Noninvasive Detection of Passively Targeted Poly(ethylene glycol) Nanocarriers in Tumors

The present studies noninvasively investigate the passive tumor distribution potential of a series of poly­(ethylene glycol) (PEG) nanocarriers using a SkinSkan spectrofluorometer and an In Vivo Imaging System (IVIS) 100. Fluorescein conjugated PEG nanocarriers of varying molecular weights (10, 20, 30, 40, and 60 kDa) were prepared and characterized. The nanocarriers were administered intravenously to female balb/c mice bearing subcutaneous 4T1 tumors. Passive distribution was measured in vivo (λ_exc, 480 nm; λ_em, 515–520 nm) from the tumor and a contralateral skin site (i.e., control site). The signal intensity from the tumor was always significantly higher than that from the contralateral site. Trends in results between the two methods were consistent with tumor distribution increasing in a molecular weight-dependent manner (10 < 20 < 30 ≪ 40 ≪ 60 kDa). The 10 kDa nanocarrier was not detected in tumors at 24 h, whereas 40–60 kDa nanocarriers were detected in tumors for up to 96 h. The 30, 40, and 60 kDa nanocarriers showed 2.1, 5.3, and 4.1 times higher passive distribution in tumors at 24 h, respectively, as compared to the 20 kDa nanocarrier. The 60 kDa nanocarrier exhibited 1.5 times higher tumor distribution than 40 kDa nanocarrier at 96 h. Thus, PEG nanocarriers (40 and 60 kDa) with molecular weights close to or above the renal exclusion limit, which for globular proteins is ≥45 kDa, showed significantly higher tumor distribution than those below it. The hydrodynamic radii of PEG polymers, measured using dynamic light scattering (DLS), showed that nanocarriers obtained from polymers with hydrodynamic radii ≥8 nm exhibited higher tumor distribution. Ex vivo mass balance studies revealed that nanocarrier tissue distribution followed the rank order tumor > lung > spleen > liver > kidney > muscle > heart, thus validating the in vivo studies. The results of the current studies suggest that noninvasive dermal imaging of tumors provides a reliable and rapid method for the initial screening of nanocarrier tumor distribution pharmacokinetics

CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse

<div><p><i>Cyclin Y-like 1</i> (<i>Ccnyl1</i>) is a newly-identified member of the cyclin family and is highly similar in protein sequences to <i>Cyclin Y</i> (<i>Ccny</i>). However, the function of <i>Ccnyl1</i> is poorly characterized in any organism. Here we found that <i>Ccnyl1</i> was most abundantly expressed in the testis of mice and was about seven times higher than the level of <i>Ccny</i>. Male <i>Ccnyl1</i>-/- mice were infertile, whereas both male and female <i>Ccny</i>-/- mice displayed normal fertility. These results suggest that <i>Ccnyl1</i>, but not <i>Ccny</i>, is indispensable for male fertility. Spermatozoa obtained from <i>Ccnyl1</i>-/- mice displayed significantly impaired motility, and represented a thinned annulus region and/or a bent head. We found that the protein, but not the mRNA, level of cyclin-dependent kinase 16 (CDK16) was decreased in the testis of <i>Ccnyl1</i>-/- mice. Further study demonstrated that CCNYL1 interacted with CDK16 and this interaction mutually increased the stability of these two proteins. Moreover, the interaction increased the kinase activity of CDK16. In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1. We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity. Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.</p></div

Identification and functional study of phosphorylation modifications of CDK16.

<p>(A) Testicular lysates from two pairs of WT and <i>Ccnyl1</i>-/- mice were incubated with or without calf intestinal phosphatase (CIP) at 37°C for 30 min and analyzed by western blotting. (B) CDK16-Flag was either expressed alone or together with CCNYL1-HA in HEK293T cells. The cell lysates were incubated with or without CIP, and analyzed by western blotting. (C) Phosphorylation sites on CDK16 identified by mass spectrometry. Sites that changed significantly in phosphorylation levels between single- (CDK16-Flag) and double- (CDK16-Flag and CCNYL1-HA) expressed groups are labeled red with asterisks. (D) Various CDK16-Flag mutants were coexpressed with CCNYL1-HA in HEK293T cells. The interactions were analyzed by CoIP experiments followed by western blot. (E) Varies CDK16-Flag mutants were either expressed alone or coexpressed with CCNYL1-HA in HEK293T cells. The CDK16-Flag protein was immunoprecipitated, and incubated with MBP as substrate in the kinase buffer. Reaction products were separated by SDS-PAGE and followed by autoradiography.</p

The interaction of CCNYL1 and CDK16 and protection of protein stability.

<p>(A) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells. Protein expression levels were measured by western blotting. (B) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM cycloheximide for the indicated times. Protein levels were measured by western blotting. (C) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 h followed by incubation with 10 μM MG132. Then, cells were harvested at 24 h for the detection of CDK16-Flag, or at 6 h for the detection of CCNYL1-HA. Protein levels were measured by western blotting. (D) CDK16-Flag and CCNYL1-HA were either expressed alone or together in HEK293T cells for 24 hours followed by incubation with 10 μM MG132 for 6 h. The CDK16-Flag and CCNYL1-HA protein were immunoprecipitated for the detection of their ubiquitination levels by western blotting.</p

Essential function of CCNYL1 for male fertility and sperm motility.

<p>(A) Measurement of CCNYL1 or CCNY protein expression in testes of WT, <i>Ccnyl1</i>-/-, and <i>Ccny</i>-/- mice, with β-actin serving as loading control. (B) Male fertility of adult WT, <i>Ccnyl1</i>-/- and <i>Ccny</i>-/- mice (n ≥ 6 per group). Pregnancy was counted in mating cages with male WT, <i>Ccnyl1</i>-/-, or <i>Ccny</i>-/- mice. (C) Number of spermatozoa in male WT, <i>Ccnyl1</i>-/-, and <i>Ccny</i>-/- mice collected from the caput and cauda epididymidis (n ≥ 6 per group). (D) HE staining of testicular sections from adult WT and <i>Ccnyl1</i>-/- mice. Scale bar: 100μm. (E-G) The percentage of total and progressive motility (E), velocity (F) and the motility distribution (G) of spermatozoa were measured by CASA (n = 5 animals per group). (H) <i>In vitro</i> fertilization assay. Oocytes were incubated with cauda epididymidal spermatozoa collected from <i>WT</i> or <i>Ccnyl1-/-</i> mice. The development of the oocytes was monitored <i>in vitro</i>. Data in bar graphs are presented as mean ± SEM. *<i>P</i> < 0.05; **<i>P</i> < 0.01.</p

Interaction of CCNYL1 with CDK16.

<p>(A) Co-immunolabeling of CCNYL1 (red) and CDK16 (green) in testicular sections of WT mice. Nuclei were labeled with DAPI (blue). Scale bar: 50 μm. (B) CDK16 protein expression was measured in membrane extracts and cytoplasmic extracts isolated from testes or germ cells of WT and <i>Ccnyl1-/-</i> mice. N-cadherin served as the membrane protein control, while β-actin served as the cytoplasmic protein control. (C) CDK16 protein was immunoprecipitated from testicular lysate with CDK16 antibody. Both CDK16 and the Co-immunoprecipitated CCNYL1 were analyzed by western blotting. (D) HEK293T cells were co-transfected with N-terminal tagged CCNYL1<i>-</i>HA and C-terminal tagged CDK16-Flag plasmids. CCNYL1-HA and CCNYL1-Flag proteins were co-immunoprecipitated using either anti-HA or anti-Flag conjugated agarose before analyzed by western blot. (E) HEK293T cells were co-transfected with CDK16-Flag and CCNYL1-HA, or CDK16-Flag and CCNYL-HA plasmids, proteins were co-immunoprecipitated by using anti-Flag conjugated agarose before analyzed by western blotting. (F) Truncated CCNYL1-HA variants were coexpressed with CDK16-Flag in HEK293T cells. The interactions were analyzed by CoIP experiments followed by western blotting. (G) CDK16-Flag variants were co-expressed with CCNYL1-HA in HEK293T cells. The interactions were analyzed by CoIP experiments followed by western blotting. PCTAIRE is the conserved sequence of CDK16 protein. PCTAIRE mutant (PCTAIRE was mutated to ACAAIAA).</p

The expression pattern of CCNYL1 in mice.

<p>(A) <i>Ccnyl1</i> and <i>Ccny</i> mRNA levels were measured in different tissues of adult WT mice (n = 4). (B) <i>Ccnyl1</i> and <i>Ccny</i> mRNA levels were measured in testes of ICR and BABL/c mouse lines (n = 4 per group). (C) Both <i>Ccnyl1</i> and <i>Ccny</i> mRNA levels were measured in testes of WT mice of different age (n = 4). (D) CCNYL1 protein levels were measured in different tissues of adult WT mice, and in testes of WT mice of different ages. β-actin served as the protein loading control. (E) Immunolabeling of CCNYL1 (red) during the spermatogenic cycle of testicular sections. Nuclei were labeled with DAPI (blue). R: round spermatids (white arrows); E: elongated spermatids (yellow arrows); spermatozoa with shed residual bodies (asterisks). Scale bar for left and middle panels: 50 μm, scale bar for right panel: 25 μm. (F) CCNYL1 protein expression was measured in membrane extracts (designated as M) and cytoplasmic extracts (designated as C) isolated from testicular or germ cells of WT and <i>Ccnyl1-/-</i> mice. N-cadherin served as the membrane protein-loading control, whereas β-actin served as the cytoplasmic protein loading control. Data in bar graphs are presented as mean ± SEM.</p

Morphological defects of <i>Ccnyl1</i>-/- spermatozoa.

<p>(A) Phase Contrast images of spermatozoa collected from the testis, caput and cauda epididymidis of adult WT and <i>Ccnyl1</i>-/- mice. <i>Ccnyl1</i>-/- spermatozoa showed a thinning of annulus (white arrows) and a bent head wrapped around the neck (red arrows). Scale bar: 20μm. The ratios of defects are summarized in the histograms below (mice: n≥ 3 animals per group). Data are presented as mean ± SEM. (B) TEM images of spermatozoa collected from the cauda epididymidis of adult WT and <i>Ccnyl1</i>-/- mice. MP: Middle Piece; PP: Principal Piece. The annulus structure (black arrows) closely linked the MP and PP in WT spermatozoa, but was distant from the MP as the thinning of the annulus region in <i>Ccnyl1</i>-/- spermatozoa. Microtubules (green arrows) were dispersed out of the PP in <i>Ccnyl1</i>-/- spermatozoa. Two <i>Ccnyl1-/-</i> spermatozoa with bent heads wrapped by cytoplasmic contents are shown (blue arrows). Scale bar for the top and middle panels: 0.5 μm, scale bar for the bottom panels: 1 μm. (C and D) Immuno-labeling of α-tubulin (C) and F-actin (D) of spermatozoa collected from cauda epididymidis of adult WT and <i>Ccnyl1</i>-/- mice. Microtubules (yellow arrows) and F-actin (pink arrows) were extruded from of this region in spermatozoa of <i>Ccnyl1</i>-/- mice, scale bar: 5 μm.</p

Gelation Chemistries for the Encapsulation of Nanoparticles in Composite Gel Microparticles for Lung Imaging and Drug Delivery

The formation of 10–40 μm composite gel microparticles (CGMPs) comprised of ∼100 nm drug containing nanoparticles (NPs) in a poly­(ethylene glycol) (PEG) gel matrix is described. The CGMP particles enable targeting to the lung by filtration from the venous circulation. UV radical polymerization and Michael addition polymerization reactions are compared as approaches to form the PEG matrix. A fluorescent dye in the solid core of the NP was used to investigate the effect of reaction chemistry on the integrity of encapsulated species. When formed via UV radical polymerization, the fluorescence signal from the NPs indicated degradation of the encapsulated species by radical attack. The degradation decreased fluorescence by 90% over 15 min of UV exposure. When formed via Michael addition polymerization, the fluorescence was maintained. Emulsion processing using controlled shear stress enabled control of droplet size with narrow polydispersity. To allow for emulsion processing, the gelation rate was delayed by adjusting the solution pH. At a pH = 5.4, the gelation occurred at 3.5 h. The modulus of the gels was tuned over the range of 5 to 50 kPa by changing the polymer concentration between 20 and 70 vol %. NP aggregation during polymerization, driven by depletion forces, was controlled by the reaction kinetics. The ester bonds in the gel network enabled CGMP degradation. The gel modulus decreased by 50% over 27 days, followed by complete gel degradation after 55 days. This permits ultimate clearance of the CGMPs from the lungs. The demonstration of uniform delivery of 15.8 ± 2.6 μm CGMPs to the lungs of mice, with no deposition in other organs, is shown, and indicates the ability to concentrate therapeutics in the lung while avoiding off-target toxic exposure