Hyperkalemia and Electrocardiogram Manifestations in End-Stage Renal Disease.

Hyperkalemia is one of the more common acute life-threatening metabolic emergencies. The aim of our study is to determine the correlation and accuracy of abnormal ECG parameters as a function of serum potassium concentration in the end-stage renal disease (ESRD) population. We performed a retrospective chart review of emergency department patients presenting with ESRD and receiving emergent hemodialysis treatment. A total of 96 patients, each with five independent ED visits, provided 480 sets of ECGs and electrolytes. Of these, four ECGs were excluded for inability to interpret, leaving a total of 476 patient encounters that met all inclusion criteria. Linear regression analysis on the limited data set for serum potassium versus T/R in V2, V3, and V4, PR, and QRS found weak correlations (r2 = 0.02 to 0.12) with statistical significance <0.05 level for T/R in V2, V3, and V4. In summary, we found that a QRS duration of 120 ms or greater is most predictive of hyperkalemia in the ESRD population. On the other hand, T/R ratio, PR interval and QRS duration have poor correlations with serum potassium and are not predictive of hyperkalemia in patients with ESRD.

Palliative Endoscopic Salvage of a Functionally Obstructed Gastrojejunostomy - Report of Technique.

Background: Gastric outlet obstruction secondary to foregut gastrointestinal malignancies can be managed with a variety of medical, endoscopic, and surgical options. Laparoscopic gastrojejunostomy is an option for those patients who are able to tolerate an operation as a long-term palliative option. This operation may be associated with some significant postoperative technical and nontechnical complications, including delayed gastric emptying. This paper describes an incision-less, endoscopic option that we propose can be used to salvage a functionally obstructed gastrojejunostomy. Case Description: A 57-year old male patient had a history of pancreatic adenocarcinoma causing gastric outlet obstruction and underwent a previously created surgical gastrojejunostomy at an outside hospital. His procedure was complicated by anastomotic leak and essentially persistent obstructive symptoms secondary to delayed gastric emptying. Though his anastomosis was demonstrably patent, these symptoms were thought to be secondary to a functional obstruction at the gastrojejunostomy. After repeated workups and many failed attempts to treat these symptoms, he ultimately underwent endoscopic placement of an uncovered colonic stent into the efferent limb of his gastrojejunostomy. This allowed for preferential drainage of gastric contents down the efferent limb, and improvement of his delayed gastric emptying. Conclusions: In a select group of patients with advanced foregut malignancy, and with high re-operative risks, salvage endoscopic stenting may be useful in the palliation of symptoms from a functionally obstructed gastrojejunostomy.

Perfluorocarbon nanodroplets can reoxygenate hypoxic tumors in vivo without carbogen breathing.

Nanoscale perfluorocarbon (PFC) droplets have enormous potential as clinical theranostic agents. They are biocompatible and are currently used in vivo as contrast agents for a variety of medical imaging modalities, including ultrasound, computed tomography, photoacoustic and 19F-magnetic resonance imaging. PFC nanodroplets can also carry molecular and nanoparticulate drugs and be activated in situ by ultrasound or light for targeted therapy. Recently, there has been renewed interest in using PFC nanodroplets for hypoxic tumor reoxygenation towards radiosensitization based on the high oxygen solubility of PFCs. Previous studies showed that tumor oxygenation using PFC agents only occurs in combination with enhanced oxygen breathing. However, recent studies suggest that PFC agents that accumulate in solid tumors can contribute to radiosensitization, presumably due to tumor reoxygenation without enhanced oxygen breathing. In this study, we quantify the impact of oxygenation due to PFC nanodroplet accumulation in tumors alone in comparison with other reoxygenation methodologies, in particular, carbogen breathing. Methods: Lipid-stabilized, PFC (i.e., perfluorooctyl bromide, CF3(CF2)7Br, PFOB) nanoscale droplets were synthesized and evaluated in xenograft prostate (DU145) tumors in male mice. Biodistribution assessment of the nanodroplets was achieved using a fluorescent lipophilic indocarbocyanine dye label (i.e., DiI dye) on the lipid shell in combination with fluorescence imaging in mice (n≥3 per group). Hypoxia reduction in tumors was measured using PET imaging and a known hypoxia radiotracer, [18F]FAZA (n≥ 3 per group). Results: Lipid-stabilized nanoscale PFOB emulsions (mean diameter of ~250 nm), accumulated in the xenograft prostate tumors in mice 24 hours post-injection. In vivo PET imaging with [18F]FAZA showed that the accumulation of the PFOB nanodroplets in the tumor tissues alone significantly reduced tumor hypoxia, without enhanced oxygen (i.e., carbogen) breathing. This reoxygenation effect was found to be comparable with carbogen breathing alone. Conclusion: Accumulation of nanoscale PFOB agents in solid tumors alone successfully reoxygenated hypoxic tumors to levels comparable with carbogen breathing alone, an established tumor oxygenation method. This study confirms that PFC agents can be used to reoxygenate hypoxic tumors in addition to their current applications as multifunctional theranostic agents.

Docetaxel-carboxymethylcellulose nanoparticles ameliorate CCl4-induced hepatic fibrosis in mice.

Chronic liver diseases have recently garnered substantial attention as a leading cause of death around the world. During the progression of liver fibrosis/cirrhosis induced by chronic liver injury, hepatic stellate cells (HSCs) play key roles in the regulation of liver fibrogenesis and can even accelerate the progression of hepatocellular carcinoma (HCC). Thus, inhibition of HSC activation or suppression of inflammatory cytokine secretion by HSCs may be an efficient therapeutic strategy to ameliorate liver fibrosis/cirrhosis. In this study, we demonstrated that Cellax NPs (Carboxymethylcellulose - docetaxel-conjugated nanoparticles), which are nanoscale Pegylated carboxymethylcellulose - DTX conjugates, selectively target activated HSCs and abrogate their fibrogenic properties in vitro. Furthermore, Cellax NPs alleviated CCl4-induced hepatic fibrosis and suppressed HCC progression in a clinically relevant HCC model associated with underlying liver fibrosis in vivo. Taken together, Cellax NPs demonstrate great therapeutic promise as a treatment for liver fibrosis and cancer.

Cabazitaxel-conjugated nanoparticles for docetaxel-resistant and bone metastatic prostate cancer.

Effective treatment of metastatic castration resistant prostate cancer (mCRPC) remains an unmet challenge. Cabazitaxel (CBZ) is approved for mCRPC after docetaxel (DTX) failure, but the improvement in survival is only moderate (∼2 months) and patients suffer from significant side effects. Here, we report the development of a polymer based delivery system for CBZ to improve its safety and efficacy against DTX-resistant mCRPC. CBZ was conjugated to a carboxymethylcellulose-based polymer (Cellax-CBZ), which self-assembled into ∼100 nm particles in saline and exhibited sustained drug release in serum at 10%/day. Cellax-CBZ delivered 157-fold higher CBZ to PC3-RES prostate tumor in mice and could be safely administered at a 25-fold higher dose compared to free CBZ, resulting in superior tumor inhibition in multiple mice models of DTX-resistant CRPC. In a metastatic bone model of CRPC, Cellax-CBZ significantly improves overall survival with a 70% long-term survival rate to day 120, while mice treated with free CBZ had a median survival of 40 days. Cellax-CBZ induced mild and reversible neutropenia in mice but no other tissue damage. Cellax-CBZ showed significant potential for improving therapy of mCRPC over clinically approved CBZ.

Docetaxel-carboxymethylcellulose nanoparticles target cells via a SPARC and albumin dependent mechanism.

Cellax, a polymer-docetaxel (DTX) conjugate that self-assembled into 120 nm particles, displayed significant enhancements in safety and efficacy over native DTX across a number of primary and metastatic tumor models. Despite these exciting preclinical data, the underlying mechanism of delivery of Cellax remains elusive. Herein, we demonstrated that serum albumin efficiently adsorbed onto the Cellax particles with a 4-fold increased avidity compared to native DTX, and the uptake of Cellax by cells was primarily driven by an albumin and SPARC (secreted protein acidic and rich in cysteine, an albumin binder) dependent internalization mechanism. In the SPARC-positive cells, a >2-fold increase in cellular internalization of Cellax was observed in the presence of albumin. In the SPARC-negative cells, no difference in Cellax internalization was observed in the presence or absence of albumin. Evaluation of the internalization mechanism using endocytotic inhibitors revealed that Cellax was internalized predominantly via a clathrin-mediated endocytotic mechanism. Upon internalization, it was demonstrated that Cellax was entrapped within the endo-lysosomal and autophagosomal compartments. Analysis of the tumor SPARC level with tumor growth inhibition of Cellax in a panel of tumor models revealed a positive and linear correlation (R(2) > 0.9). Thus, this albumin and SPARC-dependent pathway for Cellax delivery to tumors was confirmed both in vitro and in vivo.

Targeting of metastasis-promoting tumor-associated fibroblasts and modulation of pancreatic tumor-associated stroma with a carboxymethylcellulose-docetaxel nanoparticle.

Pancreatic ductal adenocarcinomas are characterized by the desmoplastic reaction, a dense fibrous stroma that has been shown to be supportive of tumor cell growth, invasion, and metastasis, and has been associated with resistance to chemotherapy and reduced patient survival. Here, we investigated targeted depletion of stroma for pancreatic cancer therapy via taxane nanoparticles. Cellax-DTX polymer is a conjugate of docetaxel (DTX), polyethylene glycol (PEG), and acetylated carboxymethylcellulose, a construct which condenses into well-defined 120nm particles in an aqueous solution, and is suitable for intravenous injection. We examined Cellax-DTX treatment effects in highly stromal primary patient-derived pancreatic cancer xenografts and in a metastatic PAN02 mouse model of pancreatic cancer, focusing on specific cellular interactions in the stroma, pancreatic tumor growth and metastasis. Greater than 90% of Cellax-DTX particles accumulate in smooth muscle actin (SMA) positive cancer-associated fibroblasts which results in long-term depletion of this stromal cell population, an effect not observed with Nab-paclitaxel (Nab-PTX). The reduction in stromal density leads to a >10-fold increase in tumor perfusion, reduced tumor weight and a reduction in metastasis. Consentingly, Cellax-DTX treatment increased survival when compared to treatment with gemcitabine or Nab-PTX in a metastatic PAN02 mouse model. Cellax-DTX nanoparticles interact with the tumor-associated stroma, selectively interacting with and depleting SMA positive cells and macrophage, effects of which are associated with significant changes in tumor progression and metastasis.

Docetaxel-carboxymethylcellulose nanoparticles display enhanced anti-tumor activity in murine models of castration-resistant prostate cancer.

Docetaxel (DTX) remains the only effective drug for prolonging survival and improving quality of life of metastatic castration resistant prostate cancer (mCRPC) patients. Despite some clinical successes with DTX-based therapies, advent of cumulative toxicity and development of drug resistance limit its long-term clinical application. The integration of nanotechnology for drug delivery can be exploited to overcome the major intrinsic limitations of DTX therapy for mCRPC. We evaluated whether reformulation of DTX by facile conjugation to carboxymethylcellulose nanoparticles (Cellax) can improve the efficacy and safety of the drug in s.c. and bone metastatic models of CRPC. A single dose of the nanoparticles completely regressed s.c. PC3 tumor xenografts in mice. In addition, Cellax elicited fewer side effects compared to native DTX. Importantly, Cellax did not increase the expression of drug resistance molecules in androgen-independent PC3 prostate cancer cells in comparison with DTX. Lastly, in a bone metastatic model of CRPC, Cellax treatment afforded a 2- to 3-fold improvement in survival and enhancements in quality-of-life of the animals over DTX and saline controls. These results demonstrate the potential of Cellax in improving the treatment of mCRPC.

Carboxymethylcellulose-based and docetaxel-loaded nanoparticles circumvent P-glycoprotein-mediated multidrug resistance.

Taxanes are a class of anticancer agents with a broad spectrum and have been widely used to treat a variety of cancer. However, its long-term use has been hampered by accumulating toxicity and development of drug resistance. The most extensively reported mechanism of resistance is the overexpression of P-glycoprotein (Pgp). We have developed a PEGylated carboxymethylcellulose conjugate of docetaxel (Cellax), which condenses into ∼120 nm nanoparticles. Here we demonstrated that Cellax therapy did not upregulate Pgp expression in MDA-MB-231 and EMT-6 breast tumor cells, whereas a significant increase in Pgp expression was measured with native docetaxel (DTX) treatment. Treatment with DTX led to 4-7-fold higher Pgp mRNA expression and 2-fold higher Pgp protein expression compared with Cellax treatment in the in vitro and in vivo system, respectively. Cellax also exhibited significantly increased efficacy compared with that of DTX in a taxane-resistant breast tumor model. Against the highly Pgp expressing EMT6/AR1 cells, Cellax exhibited a 6.5 times lower IC50 compared with that of native DTX, and in the in vivo model, Cellax exhibited 90% tumor growth inhibition, while native DTX had no significant antitumor activity.

Active targeting of block copolymer micelles with trastuzumab Fab fragments and nuclear localization signal leads to increased tumor uptake and nuclear localization in HER2-overexpressing xenografts.

Block copolymer micelles (BCMs) have been employed as effective drug delivery systems to solid tumors by virtue of their capacity to transport large therapeutic payloads and passively target tumor sites. Active targeting of nanoparticles (NPs) has been exploited as a means to increase the therapeutic efficacy of NP-based drugs by promoting their delivery to cellular sites of action. Effective whole tumor accumulation and cellular uptake constitute key objectives in the success of preclinical drug formulations, although they have seldom been investigated concurrently in vivo. The current study aims to elucidate the in vivo fate of 31-nm-sized block copolymer micelles (BCMs) targeted to the nucleus of HER2-overexpressing breast cancer cells. Pharmacokinetics, biodistribution, tumor uptake, and intratumoral distribution of BCMs were investigated in mice bearing subcutaneous BT-474 and MDA-MB-231 xenografts expressing high and low levels of HER2, respectively. Radiolabeling with (111)indium enabled quantitative assessment of BCM distribution at the whole body, tissue, and cellular levels. Surface-grafted trastuzumab Fab fragments (TmAb-Fab) facilitated binding and internalization of BCMs by HER2-positive breast cancer cells, while synthetic 13-mer nuclear localization signal (NLS) peptides conjugated to the TmAb-Fab conferred nuclear translocation capability. Active targeting of BCMs led to a 5-fold increase in tumor uptake in HER2-overexpressing BT-474 tumors, alongside a correspondingly greater level of cellular uptake and nuclear localization, relative to the nontargeted formulations. This study distinctively highlights the quantitative evaluation of active targeting on tumor, cellular and subcellular uptake of BCMs and presents a promising platform for the effective delivery of chemo- and/or radiotherapy in vivo.

Ion association in aqueous solutions probed through vibrational energy transfers among cation, anion, and water molecules.

KSCN and NH4SCN aqueous solutions were investigated with intermolecular vibrational energy transfer methods. In a KSCN/H2O (1/10 molar ratio) solution, 90% of the initial excitation of the CN stretch (~2066 cm(-1)) of the SCN(-) anion is transferred to the HOH bending mode (~1636 cm(-1)) of water molecules with an energy transfer time constant 3.1 ps. In a NH4SCN/H2O (1/10 molar ratio) solution, only 49% of the CN excitation flows to the water HOH bending mode with a time constant 6.3 ps. Most of the remaining CN excitation goes to the NH bending mode (~1460 cm(-1)) of the NH(+) cation with a time constant of 7.0 ps. The results indicate that about 50% of the energy transfer channel from the CN stretch to the HOH bending observed in the KSCN solution is overpowered by the NH4(+) cations in the NH4SCN/H2O solution. Ion concentration dependent measurements support this argument. According to the dipole/dipole approximation, the CN/OH energy transfer occurs most efficiently between SCN(-) anions and the water molecules closest to them. The experimental results therefore suggest that more than 50% of the water molecules closest to the SCN(-) anions are replaced by the NH4(+) cations in the NH4SCN/H2O (1/10 molar ratio) solution. The percentage is much larger than the NH4(+)/water ratio of 10%, indicating that the ion association between NH4(+) and SCN(-) is caused by the chemical nature of the solution rather than the statistical "forced contact" because of the high ion concentration.

Block copolymer micelles target Auger electron radiotherapy to the nucleus of HER2-positive breast cancer cells.

Intracellular trafficking of Auger electron emitting radionuclides to perinuclear and nuclear regions of cells is critical to realizing their full therapeutic potential. In the present study, block copolymer micelles (BCMs) were labeled with the Auger electron emitter indium-111 ((111)In) and loaded with the radiosensitizer methotrexate. HER2 specific antibodies (trastuzumab fab) and nuclear localization signal (NLS; CGYGPKKKRKVGG) peptides were conjugated to the surface of the BCMs to direct uptake in HER2 expressing cells and subsequent localization in the cell nucleus. Cell uptake and intracellular distribution of the multifunctional BCMs were evaluated in a panel of breast cancer cell lines with different levels of HER2 expression. Indeed cell uptake was found to be HER2 density dependent, confirming receptor-mediated internalization of the BCMs. Importantly, conjugation of NLS peptides to the surface of BCMs was found to result in a significant increase in nuclear uptake of the radionuclide (111)In. Successful nuclear targeting was shown to improve the antipoliferative effect of the Auger electrons as measured by clonogenic assays. In addition, a significant radiation enhancement effect was observed by concurrent delivery of low-dose MTX and (111)In in all breast cancer cell lines evaluated.

The effects of particle size and molecular targeting on the intratumoral and subcellular distribution of polymeric nanoparticles.

The current study describes the impact of particle size and/or molecular targeting (epidermal growth factor, EGF) on the in vivo transport of block copolymer micelles (BCMs) in athymic mice bearing human breast cancer xenografts that express differential levels of EGF receptors (EGFR). BCMs with diameters of 25 nm (BCM-25) and 60 nm (BCM-60) were labeled with indium-111 ((111)In) or a fluorescent probe to provide a quantitative and qualitative means of evaluating their whole body, intratumoral, and subcellular distributions. BCM-25 was found to clear rapidly from the plasma compared to BCM-60, leading to an almost 2-fold decrease in their total tumor accumulation. However, the tumoral clearance of BCM-25 was delayed through EGF functionalization, enabling the targeted BCM-25 (T-BCM-25) to achieve a comparable level of total tumor deposition as the nontargeted BCM-60 (NT-BCM-60). Confocal fluorescence microscopy combined with MATLAB analyses revealed that NT-BCM-25 diffuses further away from the blood vessels (D(mean) = 42 +/- 9 microm) following extravasation, compared to NT-BCM-60 which mainly remains in the perivascular regions (D(mean) = 23 +/- 4 microm). The introduction of molecular targeting imposes the "binding site barrier" effect, which retards the tumor penetration of T-BCM-25 (D(mean) = 29 +/- 7 microm, p < 0.05). The intrinsic nuclear translocation property of EGF/EGFR leads to a significant increase in the nuclear uptake of T-BCM-25 in vitro and in vivo via active transport. Overall, these results highlight the need to consider multiple design parameters in the development of nanosystems for delivery of anticancer agents.

In vivo distribution of polymeric nanoparticles at the whole-body, tumor, and cellular levels.

PURPOSE: Block copolymer micelles (BCMs) were functionalized with indium-111 and/or epidermal growth factor (EGF), which enabled investigation of the in vivo transport of passively and actively targeted BCMs. The integration of conventional and image-based techniques afforded novel quantitative means to achieve an in-depth insight into the fate of polymeric nanoparticles in vivo. METHODS: Pharmacokinetics and biodistribution studies were performed in athymic mice bearing human breast xenografts to evaluate the whole-body transport of NT-BCMs (non-targeted, EGF-) and T-BCMs (targeted, EGF+). The intratumoral distribution of BCMs was investigated using MicroSPECT/CT and autoradiographic imaging, complemented with quantitative MATLAB® analyses. Tumors were fractionated for quantifying intracellular uptake of BCMs via γ-counting. RESULTS: The intratumoral distribution of NT-BCMs and T-BCMs were found to be heterogeneous, and positively correlated with tumor vascularization (r>0.68 ± 0.04). The enhanced in vivo cell uptake and cell membrane binding of T-BCMs were found to delay their clearance from tumors overexpressing EGFR, and therefore resulted in enhanced tumor accumulation for the T-BCMs in comparison to the NT-BCMs. CONCLUSIONS: Adequate passive targeting is required in order to achieve effective active targeting. Tumor physiology has a significant impact on the transvascular and intratumoral transport of passively and actively targeted BCMs.

Noninvasive monitoring of the fate of 111In-labeled block copolymer micelles by high resolution and high sensitivity microSPECT/CT imaging.

The validation of high sensitivity and high resolution microSPECT/CT imaging for tracking the in vivo pathway and fate of an 111Indium-labeled (111In) amphiphilic diblock copolymer micelle formulation is presented. Heterobifunctional poly(ethylene glycol) was used to initiate cationic ring opening polymerization of epsilon-caprolactone, which was then conjugated to 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA) for chelation with 111In. The micelles were characterized in terms of their physicochemical properties including size, size distribution, zeta-potential, and radiochemical purity. Elimination kinetics and tissue deposition were evaluated in healthy mice following administration of 111In-micelles, 111In-DTPA-b-PCL unimers (i.e., administered under the critical micelle concentration) or 111In-Bn-DTPA. Healthy and MDA-MB-231 tumor-bearing mice were imaged using microSPECT/CT following iv administration of 111In-micelles or 111In-Bn-DTPA. Overall, incorporation of 111In onto the surface of thermodynamically stable micelles results in long plasma residence times for the radionuclide and preferential localization within the spleen (22 +/- 5% i.d/g), liver (13 +/- 3% i.d./g), and tumor (9 +/- 2% i.d./g). MicroSPECT/CT imaging provided noninvasive longitudinal visualization of circulation dynamics and tissue deposition. A strong correlation between image-based region of interest (ROI) analysis and biodistribution data was found, implying that nuclear imaging can be used as a noninvasive tool to accurately quantify tissue distribution. As well, the image-based assessment provided unique insight into the intratumoral distribution of the micelles in vivo.

Morphological control of poly(ethylene glycol)-block-poly(epsilon-caprolactone) copolymer aggregates in aqueous solution.

In aqueous solution, it was found that the amphiphilic copolymer poly(ethylene glycol)-b-poly(caprolactone) (PEG(5000)-b-PCL(4100)) formed different morphologies, including long rod-like, short rod-like, or spherical aggregates, when the copolymer concentration was increased. Nearly identical morphologies were observed with the addition of increasing amounts of PEG(2000)-distearoylphosphoethanolamine (PEG(2000)-DSPE) to the copolymer. The morphologies of the aggregates in solution were confirmed by negative stain transmission electron microscopy (TEM) and cryogenic-TEM (cryo-TEM). The critical micelle concentrations of the PEG(5000)-b-PCL(4100) copolymer, PEG(2000)-DSPE and a mixture of the two materials (PEG(5000)-b-PCL 4100/PEG(2000)-DSPE) were evaluated to determine the thermodynamic stability of the aggregates. Differential scanning calorimetry was performed to gain insight into the degree of mixing of PEG(5000)-b-PCL(4100) and PEG(2000)-DSPE. Overall, combining PEG(5000)-b-PCL(4100) and PEG(2000)-DSPE produced a single population of mixed micelles with rod-like or spherical morphologies depending on the material composition and concentration.

An optimized method for histological detection of dopaminergic neurons in Drosophila melanogaster.

Parkinson's disease (PD) affects >1 million Americans and is marked by the loss of dopaminergic neurons in the substantia nigra. PD has been linked to two causative factors: genetic risks (hereditary PD) and environmental toxins (idiopathic PD). In recent years, considerable effort has been devoted to the development of a Drosophila model of human PD that might be useful for examining the cellular mechanisms of PD pathology by genetic screening. In 2000, Feany and Bender reported a Drosophila model of PD in which transgenic flies expressing human mutant alpha-synuclein exhibited shortened life spans, dopaminergic losses, Parkinsonian behaviors, and Lewy bodies in surviving dopaminergic neurons. Since then, a number of studies have been published that validate the model or build on it; conversely, a number report an inability to replicate the results and suggest that most protocols for dopaminergic histology underreport the actual numbers of dopaminergic neurons in the insect brain. Here we report the optimization of dopaminergic histology in Drosophila and identification of new dopaminergic neurons, show the remarkable dendritic complexity of these neurons, and provide an updated count of these neurons in adult brains. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.