Optimizing Nanosuspension Drug Release and Wound Healing Using a Design of Experiments Approach: Improving the Drug Delivery Potential of NDH-4338 for Treating Chemical Burns.

NDH-4338 is a highly lipophilic prodrug comprising indomethacin and an acetylcholinesterase inhibitor. A design of experiments approach was used to synthesize, characterize, and evaluate the wound healing efficacy of optimized NDH-4338 nanosuspensions against nitrogen mustard-induced skin injury. Nanosuspensions were prepared by sonoprecipitation in the presence of a Vitamin E TPGS aqueous stabilizer solution. Critical processing parameters and material attributes were optimized to reduce particle size and determine the effect on dissolution rate and burn healing efficacy. The antisolvent/solvent ratio (A/S), dose concentration (DC), and drug/stabilizer ratio (D/S) were the critical sonoprecipitation factors that control particle size. These factors were subjected to a Box-Behnken design and response surface analysis, and model quality was assessed. Maximize desirability and simulation experiment optimization approaches were used to determine nanosuspension parameters with the smallest size and the lowest defect rate within the 10-50 nm specification limits. Optimized and unoptimized nanosuspensions were prepared and characterized. An established depilatory double-disc mouse model was used to evaluate the healing of nitrogen mustard-induced dermal injuries. Optimized nanosuspensions (A/S = 6.2, DC = 2% w/v, D/S = 2.8) achieved a particle size of 31.46 nm with a narrow size range (PDI = 0.110) and a reduced defect rate (42.2 to 6.1%). The optimized nanosuspensions were stable and re-dispersible, and they showed a ~45% increase in cumulative drug release and significant edema reduction in mice. Optimized NDH-4338 nanosuspensions were smaller with more uniform sizes that led to improved physical stability, faster dissolution, and enhanced burn healing efficacy compared to unoptimized nanosuspensions.

Depilatory double-disc mouse model for evaluation of vesicant dermal injury pharmacotherapy countermeasures.

BACKGROUND: Sulfur mustard (SM) is a chemical warfare vesicant that severely injures exposed eyes, lungs, and skin. Mechlorethamine hydrochloride (NM) is widely used as an SM surrogate. This study aimed to develop a depilatory double-disc (DDD) NM skin burn model for investigating vesicant pharmacotherapy countermeasures. METHODS: Hair removal method (clipping only versus clipping followed by a depilatory), the effect of acetone in the vesicant administration vehicle, NM dose (0.5-20 µmol), vehicle volume (5-20 µl), and time course (0.5-21 days) were investigated using male and female CD-1 mice. Edema, an indicator of burn response, was assessed by biopsy skin weight. The ideal NM dose to induce partial-thickness burns was assessed by edema and histopathologic evaluation. The optimized DDD model was validated using an established reagent, NDH-4338, a cyclooxygenase, inducible nitric oxide synthase, and acetylcholinesterase inhibitor prodrug. RESULTS: Clipping/depilatory resulted in a 5-fold higher skin edematous response and was highly reproducible (18-fold lower %CV) compared to clipping alone. Acetone did not affect edema formation. Peak edema occurred 24-48 h after NM administration using optimized dosing methods and volume. Ideal partial-thickness burns were achieved with 5 µmol of NM and responded to treatment with NDH-4338. No differences in burn edematous responses were observed between males and females. CONCLUSION: A highly reproducible and sensitive partial-thickness skin burn model was developed for assessing vesicant pharmacotherapy countermeasures. This model provides clinically relevant wound severity and eliminates the need for organic solvents that induce changes to the skin barrier function.

Primary Osteosarcoma of the Breast: A Rare Case Report and Literature Review.

Background: Primary osteosarcoma of the breast (POB) is an extremely aggressive and heterogeneous neoplasm that originates from nonepithelial elements of the mammary gland and accounts for fewer than 1% of breast cancers and fewer than 5% of all sarcomas. Case Presentation: An 83-year-old Chinese woman went to our hospital because of a palpable mass she had had for 8 months in the left breast accompanied by persistent dull pain for 10 days. This mass was initially misdiagnosed as a degenerating fibroadenoma and was graded as probably benign (BI-RADS category 3) by ultrasonography (US) and computed tomography (CT) plain scan and contrast enhancement of chest. Eight months later, it was presumed to be highly malignant and graded as BI-RADS category 4C because of its rapid growth and more calcifications by US and CT. 99mTc-MDP whole-body bone imaging showed that there was a mass-like abnormal radioactive concentration of Tc-99m outside the bone of the left chest. The lumpectomy of the left breast was indicated, and the pathological findings were POB. She succumbed to respiratory failure caused by multiple lung metastases 4 months after the operation. Conclusion: POB is rare, and US and CT cannot reliably distinguish the causes of calcified breast masses between benign and malignant tumors. It can be diagnosed by pathology when metaplastic carcinoma, malignant phyllodes tumor, or carcinosarcoma containing osteoid and bone is excluded. This case could help clinicians to improve the prognosis and treatment of this disease.

Effects of Rapid Igneous Intrusion Heating on the Geochemistry, Petrography, and Microcrystalline Structure of Coals from Huainan, China.

Igneous intrusion into coal-bearing strata may change the geochemical, petrographic, and microcrystalline structural characteristics of coal. Here, a series of coal samples affected by igneous intrusion were analyzed by petrography, geochemistry, and X-ray diffraction. In addition, the trend observed in altered coal with normal burial maturity is compared to evaluate whether the intrusive coal follows another maturity path. A petrographic analysis shows that the R 0 value increased rapidly and lost the ability to distinguish liptinite. Pyrolytic carbon and isotropic and anisotropic coke with a fine-grained circular mosaic structure are formed at the intrusion. Moreover, the degree of structural order of coal samples increases in an approach to the intrusion. There are transition phases with different structural orders due to different degrees of metamorphism. Petrographic and geochemical data indicate that intrusive coals may follow a maturation pathway other than that from normal burial maturation, which may be related to the rapid geological thermal event related to the intrusion. However, the results of XRD data suggest that the microcrystalline structure of igneous intrusion coals is consistent with a growth in the trend of normal burial. This study of geochemical petrography and microcrystalline structure of surrounding coal seams by rapid intrusive heating of igneous intrusions not only greatly improves the natural coke industrial utilization but also provides an important theoretical basis for the generation and enrichment of coalbed methane in igneous thermal abnormal coal reservoirs.

Breast intraductal nanoformulations for treating ductal carcinoma in situ II: Dose de-escalation using a slow releasing/slow bioconverting prodrug strategy.

Ductal carcinoma in situ (DCIS) represents approximately 20-25% of newly diagnosed breast cancers. DCIS is treated by surgery and possibly radiotherapy. Chemotherapy is only used as adjuvant or neoadjuvant therapy but not as primary therapy. The present study investigated the intraductal administration of Ciclopirox (CPX) formulated in nanosuspensions (NSs) or nanoparticles (NPs) to treat DCIS locally in a Fischer 344 rat model orthotopically implanted with 13762 Mat B III cells. Slow converting esterase responsive CPX prodrugs (CPDs) were successfully synthesized at high purity (> 95%) by directly acetylating the hydroxyl group or by appending a self-immolative linker between CPX and a phenolic ester. Direct esterification CPDs were not sufficiently stable so self-immolative CPDs were formulated in NSs and NPs. Prodrug release was evaluated from poly(lactic-co-glycolic acid) NPs, and CPD4 demonstrated the slowest release rate with the rank order of CPD2 (R = methyl) > CPD3 (R = t-butyl) > CPD4 (R = phenyl). Intraductally administered CPX NS, CPD4 NS, and an innovative mixture of CDP4 NS and NPs (at 1 mg CPX equivalent/duct) demonstrated significant (p < 0.05) in vivo anti-tumor efficacy compared with immediate release (IR) CPX NS and non-treated controls. CPX mammary persistence at 6 h and 48 h after CPD4 NS or NP administration was also greater than after the immediate release CPX NS. A strong correlation between CPX mammary persistence and efficacy is demonstrated. In conclusion, nanoformulations utilizing a slow releasing/slow bioconverting CPX prodrug delivery strategy resulted in significant dose de-escalation (~ five fold) while maintaining anti-tumor efficacy.

The Chemical and Alignment Structural Properties of Coal: Insights from Raman, Solid-State 13C NMR, XRD, and HRTEM Techniques.

The chemical and alignment structures of coal impacts coalbed methane behavior: adsorption, desorption, and diffusion. Recently, the research on accurate characterization techniques for coal structure has received widespread attention. In particular, spatial alignment is critical for the molecular modeling of coal. However, due to the great challenges of quantification, spatial alignment has often been ignored in previous studies. In this study, high-resolution transmission electron microscopy (HRTEM) was employed to quantitatively characterize the fringe length, orientation, and stacking distributions of these five coal samples with different ranks. Raman spectroscopy was utilized to investigate the overall structural disorder of the coal molecules. 13C nuclear magnetic resonance (13C NMR) was conducted to characterize the chemical structures of coals, and XRD experiments recorded the transition of the microcrystallite structure. The results show that in the range of %R o = 0.39-2.07%, the distributions of the aromatic structural units were similar: mainly composed of fringes of size equivalent to naphthalene and 2 × 2 and 3 × 3 rings. When %R o > 2.07%, the distribution shifted to longer fringes. Moreover, all the samples showed a regional orientation, and when %R o > 2.07%, there was significantly higher alignment. The degree of stacking of fringes were limited, most of which appeared in the form of a single layer. When %R o < 2.07%, the stacking appeared in the form of two or three layers. However, five-layer stacking merely appeared in the sample with %R o = 2.47%. In addition, based on the Raman data, the evolution of carbon disorder was divided into three stages: %R o = 0.39-1.23%, 1.23-2.07%, and 2.07-2.47%, and aromatization caused the overall disorder to decrease. The 13C NMR data indicated that the chemical structure also transitioned in stages, with aliphatic carbon and oxygen-containing groups gradually decreasing and aromatic carbon increasing. Meanwhile, the XRD data supported increased organization (lower d 002 values) with maturities. Thus, this study provides quantitative information about the spatial alignment and the size of aromatic rings, which helps to improve a comprehensive understanding of the chemical structure of coal and coalbed methane behaviors.

Breast intraductal nanoformulations for treating ductal carcinoma in situ I: Exploring metal-ion complexation to slow ciclopirox release, enhance mammary persistence and efficacy.

INTRODUCTION: Ductal Carcinoma In Situ (DCIS) represents a significant fraction (~20-25%) of all newly diagnosed breast cancer cases and, if left untreated, a significant fraction of patients will progress to invasive disease. Surgery is the only treatment option. Ciclopirox (CPX), an FDA-approved antifungal drug, has exhibited promising antitumor activity by down-regulating the expression of vital antiapoptotic cellular proteins and inhibiting the genetic expression of several oncogenic pathways. In this study, the feasibility of using nanoscale delivery systems to control release and prolong mammary tissue persistence of a lipophilic metal complex of CPX and Zinc (CPXZn) after intraductal administration was investigated. METHODS: CPX and CPX-Zn nanosuspensions (NSs) were prepared using an evaporative nanoprecipitation-ultra-sonication method. Flash nanoprecipitation was used to prepare PLGA nanoparticles (NPs) loaded with CPXZn. Our established orthotopic DCIS rat model was used to evaluate efficacy. Briefly, two days after 13762 Mat B III cell intraductal inoculation, rats were divided into treatment groups and a single intraductal injection of CPX NS, CPX-Zn NS or CPX-Zn NPs was administered. In the first study arm, the efficacy of CPX NS (1, 3, 5 mg/duct) was evaluated. In the second arm, the in vivo efficacy of CPX NS, CPX-Zn NS and CPX-Zn loaded NPs was evaluated and compared at equivalent CPX doses. The mammary persistence of CPX from CPX NS, CPX-Zn NS, and CPX-Zn PLGA NPs was also assessed. RESULTS: CPX-Zn complex was successfully synthesized and characterized by several spectral analyses. CPX release was slowed from the CPX-Zn NS and further slowed by incorporating CPX-Zn into PLGA NPs as compared to the CPX NS with release half times following the order: CPX NS < CPX-Zn NS << CPX-Zn NP. Intraductal CPX NS administration was dose and time dependent in suppressing tumor initiation suggesting prolonged mammary exposure may improve efficacy. In the second arm, mammary tissue persistence of CPX followed the rank order CPX NS < CPX-Zn NS << CPX-Zn NP at 6 h and 48 h post-administration. Prolonged mammary CPX exposure was highly correlated to improved efficacy. Prolonged CPX tissue persistence, attributed to slower release from the zinc complex and the PLGA NPs, resulted in a 5-fold dose reduction compared to the CPX NS. CONCLUSIONS: The current results demonstrate that slowing drug release in the mammary duct after intraductal administration overcomes the rapid ductal clearance of CPX, prolongs mammary tissue persistence, improves efficacy against DCIS lesions in vivo, and requires 5-fold less CPX to achieve equivalent efficacy. The studies also provide a strategic path forward for developing a locally administered drug delivery system for treating DCIS, for which no primary chemotherapy option is available.

Evaluation of intraductal delivery of poly(ethylene glycol)-doxorubicin conjugate nanocarriers for the treatment of ductal carcinoma in situ (DCIS)-like lesions in rats.

Ductal carcinoma in situ is the most commonly diagnosed early stage breast cancer. The efficacy of intraductally delivered poly(ethylene glycol)-doxorubicin (PEG-DOX) nanocarriers, composed of one or more DOX conjugated to various PEG polymers, was investigated in an orthotopic ductal carcinoma in situ-like rat model. In vitro cytotoxicity was evaluated against 13762 Mat B III cells using MTT assay. The orthotopic model was developed by inoculating cancer cells into mammary ducts of female Fischer 344 retired breeder rats. The ductal retention and in vivo antitumour efficacy of two of the six nanocarriers (5 kDa PEG-DOX and 40 kDa PEG-(DOX)4) were investigated based on in vitro results. Mammary retention of DOX and PEG-DOX nanocarriers was quantified using in vivo imaging. Histopathologic effects of DOX and PEG-DOX nanocarriers on mammary ductal structure were also investigated. Cytotoxicities of small linear PEG-DOX nanocarriers (5 and 10 kDa) were not different from DOX whereas larger PEG-DOX nanocarriers showed reduced potency. The order of mammary retention was 40 kDa PEG-(DOX)4 > 5 kDa PEG-DOX >> DOX, in normal and tumour-bearing rats. Intraductally administered PEG-DOX nanocarriers and DOX were effective in reducing tumour incidence and increasing survival rate, with no significant differences found among the three treatment groups. However, nanocarriers administered intravenously at the same doses were not effective, and intraductally administered free DOX caused severe local toxicity. Intraductal administration of PEG-DOX nanocarriers is effective and less toxic than that of free DOX, as well as IV DOX/PEG-DOX. Furthermore, PEG-DOX nanocarriers demonstrate the added benefit of prolonging DOX ductal retention, which would necessitate less frequent dosing.

The effect of size and polymer architecture of doxorubicin-poly(ethylene) glycol conjugate nanocarriers on breast duct retention, potency and toxicity.

Although systemic administration of chemotherapeutic agents is routinely used for treating invasive breast cancer, the only therapeutic options for ductal carcinoma in situ (DCIS) are surgery and radiation. Treating DCIS by delivering drugs locally to the affected milk duct offers significant advantages over systemic administration, including reduced systemic and breast toxicities, as well as a greatly reduced need for surgery and radiation. In this study, mammary gland retention and toxicity of intraductally administered poly(ethylene) glycol-doxorubicin (PEG-DOX) polymeric conjugate nanocarriers of varying molecular sizes and architectures were investigated. Nanocarriers were formed by conjugating one or more copies of doxorubicin to PEG polymers, of varying molecular weights (5, 10, 20, and 40 kDa) and architectures (linear, four-arm and eight-arm). Cytotoxicity against MCF7 cells, a human breast cancer cell line, was assessed, and IC50 values were calculated. The nanocarriers were intraductally administered into the mammary glands of female retired breeder Sprague-Dawley rats. Whole body images were captured using in vivo optical imaging, and changes in ductal structure as well local inflammation were monitored. Fluorescence intensities were monitored, over time, to evaluate nanocarrier mammary gland retention half-lives (t1/2). The IC50 values of PEG-DOX nanocarriers against MCF7 cells were 40 kDa PEG-(DOX)4 (1.23 µM) < 5 kDa PEG-DOX (1.76 µM) < 40 kDa PEG-(DOX)8 (3.49 µM) < 10 kDa PEG-DOX (3.86 µM) < 20 kDa PEG-DOX (8.96 µM) < 40 kDa PEG-DOX (18.11 µM), whereas the IC50 of free DOX was only 0.14 µM. The t1/2 of linear 5, 20, and 40 kDa nanocarriers were 2.2 ±â€¯0.3, 3.6 ±â€¯0.6, and 13.1 ±â€¯3.4 h, whereas the retention t1/2 of 4- and 8-arm 40 kDa nanocarriers were 14.9 ±â€¯5.6 h and 11.9 ±â€¯2.9 h, respectively. The retention t1/2 of free doxorubicin was 2.0 ±â€¯0.4 h, which was significantly shorter than that of the linear and branched 40 kDa PEG-DOX nanocarriers. Increased molecular weight and decreased branching both demonstrated a strong correlation to enhanced mammary gland retention. Intraductally administered free doxorubicin resulted in ductal damage, severe inflammation and generation of atypical cell neoplasms, whereas PEG-DOX nanocarriers induced only minor and transient inflammation (i.e., damaged epithelial cells and detached cellular debris). The 40 kDa 4-arm PEG-DOX nanocarrier demonstrated the longest ductal retention half-life, the lowest IC50 (i.e., most potent), and minimal ductal damage and inflammation. The current results suggest that PEG-DOX nanocarriers with prolonged ductal retention may present the best option for intraductal treatment of DCIS, due to their low local toxicity and potential for sustained therapeutic effect.

Single-Step Assembly of Multimodal Imaging Nanocarriers: MRI and Long-Wavelength Fluorescence Imaging.

Magnetic resonance imaging (MRI)- and near-infrared (NIR)-active, multimodal composite nanocarriers (CNCs) are prepared using a simple one-step process, flash nanoprecipitation (FNP). The FNP process allows for the independent control of the hydrodynamic diameter, co-core excipient and NIR dye loading, and iron oxide-based nanocrystal (IONC) content of the CNCs. In the controlled precipitation process, 10 nm IONCs are encapsulated into poly(ethylene glycol) (PEG) stabilized CNCs to make biocompatible T2 contrast agents. By adjusting the formulation, CNC size is tuned between 80 and 360 nm. Holding the CNC size constant at an intensity weighted average diameter of 99 ± 3 nm (PDI width 28 nm), the particle relaxivity varies linearly with encapsulated IONC content ranging from 66 to 533 × 10(-3) m(-1) s(-1) for CNCs formulated with 4-16 wt% IONC. To demonstrate the use of CNCs as in vivo MRI contrast agents, CNCs are surface functionalized with liver-targeting hydroxyl groups. The CNCs enable the detection of 0.8 mm(3) non-small cell lung cancer metastases in mice livers via MRI. Incorporating the hydrophobic, NIR dye tris-(porphyrinato)zinc(II) into CNCs enables complementary visualization with long-wavelength fluorescence at 800 nm. In vivo imaging demonstrates the ability of CNCs to act both as MRI and fluorescent imaging agents.

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.

The PCP protein Vangl2 regulates migration of hindbrain motor neurons by acting in floor plate cells, and independently of cilia function.

Vangl2, a core component of the Planar Cell Polarity pathway, is necessary for the caudal migration of Facial Branchiomotor (FBM) neurons in the vertebrate hindbrain. Studies in zebrafish suggest that vangl2 functions largely non-cell autonomously to regulate FBM neuron migration out of rhombomere 4 (r4), but the cell-type within which it acts is not known. Here, we demonstrate that vangl2 functions largely in floor plate cells to regulate caudal neuronal migration. Furthermore, FBM neurons fail to migrate caudally in the mouse Gli2 mutant that lacks the floor plate, suggesting an evolutionarily conserved role for this cell type in neuronal migration. Although hindbrain floor plate cilia are disorganized in vangl2 mutant embryos, cilia appear to be dispensable for neuronal migration. Notably, Vangl2 is enriched in the basolateral, but not apical, membranes of floor plate cells. Taken together, our data suggest strongly that Vangl2 regulates FBM neuron migration by acting in floor plate cells, independently of cilia function.

Influence of molecular size on the retention of polymeric nanocarrier diagnostic agents in breast ducts.

PURPOSE: To investigate the influence of nanocarrier molecular size and shape on breast duct retention in normal rats using a non-invasive optical imaging method. METHODS: Fluorescein-labeled PEG nanocarriers of different molecular weights and shapes (linear, two-arm, four-arm, and eight-arm) were intraductally administered (50 nmol) to female Sprague-Dawley rats. Whole body images were obtained non-invasively. Fluorescence intensities (i.e., amount remaining in duct) were plotted against time to estimate the nanocarrier ductal retention half-lives (t(1/2)). Plasma samples were taken and the pharmacokinetics (Tmax, Cmax) of absorbed nanocarriers was also assessed. RESULTS: The t(1/2) of linear 12, 20, 30, 40, and two-arm 60 kDa nanocarriers were 6.7 ± 0.9, 16.1 ± 4.1, 16.6 ± 3.4, 21.5 ± 2.7, and 19.5 ± 6.1 h, whereas the four-arm 20, 40, and eight-arm 20 kDa had t(1/2) of 9.0 ± 0.5, 11.5 ± 1.9, and 12.6 ± 3.0 h. The t(1/2) of unconjugated fluorescein was significantly lower (14.5 ± 1.4 min). The Tmax for 12, 40, 60 kDa nanocarriers were 1, 24, and 32 h, respectively, and only 30 min for fluorescein. CONCLUSIONS: Since normal breast ducts are highly permeable, the use of nanocarriers may be helpful in prolonging ductal retention of diagnostic and/or therapeutic agents.

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.

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord.

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.

Wnt signaling inhibitors regulate the transcriptional response to morphogenetic Shh-Gli signaling in the neural tube.

Shh-Gli signaling controls cell fates in the developing ventral neural tube by regulating the patterned expression of transcription factors in neural progenitors. However, the molecular mechanisms that limit target gene responses to specific domains are unclear. Here, we show that Wnt pathway inhibitors regulate the threshold response of a ventral Shh target gene, Nkx2.2, to establish its restricted expression in the ventral spinal cord. Identification and characterization of an Nkx2.2 enhancer reveals that expression is directly regulated by positive Shh-Gli signaling and negative Tcf repressor activity. Our data indicate that the dorsal limit of Nkx2.2 is controlled by Tcf4-mediated transcriptional repression, and not by a direct requirement for high-level Shh-Gli signaling, arguing against a simple model based on differential Gli factor affinities in target genes. These results identify a transcriptional mechanism that integrates graded Shh and Wnt signaling to define progenitor gene expression domains and cell fates in the neural tube.

Transduction of graded Hedgehog signaling by a combination of Gli2 and Gli3 activator functions in the developing spinal cord.

The three vertebrate Gli proteins play a central role in mediating Hedgehog (Hh)-dependent cell fate specification in the developing spinal cord; however, their individual contributions to this process have not been fully characterized. In this paper, we have addressed this issue by examining patterning in the spinal cord of Gli2;Gli3 double mutant embryos, and in chick embryos transfected with dominant activator forms of Gli2 and Gli3. In double homozygotes, Gli1 is also not expressed; thus, all Gli protein activities are absent in these mice. We show that Gli3 contributes activator functions to ventral neuronal patterning, and plays a redundant role with Gli2 in the generation of V3 interneurons. We also show that motoneurons and three classes of ventral neurons are generated in the ventral spinal cord in double mutants, but develop as intermingled rather than discrete populations. Finally, we provide evidence that Gli2 and Gli3 activators control ventral neuronal patterning by regulating progenitor segregation. Thus, multiple ventral neuronal types can develop in the absence of Gli function, but require balanced Gli protein activities for their correct patterning and differentiation.