LAT1-Targeting Thermoresponsive Fluorescent Polymer Probes for Cancer Cell Imaging.

L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of l-[³H]-leucine, one of the substrates for LAT1 uptake, was investigated. l-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of l-[³H]-leucine, while P(NIPAAm-co-DMAAm) and l-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that l-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that l-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.

TMEM225: a possible protein phosphatase 1γ2 (PP1γ2) regulator localizes to the equatorial segment in mouse spermatozoa.

Tmem225 encodes a putative four-transmembrane domain protein that has an RVxF motif, which is known to be a consensus site for interacting with serine/threonine protein phosphatase 1 (PP1). We previously identified Tmem225 as one of 53 spermatogenesis-associated transmembrane protein genes, with no known physiological function. In this study, we investigated the expression and molecular characteristics of TMEM225 in mice. Tmem225 production was found to be specific to testicular germ cells, with expression increasing during spermatogenesis. In mature spermatozoa, TMEM225 is localized to the equatorial segment of the acrosome but not to the midpiece or tail. TMEM225 appears to be an outer and/or inner acrosomal membrane protein that is lost from the dorsal region of the acrosome after the acrosome reaction. TMEM225 interacts with PP1 in vivo, and a pull-down assay revealed that the carboxy-terminal region of TMEM225 can bind to PP1γ2, the predominant isoform of PP1 in male germ cells. In addition, TMEM225 inhibited PP1γ2 activity in vitro via its RVxF motif. Our results suggest that in mice, TMEM225 is involved in the differentiation and function of spermatozoa through the regulation of PP1γ2 activity, which is necessary for normal spermatogenesis as well as spermatozoa capacitation and motility.

Performance and usability evaluation of novel intradermal injection device Immucise™ and reanalysis of intradermal administration trials of influenza vaccine for the elderly.

Under the pandemic situation, there is an urgent need to produce and acquire sufficient quantities of prophylactic vaccines. It becomes important to devise a way to achieve reliable immunity with lower doses to distribute limited supplies of vaccines to maximum number of people very quickly. Intradermal (ID) vaccination is one such method to increase the effectiveness of vaccines. However, this method has not been widely used in general clinical practice because it is technically difficult to inject vaccines precisely into the ID tissue. Therefore, new ID delivery systems that allow reliable ID administration are under development. In this paper, we summarize its design and present the results of performance and usability testing for the Immucise™ Intradermal Injection System (Immucise™). This study showed that Immucise™ can reduce dead volume and inject drugs precisely into the ID tissues of subjects from infants to the elderly and can be used correctly and safely by healthcare professionals. This randomized controlled trial compared ID administration with Immucise™ and standard subcutaneous (SC) administration of seasonal influenza vaccine by analyzing the efficacy of the vaccine in the elderly group at 90 days and 180 days after administration. It was found that the vaccine for the ID group was as effective or more effective than that for the SC group up to 180 days later. It was also found that the geometric mean titer values, especially for B strains, were higher in the two-dose ID group than in the two-dose SC group. These findings suggest that Immucise™ is one of the best devices to distribute a small amount of vaccine quickly and widely to a larger number of people with little loss of vaccine during a pandemic.

Temperature-Responsive Fluorescence Polymer Probes with Accurate Thermally Controlled Cellular Uptakes.

Poly(N-isopropylacrylamide) (PNIPAAm)-based temperature-responsive fluorescence polymer probes were developed using radical polymerization, with 3-mercaptopropionic acid as the chain-transfer agent, followed by activation of terminal carboxyl groups with N-hydroxysuccinimide and reaction with 5-aminofluorescein (FL). The lower critical solution temperatures (LCSTs) of the resulting fluorescent polymer probes differed depending on the copolymer composition, and had a sharp phase-transition (hydrophilic/hydrophobic) boundary at the LCST. The cellular uptakes of the fluorescent polymer probes were effectively suppressed below the LCST, and increased greatly above the LCST. In particular, the cellular uptake of a copolymer with N,N-dimethylaminopropylacrylamide, P(NIPAAm-co-DMAPAAm2%)-FL (LCST: 37.4 °C), can be controlled within only 1 °C near body temperature, which is suitable for biological applications. These results indicated that the cellular uptakes of thermoresponsive polymers could be accurately controlled by the temperature, and such polymers have potential applications in discriminating between normal and pathological cells, and in intracellular drug delivery systems with local hyperthermia.