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Robert J Lee

Publications and source records attributed to Robert J Lee.

At least 19 recordsLinked to original sources
PubMed2006

Construction of anti-EGFR immunoliposomes via folate-folate binding protein affinity.

A novel method for synthesis of anti-EGFR immunoliposomes using folate-folate binding protein (FBP) affinity is described. An anti-EGFR antibody (cetuximab or C225) was covalently linked to FBP via a thioether bond. Liposomes incorporating a lipophilic folate derivative (folate-PEG-cholesterol) were prepared by polycarbonate membrane extrusion. Anti-EGFR immunoliposomes were then obtained by combining FBP-C225 and folate-liposomes and evaluated for uptake and cytotoxicity in EGFR-overexpressing U87 human glioblastoma cells. Anti-EGFR immunoliposomes constructed via folate-FBP affinity exhibited excellent stability under physiological pH, and quickly released the bound FBP-C225 upon low pH (pH 3.5) treatment. Flow cytometry and fluorescence microscopy showed similar receptor-specific binding and internalization for both folate-FBP affinity-coupled and covalently coupled C225-immunoliposomes, but not for the non-targeted IgG-immunoliposomes. C225-immunoliposomes loaded with anticancer drug doxorubicin were more cytotoxic than non-targeted immunoliposomes in EGFR-overexpressing U87 glioma cells. Folate-FBP affinity is a potential method for construction of immunoliposomes and may have applications in synthesis of targeted drug carriers in general.

Antibodies, Monoclonal↗
PubMed2006

The prevalence and characteristics of anaemia at discharge home after intensive care.

OBJECTIVE: To document the prevalence of anaemia among ICU survivors at the time of discharge home and to document red cell morphology among anaemic patients. DESIGN AND SETTING: Observational cohort study in a single-centre teaching hospital. PATIENTS AND PARTICIPANTS: Three cohorts of ICU admissions over a 3-year period managed with restrictive ICU transfusion practice. The study group comprised the 283 patients who survived and were discharged directly home from our hospital. MEASUREMENTS AND RESULTS: The median time from ICU discharge to hospital discharge was 13[Symbol: see text]days (IQR 6-22, range 1-119). Using the last recorded Hb concentration 77.4% (95% CI 72.2-82.1) of patients were anaemic, 32.5% (27.3-38.2) had Hb less than 100 g/l, and 11.3% (8.1-15.5) had Hb less than 90 g/l at hospital discharge. Patients who spent longer in intensive care and in hospital after ICU discharge were more likely to be discharged home anaemic. The strongest predictor of discharge home with Hb less than 100 g/l was Hb at the time of ICU discharge. Multivariate regression analysis showed patient age, gender, APACHE II score, and ICU length of stay not to be independent predictors after including the ICU discharge Hb. Among anaemic patients 82% had normochromic normocytic red cell indices, but 12% had red cell hypochromasia and/or microcytosis, which may indicate iron deficiency. CONCLUSIONS: Anaemia is highly prevalent among survivors of critical illness and persists until hospital discharge. Most patients have red cell morphology similar to "anaemia of chronic disease".

Adult↗
PubMed2006

Nanoparticles as image enhancing agents for ultrasonography.

Nanoparticles have drawn great attention as targeted imaging and/or therapeutic agents. The small size of the nanoparticles allows them to target cells that are beyond capillary vasculature, such as cancer cells. We investigated the effect of solid nanoparticles for enhancing ultrasonic grey scale images in tissue phantoms and mouse livers in vivo. Silica nanospheres (100 nm) were dispersed in agarose at 1-2.5% mass concentration and imaged by a high-resolution ultrasound imaging system (transducer centre frequency: 30 MHz). Polystyrene particles of different sizes (500-3000 nm) and concentrations (0.13-0.75% mass) were similarly dispersed in agarose and imaged. Mice were injected intravenously with nanoparticle suspensions in saline. B-mode images of the livers were acquired at different time points after particle injection. An automated computer program was used to quantify the grey scale changes. Ultrasonic reflections were observed from nanoparticle suspensions in agarose gels. The image brightness, i.e., mean grey scale level, increased with particle size and concentration. The mean grey scale of mouse livers also increased following particle administration. These results indicated that it is feasible to use solid nanoparticles as contrast enhancing agents for ultrasonic imaging.

Animals↗
PubMed2006

A folate receptor-targeted liposomal formulation for paclitaxel.

A novel liposomal formulation of paclitaxel targeting the folate receptor (FR) was synthesized and characterized. This formulation was designed to overcome vehicle toxicity associated with the traditional Cremophor EL-based formulation and to provide the added advantages of prolonged systemic circulation time and selective targeting of the FR, which is frequently overexpressed on epithelial cancer cells. The formulation had the composition of dipalmitoyl phosphatidylcholine/dimyristoyl phosphatidylglycerol/monomethoxy-polyethylene glycol (PEG)2000-distearoyl phosphatidylethanolamine/folate-PEG3350-distearoyl phosphatidylethanolamine (DPPC/DMPG/mPEG-DSPE/folate-PEG-DSPE) at molar ratios of (85.5:9.5:4.5:0.5) and a drug-to-lipid molar ratio of 1:33. The liposomes were prepared by polycarbonate membrane extrusion. The mean particle size of the liposomes was 97.1 nm and remained stable for at least 72 h at 4 degrees C. FR-targeted liposomes of the same lipid composition entrapping calcein were shown to be efficiently taken up by KB oral carcinoma cells, which are highly FR+. FR-targeted liposomes containing paclitaxel showed 3.8-fold greater cytotoxicity compared to non-targeted control liposomes in KB cells. Plasma clearance profiles of paclitaxel in the liposomal formulations were then compared to paclitaxel in Cremophor EL formulation. The liposomal formulations showed much longer terminal half-lives (12.33 and 14.23 h for FR-targeted and non-targeted liposomes, respectively) than paclitaxel in Cremophor EL (1.78 h). In conclusion, the paclitaxel formulation described in this study has substantial stability and favorable pharmacokinetic properties. The FR-targeted paclitaxel formulation is potentially useful for treatment of FR+ tumors and warrants further investigation.

Animals↗
PubMed2006

Efficient delivery of a Bcl-2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-targeted liposomes.

A novel transferrin receptor (TfR)-targeted liposomal formulation was synthesized and evaluated for the delivery of a phosphorothioate antisense oligodeoxyribonucleotide (ODN) (G3139, oblimerson sodium, or Genasense) to Bcl-2 in K562 leukemia cells. Liposomes composed of DC-Chol/egg PC/PEG-DSPE (25:73.5:1.5, mol/mol/mol) were loaded with G3139 with high efficiency (70-80%). To prepare targeted liposomes, transferrin was first coupled to PEG-DSPE and then incorporated into the bilayer by post-insertion. The liposomes had a mean diameter of 100 to 150 nm and exhibited colloidal stability for up to 8 weeks. Uptake of Tf-conjugated G3139-containing liposomes in TfR positive K562 cells was found to be more efficient than that of the non-targeted control formulation and could be blocked by excess free Tf. Treatment with Tf-conjugated liposomes resulted in Bcl-2 protein downregulation in K562 cells that was approximately 2-fold greater than with non-targeted liposomes (p<0.05) and 10-fold greater than with free G3139. Treatment with 2 microM G3139 in Tf-conjugated liposomes resulted in >80% reduction in Bcl-2 transcript. In addition, Tf-conjugated liposomal G3139-sensitized K562 cells to daunorubicin, lowering IC50 from 1.8 microM to 0.18 microM. In conclusion, Tf-conjugated liposomes are effective delivery vehicles for G3139 antisense oligos in TfR positive K562 cells and warrant further investigation as an in vivo oligo delivery vehicle.

Blotting, Western↗
PubMed2006

Boron containing macromolecules and nanovehicles as delivery agents for neutron capture therapy.

Boron neutron capture therapy (BNCT) is based on the nuclear capture and fission reactions that occur when non-radioactive boron-10 is irradiated with low energy thermal neutrons to yield high linear energy transfer (LET) alpha particles ((4)He) and recoiling lithium -7((7)Li) nuclei. For BNCT to be successful, a sufficient number of (10)B atoms ( approximately 10(9) atoms/cell) must be selectively delivered to the tumor and enough thermal neutrons must be absorbed by them to sustain a lethal (10)B(n, alpha) (7)Li capture reaction. BNCT primarily has been used to treat patients with brain tumors, and more recently those with head and neck cancer. Two low molecular weight (LMW) boron delivery agents currently are being used clinically, sodium borocaptate and boronophenylalanine. However, a variety of high molecular weight (HMW) agents consisting of macromolecules and nanovehicles have been developed. This review will focus on the latter which include: monoclonal antibodies, dendrimers, liposomes, dextrans, polylysine, avidin, folic acid, and epidermal and vascular endothelial growth factors (EGF and VEGF). Procedures for introducing boron atoms into these HMW agents and their chemical properties will be discussed. In vivo studies on their biodistribution will be described, and the efficacy of a subset of them, which have been used for BNCT of tumors in experimental animals, will be discussed. Since brain tumors currently are the primary candidates for treatment by BNCT, delivery of these HMW agents across the blood-brain barrier presents a special challenge. Various routes of administration will be discussed including receptor-facilitated transcytosis following intravenous administration, direct intratumoral injection and convection enhanced delivery by which a pump is used to apply a pressure gradient to establish bulk flow of the HMW agent during interstitial infusion. Finally, we will conclude with a discussion relating to issues that must be addressed if these HMW agents are to be used clinically.

Animals↗
PubMed2005

Anemia during and at discharge from intensive care: the impact of restrictive blood transfusion practice.

OBJECTIVE: To document the prevalence of anemia among patients admitted to intensive care (ICU) and, among survivors, at ICU discharge when restrictive transfusion practice was used. DESIGN: This was an observational cohort study. SETTING: Ten of the 26 general ICUs in Scotland. PATIENTS AND PARTICIPANTS: One thousand twenty-three sequential ICU admissions over 100 days, representing 44% of all ICU admissions in Scotland during the study period, studied daily from admission to discharge or death in the ICU. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: The median transfusion trigger used, in the absence of bleeding, was 78 g/l (interquartile range 73-84); <2% of transfusion triggers were above the upper limit of the national transfusion trigger guideline (100 g/l). Overall, 25% of admissions had a hemoglobin concentration <90 g/l at ICU admission. Seven hundred sixty-six patients admitted survived to ICU discharge. Among these, the prevalence of anemia (male <130 g/l; female <115 g/l) at ICU discharge was 87.0 (95% CI: 83.6 to 89.9)% for males and 79.6 (74.8 to 83.7)% for females. Of the male survivors 24.1 (20.3 to 28.3)% and of the female 27.9 (23.4 to 33.2)% had a hemoglobin <90 g/l at ICU discharge. The prevalence was similar for patients with and without pre-existing ischemic heart disease. Logistic regression found independent associations between having a hemoglobin concentration <90 g/l at ICU discharge and the first measured hemoglobin in ICU, the presence of acute renal failure and thrombocytopenia during ICU stay. CONCLUSIONS: Anemia is highly prevalent in ICUs that use restrictive transfusion triggers. The impact of anemia on functional recovery after intensive care requires investigation.

Anemia↗
PubMed2005

Reconstitution of endoplasmic reticulum-associated degradation using yeast membranes and cytosol.

The first compartment encountered by newly synthesized secreted proteins is the endoplasmic reticulum (ER). Before secreted proteins can traffic beyond the ER they must fold into their final conformations, and components of multiprotein complexes must assemble. Not surprisingly, then, the ER lumen houses a high concentration of molecular chaperones, factors that facilitate protein folding. However, if misfolded secreted proteins arise they may be selected and proteolyzed. This process, which removes potentially toxic proteins from the cell, has been termed ER-associated degradation (ERAD). Surprisingly, ERAD substrates are not degraded within the ER after being selected but are retrotranslocated to the cytoplasm and destroyed by the 26S proteasome. Thus, the ERAD pathway comprises substrate selection, substrate export from the ER, and substrate degradation, and each step in this pathway has been elucidated in part through an in vitro ERAD assay using reagents prepared from a model eukaryote, the yeast Saccharomyces cerevisiae. This chapter describes this in vitro ERAD assay in detail, and special considerations when performing the assay are noted.

Cell-Free System↗
PubMed2004

Tumor-targeted gene delivery via anti-HER2 antibody (trastuzumab, Herceptin) conjugated polyethylenimine.

A series of novel nonviral vectors targeting the HER-2/neu gene product human epidermal growth factor receptor-2 (HER2) were constructed and evaluated in breast cancer cell lines to optimize vector formulation for receptor-specific gene transfer. These vectors were DNA/polycation complexes (polyplexes) prepared by mixing, at varying ratios, plasmid DNA carrying a luciferase reporter gene to HerPEI, which is a conjugate of linear polyethylenimine (PEI), a cationic polymer, and trastuzumab (Herceptin), a HER2-specific monoclonal antibody. Transfection studies were carried out in both HER2 overexpressing Sk-Br-3 and HER2 low-expressing MDA-MB-231 breast cancer cells. The HerPEI polyplexes showed significantly greater transfection activity up to 20-folds than nonderivatized PEI-based polyplexes in the Sk-Br-3 cells. The transfection efficiency of targeted polyplexes was dependent on the trastuzumab:PEI ratio and can be blocked by excess free trastuzumab, suggesting HER2-mediated gene delivery. In contrast, no significant difference in transfection activities was observed between HER2-targeted and nontargeted polyplexes in the HER2 low-expressing MDA-MB-231 cells. The transfection efficiency of HerPEI polyplexes was retained in serum-containing medium. In summary, HerPEI polyplexes have shown promising HER2 receptor-specific gene transfer properties and warrant further evaluation as a tumor-targeted vector for gene therapy.

Antibodies, Monoclonal↗
PubMed2004

Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein.

Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD). To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATP-dependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle.

Animals↗
PubMed2004

Tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor.

Gene therapy is a promising approach for the treatment of cancer. The main obstacle for the clinical application of cancer gene therapy is the lack of gene transfer vectors that are safe, efficacious, and tumor-selective. In recent years, targeted gene delivery through cellular receptors, using either viral or nonviral vectors, is emerging as a novel approach to enhance the efficacy of tumor-selective gene delivery. The folate receptor (FR), which is absent in most normal tissues and elevated in over 90% of ovarian carcinomas and at a high frequency in other human malignancies, is an attractive tumor-selective target. FR-targeted vectors include folate-derivatized adenoviruses, cationic polymers, cationic liposomes, and pH-sensitive liposomes. In addition, FR-targeted liposomes have been evaluated for the targeted delivery of antisense oligodeoxyribonucleotides (ODNs). These vectors have invariably shown impressive FR-selectivity in cell culture assays and, in addition, shown promising tumor-specific gene transfer activity in several in vivo models. There are important theoretical advantages for FR-targeted vectors over traditional non-targeted vectors in therapeutic gene and oligodeoxyribonucleotides delivery in vivo to cancer cells. Further preclinical characterization of these vectors is, therefore, warranted to determine their potential utility in cancer gene therapy.

Animals↗
PubMed2004

A folate receptor-targeted lipid nanoparticle formulation for a lipophilic paclitaxel prodrug.

PURPOSE: The anticancer drug paclitaxel has poor aqueous solubility and is difficult to formulate in a lipid-based formulation due to its limited lipid solubility. Paclitaxel-7-carbonyl-cholesterol (Tax-Chol), a prodrug of paclitaxel with increased lipophilicity, was therefore synthesized and evaluated for incorporation into a lipid nanoparticle (LN) formulation, which also contained folate-polyethylene glycolcholesterol (f-PEG-Chol) as a ligand that targets the tumor marker folate receptor (FR). This novel formulation was designed for prolonged systemic circulation and selective targeting of tumor cells with amplified FR expression. METHODS: Tax-Chol was synthesized. FR-targeted LNs, composed of distearoyl phosphatidylcholine (DSPC)/triolein/Chol oleate/PEG-Chol/f-PEG-Chol (40:40:18:2.0:0.5, mole/mole), were then prepared by solvent dilution followed by diafiltration. FR-targeted LNs containing Tax-Chol were then evaluated for cytotoxicity in KB, a human oral carcinoma cell line, and M109, a murine lung carcinoma cell line, both of which are FR(+) and in FR(-) Chinese hamster ovary (CHO) cells. Furthermore, tumor growth inhibition and animal survival in response to treatment with FR-targeted LNs and control formulations were evaluated in BALB/c mice bearing subcutaneously engrafted M109 tumors. RESULTS: The LNs had a mean diameter of 130 nm and Tax-Chol incoporation efficiency of greater than 90% and exhibited excellent colloidal stability. FR-targeted LNs showed greater uptake and cytotoxicity in FR(+) KB and M109 cells than nontargeted LNs. Furthermore, treatment of mice bearing M109 tumors with FR-targeted LNs resulted in significantly greater tumor growth inhibition and animal survival compared to treatment with nontargeted LNs or paclitaxel formulated in Cremophor EL. CONCLUSION: FR-targeted LNs containing Tax-Chol are a promising novel formulation for the treatment of FR(+) tumors and further preclinical studies are warranted.

Animals↗
PubMed2004

Optical characteristics of the canine prostate at 665 nm sensitized with tin etiopurpurin dichloride: need for real-time monitoring of photodynamic therapy.

PURPOSE: Photodynamic therapy (PDT) is an emerging, minimally invasive therapy for prostate cancer that depends on the sequestration of a photosensitizing drug within targeted tissue. The photosensitizer is subsequently activated by light of a specific wavelength, resulting in destruction of the targeted tissue. Successful treatment requires knowledge of the optical properties of the target tissue, a critical element for therapy. MATERIALS AND METHODS: Adult canines were injected with tin etiopurpurin dichloride (1.0 mg/kg) as a liposome emulsion vehicle in saline 24 hours prior to light treatment. Laser light was delivered to the prostate via a 400 microm optical fiber fitted with a 2.0 cm cylindrical diffuser and optical properties of the prostate were measured. RESULTS: In this study we determined the attenuation coefficient and critical fluence in the canine prostate. Our studies shown that the attenuation coefficient is not uniform but higher at the base (average for all animals 2.59 to 2.79 cm-1) than in the mid section or apex of the prostate (1.71 to 1.90 cm-1). Significant differences among dogs (0.11 to 12.70 cm-1) were found. In some cases we observed a fluctuation of the attenuation coefficient during treatment. We also established experimentally the minimum energy (1449 mJ/cm2) needed (critical fluence) to produce necrosis. Experimentally establishing the values of effective attenuation and critical fluence is necessary to predict the area of ablation during PDT and protect surrounding organs from over treatment. CONCLUSIONS: Based on our results it is evident that for PDT of the prostate to be successful the optical parameters of the prostate must be measured and monitored during treatment. We suggest that the optimum way of doing this is real-time computerized monitoring combined with simulation PDT.

Animals↗
PubMed2004

Red cell requirements for intensive care units adhering to evidence-based transfusion guidelines.

BACKGROUND: Anemia commonly complicates critical illness. Restrictive transfusion triggers are appropriate in this setting, but no large studies have measured red cell (RBC) requirements for intensive care patients when evidence-based transfusion guidelines are followed consistently. STUDY DESIGN AND METHODS: Data were recorded daily for 1023 of 1042 sequential admissions to 10 intensive care units (ICUs) over 100 days. The sample comprised 44 percent of all ICU admissions in Scotland during this period. RBC transfusions and the occurrence of clinically significant hemorrhage were recorded for every ICU day. Transfusion episodes were classified as either associated with or not associated with hemorrhage. Measures of RBC use were derived for the cohort and for Scotland with national audit data. RESULTS: A total of 39.5 percent (95% confidence interval [CI], 36.5%-42.5%) of admissions received transfusions. Eighteen percent of admissions received at least one transfusion associated with hemorrhage and 26 percent received at least one transfusion not associated with hemorrhage. The median (interquartile range) transfusion trigger in the absence of hemorrhage was 78 (73-78) g/L. The overall mean RBC use was 1.87 (95% CI, 1.79-1.96) units per admission or 0.34 (95% CI, 0.33-0.36) units per ICU-day. Forty-seven percent of RBCs administered were not associated with clinically significant hemorrhage. Mean RBC requirements for intensive care in Scotland were estimated to be 3950 (95% CI, 3780-4140) per million-adult-population per year. This represented 7 to 8 percent of the Scottish blood supply. CONCLUSIONS: Despite evidence-based transfusion practice, 40 percent of ICU patients receive transfusions, which account for 7 to 8 percent of the national blood supply.

Adult↗
PubMed2004

Tumour-selective drug delivery via folate receptor-targeted liposomes.

Tumour cell-targeted liposomal delivery has the potential to enhance the therapeutic efficacy and reduce the toxicity of anticancer agents. Folate receptor (FR) expression is frequently amplified among human malignancies. FR is, therefore, potentially useful as a tumour marker for targeted drug delivery. FR-mediated liposomal delivery has been shown to enhance the antitumour efficacy of doxorubicin both in vitro and in vivo, and to overcome P-glycoprotein-mediated multi-drug resistance. In addition, FR-targeted liposomes have shown utility as effective delivery vehicles of genes and antisense oligodeoxyribonucleotides to FR(+) tumour cells. Both solid tumours and leukaemias can potentially benefit from FR-targeted drug delivery. Multiple mechanisms might contribute to greater therapeutic efficacy for FR-targeted liposomes, such as FR-dependent cytotoxicity and antiangiogenic activity. Further investigation of this promising drug delivery strategy is clearly warranted.

Antineoplastic Agents↗