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Enzymic hydrolyzed chitosan was employed to prepare chitosan-tripolyphosphate and chitosan-polyphosphoric acid gel beads using a polyelectrolyte complexation method for the sustained-release of anticancer agent, 6-mercaptopurine (6-MP). pH responsive swelling ability, drug-release characteristics, and morphology of the chitosan gel bead depends on polyelectrolyte complexation mechanism and molecular weight of the enzymic hydrolyzed chitosan. The complexation mechanism of chitosan beads gelled in pentasodium tripolyphosphate or polyphosphoric acid solution was ionotropic crosslinking or interpolymer complex, respectively. The drug-release patterns of all chitosan gel beads in pH 6.8 seemed to be diffusional based, which might be in accordance with the Higuchi model, whereas release profiles of the chitosan-tripolyphosphate gel beads in pH 1.2 medium seemed to be non-Fickian diffusion controlled due to the swelling or matrix erosion of the beads. The rate of 6-MP releasing from chitosan-tripolyphosphate or chitosanpolyphosphoric acid gel matrix were significantly increased with the decreased molecular weight of enzymic hydrolyzed chitosan. However, the dissolution rates of 6-MP entraped in chitosan-tripolyphosphate and chitosan-polyphosphoric acid gel matrix were significantly slower than the dissolution rate of the original drug. These results indicate that the chitosan-polyphosphoric acid gel bead is a better polymer carrier for the sustained release of anticancer drugs in simulated intestinal and gastric juice medium than the chitosantripolyphosphate gel beads.

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The aim of this study was to establish a basic manipulation protocol of preantral follicles for deriving developmentally competent oocytes. Primary, early and late secondary follicles retrieved from the ovaries of 14-day-old F1 (C57BL/6 x DBA2) female mice mechanically or enzymatically were cultured singly and in vitro growth of the follicles and maturation of intrafollicular oocytes were subsequently monitored. A mechanical method retrieved more (p < 0.0001) follicles (339 +/- 48 vs. 202 +/- 28) than an enzymatic method. However, the enzymatic method collected more singly isolated follicles that could be provided for subsequent culture (102 +/- 26 vs. 202 +/- 28). When an enzymatic method was employed, early and late secondary follicles required 9 and 6 days for reaching the maximal incidence of the pseudoantral stage. However, primary follicles were not possible to develop into the pseudoantral stage. The optimal duration of oocyte maturation from the onset of follicle culture was 7 days and 5-7 days for early and late secondary follicles, respectively. A general decrease in oocyte diameter (65.2-65.53 microm vs. 75 microm) and zona thickness (5.41-5.74 microm vs. 7.76 microm) was detected in in vitro-derived compared with in vivo-derived matured oocytes. Pronuclear formation was detected in 86-94% of mature oocytes after parthenogenetic activation and no significant difference was detected among groups. These results showed that preantral follicles retrieved by an enzymatic method underwent step-by-step growth in vitro, which could yield mature oocytes.

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While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB+SSEA1+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB− cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.