiometal-based imaging agents are advantageous for tumor imaging because radiometals (i) have a wide range of half-lives and (ii) are easily incorporated into imaging probes via a mild, rapid chelation event with a bifunctional chelator (BFC). Microfluidic platforms hold promise for synthesis of radiotracers because they can easily handle minute volumes, reduce consumption of expensive reagents, and minimize personnel exposure to radioactive compounds. Here we demonstrate the use of a "click chip" with an immobilized Cu(I) catalyst to facilitate the "click chemistry" conjugation of BFCs to biomolecules (BMs); a key step in the synthesis of radiometal-based imaging probes. The "click chip" was used to synthesize three different BM-BFC conjugates with minimal amounts of copper present in reaction solutions (∼20 ppm), which reduces or obviates the need for a copper removal step. These initial results are promising for future endeavors of synthesizing radiometal-based imaging agents completely on chip.

Development of a microfluidic "click chip" incorporating an immobilized Cu(I) catalyst.

We have developed a microfluidic "click chip" incorporating an immobilized Cu(I) catalyst for click reactions. The microfluidic device was fabricated from polydimethylsiloxane (PDMS) bonded to glass and featured ~14,400 posts on the surface to improve catalyst immobilization. This design increased the immobilization efficiency and reduces the reagents' diffusion time to active catalyst site. The device also incorporates five reservoirs to increase the reaction volume with minimal hydrodynamic pressure drop across the device. A novel water-soluble tris-(benzyltriazolylmethyl)amine (TBTA) derivative capable of stabilizing Cu(I), ligand 2, was synthesized and successfully immobilized on the chip surface. The catalyst immobilized chip surface was characterized by X-ray photoelectron spectroscopy (XPS). The immobilization efficiency was evaluated via radiotracer methods: the immobilized Cu(I) was measured as 1136±272 nmol and the surface immobilized Cu(I) density was 81±20 nmol cm-2. The active Cu(I)-ligand 2 could be regenerated up to five times without losing any catalyst efficiency. The "click" reaction of Flu568-azide and propargylamine was studied on chip for proof-of-principle. The on-chip reaction yields were ca. 82% with a 50 min reaction time or ca. 55% with a 15 min period at 37 °C, which was higher than those obtained in the conventional reaction. The on-chip "click" reaction involving a biomolecule, cyclo(RGDfK) peptide was also studied and demonstrated a conversion yield of ca. 98%. These encouraging results show promise on the application of the Cu(I) catalyst immobilized "click chip" for the development of biomolecule based imaging agents.

Triazine-based tool box for developing peptidic PET imaging probes: syntheses, microfluidic radiolabeling, and structure-activity evaluation.

This study was aimed at developing a triazine-based modular platform for targeted PET imaging. We synthesized mono- or bis-cyclo(RGDfK) linked triazine-based conjugates specifically targeting integrin αvß3 receptors. The core molecules could be easily linked to targeting peptide and radiolabeled bifunctional chelator. The spacer core molecule was synthesized in 2 or 3 steps in 64-80% yield, and the following conjugation reactions with cyclo(RGDfK) peptide or bifunctional chelator were accomplished using "click" chemistry or amidation reactions. The DOTA-TZ-Bis-cyclo(RGDfK) 13 conjugate was radiolabeled successfully with (64)Cu(OAc)2 using a microfluidic method, resulting in higher specific activity with above 95% labeling yields compared to conventional radiolabeling (SA ca. 850 vs 600 Ci/mmol). The dimeric cyclo(RGDfK) peptide was found to display significant bivalency effect using I(125)-Echistatin binding assay with IC50 value as 178.5 ± 57.1 nM, which displayed a 3.6-fold enhancement of binding affinity compared to DOTA-TZ-cyclo(RGDfK) 14 conjugate on U87MG human glioblastoma cell. Biodistribution of all four conjugates in female athymic nude mice were evaluated. DOTA-"Click"-cyclo(RGDfK) 15 had the highest tumor uptake among these four at 4 h p.i. with 1.90 ± 0.65%ID/g, while there was no clear bivalency effect for DOTA-TZ-BisRGD in vivo, which needs further experiments to address the unexpected questions.