SEW2871 treatment promotes leptomeningeal collateral growth under shear stress conditions.

<p>(A) Changes in cerebral blood flow (CBF) in the border zone between the anterior cerebral artery (ACA) and middle cerebral artery (MCA) in each group (n = 11 for vehicle, 11 for SEW, and 8 for SEW+VPC; ***<i>P</i> < 0.001 compared with vehicle, ^†††<i>P</i> < 0.001 compared with SEW+VPC group; repeated-measures ANOVA followed by Tukey–Kramer post hoc test.) (B) Representative images of superficial vessels on ipsilateral hemisphere after left common carotid artery occlusion (LtCCAO), as assessed after latex perfusion. Magnified images of boxes in the upper panels are shown in the lower panels. Arrows indicate leptomeningeal anastomoses between the ACA and MCA (bars = 100 μm). (C) Average diameters and average numbers of leptomeningeal anastomoses between the ACA and MCA, as assessed after latex perfusion. (n = 7 for vehicle, 7 for SEW, 6 for SEW without CCAO, and 4 for SEW+VPC ***<i>P</i> < 0.001 compared with vehicle group; one-way ANOVA followed by Tukey–Kramer post hoc test). SEW, sphingosine-1-phosphate receptor-1 (S1PR1) selective agonist; VPC, S1PR1 inverse agonist.</p

Sphingosine-1-Phosphate Receptor-1 Selective Agonist Enhances Collateral Growth and Protects against Subsequent Stroke

<div><p>Background and Purpose</p><p>Collateral growth after acute occlusion of an intracranial artery is triggered by increasing shear stress in preexisting collateral pathways. Recently, sphingosine-1-phosphate receptor-1 (S1PR1) on endothelial cells was reported to be essential in sensing fluid shear stress. Here, we evaluated the expression of S1PR1 in the hypoperfused mouse brain and investigated the effect of a selective S1PR1 agonist on leptomeningeal collateral growth and subsequent ischemic damage after focal ischemia.</p><p>Methods</p><p>In C57Bl/6 mice (n = 133) subjected to unilateral common carotid occlusion (CCAO) and sham surgery. The first series examined the time course of collateral growth, cell proliferation, and S1PR1 expression in the leptomeningeal arteries after CCAO. The second series examined the relationship between pharmacological regulation of S1PR1 and collateral growth of leptomeningeal anastomoses. Animals were randomly assigned to one of the following groups: LtCCAO and daily intraperitoneal (ip) injection for 7 days of an S1PR1 selective agonist (SEW2871, 5 mg/kg/day); sham surgery and daily ip injection for 7 days of SEW2871 after surgery; LtCCAO and daily ip injection for 7 days of SEW2871 and an S1PR1 inverse agonist (VPC23019, 0.5 mg/kg); LtCCAO and daily ip injection of DMSO for 7 days after surgery; and sham surgery and daily ip injection of DMSO for 7 days. Leptomeningeal anastomoses were visualized 14 days after LtCCAO by latex perfusion method, and a set of animals underwent subsequent permanent middle cerebral artery occlusion (pMCAO) 7days after the treatment termination. Neurological functions 1hour, 1, 4, and 7days and infarction volume 7days after pMCAO were evaluated.</p><p>Results</p><p>In parallel with the increase in S1PR1 mRNA levels, S1PR1 expression colocalized with endothelial cell markers in the leptomeningeal arteries, increased markedly on the side of the CCAO, and peaked 7 days after CCAO. Mitotic cell numbers in the leptomeningeal arteries increased after CCAO. Administration of the S1PR1 selective agonist significantly increased cerebral blood flow (CBF) and the diameter of leptomeningeal collateral vessels (42.9 ± 2.6 μm) compared with the controls (27.6 ± 5.7 μm; <i>P</i> < 0.01). S1PR1 inverse agonist administration diminished the effect of the S1PR1 agonist (<i>P</i> < 0.001). After pMCAO, S1PR1 agonist pretreated animals showed significantly smaller infarct volume (17.5% ± 4.0% vs. 7.7% ± 4.0%, <i>P</i> < 0.01) and better functional recovery than vehicle-treated controls.</p><p>Conclusions</p><p>These results suggest that S1PR1 is one of the principal regulators of leptomeningeal collateral recruitment at the site of increased shear stress and provide evidence that an S1PR1 selective agonist has a role in promoting collateral growth and preventing of ischemic damage and neurological dysfunction after subsequent stroke in patients with intracranial major artery stenosis or occlusion.</p></div

Domino-Like Intercellular Delivery of Undecylenic Acid-Conjugated Porous Silicon Nanoparticles for Deep Tumor Penetration

Improving the intratumoral distribution of anticancer agents remains the critical challenge for developing efficient cancer chemotherapy. Luminescent porous silicon nanoparticles (PSiNPs) have attracted considerable attention in the biomedical field especially in drug delivery. Here, we described the lysosomal exocytosis-mediated domino-like intercellular delivery of undecylenic acid-conjugated PSiNPs (UA-PSiNPs) for deep tumor penetration. UA-PSiNPs with significantly improved stability in physiological conditions were internalized into tumor cells by macropinocytosis-, caveolae-, and clathrin-mediated endocytosis and mainly colocalized with Golgi apparatus and lysosomes. Substantial evidence showed that UA-PSiNPs was excreted from cells via lysosomal exocytosis after cellular uptake. The exocytosed UA-PSiNPs induced a domino-like infection of adjacent cancer cells and allowed encapsulated doxorubicin (DOX) to deeply penetrate into both three-dimensional tumor spheroids and <i>in vivo</i> tumors. In addition, DOX-loaded UA-PSiNPs exhibited strong antitumor activity and few side effects <i>in vivo</i>. This study demonstrated that UA-PSiNPs as a drug carrier might be applied for deep tumor penetration, offering a new insight into the design of more efficient delivery systems of anticancer drugs

Ischemic surgery and treatment schedule.

<p>In the protocol I, animals were euthanized 1, 4, 7, or 14 days after surgery (A). In protocol II (B) and III (C). SEW, SEW+VPC, or control vehicle (DMSO) were administered intraperitoneally for 7 days after surgery.</p

Smart pH/Redox Dual-Responsive Nanogels for On-Demand Intracellular Anticancer Drug Release

Efficient accumulation and intracellular drug release in cancer cells remain a crucial challenge in developing ideal anticancer drug delivery systems. Here, poly­(<i>N</i>-isopropylacrylamide)-<i>ss</i>-acrylic acid (P­(NIPAM-<i>ss</i>-AA)) nanogels based on NIPAM and AA cross-linked by <i>N,N’</i>-bis­(acryloyl)­cystamine (BAC) were constructed by precipitation polymerization. The nanogels exhibited pH/redox dual responsive doxorubicin (DOX) release behavior in vitro and in tumor cells, in which DOX release from nanogels was accelerated in lysosomal pH (pH 4.5) and cytosolic reduction (10 mM GSH) conditions. Moreover, intracellular tracking of DOX-loaded nanogels confirmed that after the nanogels and the loaded DOX entered the cells simultaneously mainly via lipid raft/caveolae-mediated endocytosis, DOX-loaded nanogels were transported to lysosomes and then the loaded DOX was released to nucleus triggered by lysosomal pH and cytoplasmic high GSH. MTT analysis showed that DOX-loaded nanogels could efficiently inhibit the proliferation of HepG2 cells. In vivo animal studies demonstrated that DOX-loaded nanogels were accumulated and penetrated in tumor tissues more efficiently than free DOX. Meanwhile, DOX-loaded nanogels exhibited stronger tumor inhibition activity and fewer side effects. This study indicated that pH/redox dual-responsive nanogels might present a prospective platform for intracellular drug controlled release in cancer therapy

Increase in expression of sphingosine-1-phosphate receptor-1 (S1PR1) in ipsilateral leptomeningeal arteries after LtCCAO.

<p>(A) Confocal immunofluorescence double-labeling images with anti-CD31 (green) and anti-S1PR1 (red) antibodies in the ipsilateral leptomeningeal arteries 7 and 14 days after LtCCAO and in the sham-operated control. The strong S1PR1 signals (red) were detected at 7 days, but weak at 14 days after LtCCAO (bars = 10 μm). (B) Average percentage of the CD31/S1PR1 double-positive area of the total CD31 positive area after LtCCAO increased until 7 days and then decreased. (n = 6 for each group; *<i>P</i> < 0.05, ***<i>P</i> < 0.001 compared with sham-operated controls; one-way ANOVA followed by Tukey–Kramer post hoc test). (C) Confocal immunofluorescence double-labeling images with anti-CD31 (green) and anti-S1PR1 (red) antibodies in the ipsilateral parenchyma 7 days after LtCCAO and in the sham-operated control (bars = 50 μm). (D) Measurement of S1PR1 mRNA levels in ipsilateral cortex of sham-operated and ltCCAO animals. (n = 2 for sham, 3 for each CCAO group; **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with sham-operated controls; one-way ANOVA followed by Tukey–Kramer post hoc test).</p

Changes in CBF and leptomeningeal anastomosis after LtCCAO.

<p>(A) Changes in CBF in the border zone between the middle cerebral artery (MCA) and anterior cerebral artery (ACA) after CCAO surgery (n = 7 for each group; ***<i>P</i> < 0.001 compared with sham; repeated-measures ANOVA followed by Tukey–Kramer post hoc test). (B) Representative images of superficial vessels on the ipsilateral hemisphere after LtCCAO, as assessed after latex perfusion (bars = 100 μm). Magnified images of the boxes in the upper panels are shown in the lower panels. Leptomeningeal artery between the ACA and MCA is indicated by arrows. The bar graphs indicates average diameter and average number of leptomeningeal anastomoses in the area between the ACA and MCA, as assessed after latex perfusion. CBF, cerebral blood flow; LtCCAO, left common cerebral artery occlusion.</p

Significant increase in the number of proliferating cells in the ipsilateral leptomeningeal arteries after LtCCAO.

<p>Representative images of α-smooth muscle actin staining (A) of the ipsilateral leptomeningeal artery 2 weeks after sham surgery or LtCCAO surgery (scale bars = 10 μm). Dot line indicates the surface of cerebral cortex. There was no significant increase in the average area of ipsilateral leptomeningeal arteries after LtCCAO (B). Representative images of Ki-67 staining of ipsilateral leptomeningeal arteries (bars = 10 μm). The average number of Ki-67-positive cells in the leptomeningeal arteries (per section) after LtCCAO surgery increased until 7 days and then decreased. (n = 6 for each group; **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with sham-operated controls; one-way ANOVA followed by Tukey–Kramer post hoc test).</p

Zwitterionic Temperature/Redox-Sensitive Nanogels for Near-Infrared Light-Triggered Synergistic Thermo-Chemotherapy

Ideal anticancer nano drug delivery systems (NDDSs) need to overcome a series of physiological barriers including blood circulation, tumor accumulation, tumor penetration, internalization by cancer cells, lysosomal escape, and on-demand intracellular drug release following systemic administration. However, it remains a big challenge to construct NDDSs that can overcome all the barriers at the same time. Here, we develop zwitterionic temperature/redox-sensitive nanogels loaded with near-infrared (NIR) dye Indocyanine green (ICG) and anticancer drug doxorubicin (I/D@NG). I/D@NG exhibits enhanced photothermal effects, and NIR irradiation markedly decreases its diameter. NIR irradiation at tumor sites significantly enhances tumor accumulation, tumor penetration, and cellular uptake of I/D@NG with prolonged blood circulation time. Furthermore, I/D@NG can effectively escape from lysosomes by singlet oxygen-induced lysosomal disruption, and DOX is then sufficiently released from the nanogels to the nucleus in response to high intracellular GSH and photothermal effects. This nanoplatform for thermo-chemotherapy not only efficiently exerts synergistic cytotoxicity but also overcomes all the physiological barriers of therapeutic agent, thereby providing a substantial in vivo anticancer effect. The multiple functions of I/D@NG provide new insights into designing nanoplatforms for synergistic cancer therapy

Efficacy, but Not Antibody Titer or Affinity, of a Heroin Hapten Conjugate Vaccine Correlates with Increasing Hapten Densities on Tetanus Toxoid, but Not on CRM₁₉₇ Carriers

Vaccines against drugs of abuse have induced antibodies in animals that blocked the biological effects of the drug by sequestering the drug in the blood and preventing it from crossing the blood-brain barrier. Drugs of abuse are too small to induce antibodies and, therefore, require conjugation of drug hapten analogs to a carrier protein. The efficacy of these conjugate vaccines depends on several factors including hapten design, coupling strategy, hapten density, carrier protein selection, and vaccine adjuvant. Previously, we have shown that <b>1</b> (MorHap), a heroin/morphine hapten, conjugated to tetanus toxoid (TT) and mixed with liposomes containing monophosphoryl lipid A [L­(MPLA)] as adjuvant, partially blocked the antinociceptive effects of heroin in mice. Herein, we extended those findings, demonstrating greatly improved vaccine induced antinociceptive effects up to 3% mean maximal potential effect (%MPE). This was obtained by evaluating the effects of vaccine efficacy of hapten <b>1</b> vaccine conjugates with varying hapten densities using two different commonly used carrier proteins, TT and cross-reactive material 197 (CRM₁₉₇). Immunization of mice with these conjugates mixed with L­(MPLA) induced very high anti-<b>1</b> IgG peak levels of 400–1500 μg/mL that bound to both heroin and its metabolites, 6-acetylmorphine and morphine. Except for the lowest hapten density for each carrier, the antibody titers and affinity were independent of hapten density. The TT carrier based vaccines induced long-lived inhibition of heroin-induced antinociception that correlated with increasing hapten density. The best formulation contained TT with the highest hapten density of ≥30 haptens/TT molecule and induced %MPE of approximately 3% after heroin challenge. In contrast, the best formulation using CRM₁₉₇ was with intermediate <b>1</b> densities (10–15 haptens/CRM₁₉₇ molecule), but the %MPE was approximately 13%. In addition, the chemical synthesis of <b>1</b>, the optimization of the conjugation method, and the methods for the accurate quantification of hapten density are described