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J. Inorganic Biochemistry. (2007) 101:594-602.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 725 Rose Street, Lexington, KY 40536-0082, USA.

D-Penicillamine is a potent copper (Cu) chelating agent. D-Pen reduces Cu(II) to Cu(I) in the process of chelation while at the same time being oxidized to D-penicillamine disulfide. It has been proposed that hydrogen peroxide is generated during this process. However, definitive experimental proof that hydrogen peroxide is generated remains lacking. Thus, the major aims of these studies were to confirm and quantitatively assess the in vitro production of hydrogen peroxide during copper catalyzed D-penicillamine oxidation. The potential cytotoxic effect of hydrogen peroxide generation was also investigated in vitro against MCF-7 human breast cancer cells. Cell cytotoxicity resulting from the incubation of D-penicillamine with copper was compared to that of D-penicillamine, copper and hydrogen peroxide. The mechanism of copper catalyzed D-penicillamine oxidation and simultaneous hydrogen peroxide production was investigated as a function of time, concentration of cupric sulfate or ferric chloride, temperature, pH, anaerobic condition and chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid. A simple, sensitive and rapid HPLC assay was developed to simultaneously detect D-penicillamine, its major oxidation product D-penicillamine disulfide, and hydrogen peroxide in a single run. Hydrogen peroxide was shown to be generated in a concentration dependent manner as a result of D-penicillamine oxidation in the presence of cupric sulfate. Chelators such as ethylenediaminetetraacetic acid and
bathocuproinedisulfonic acid were able to inhibit D-penicillamine oxidation. The incubation of MCF-7 human breast cancer cells with D-penicillamine plus cupric sulfate resulted in the production of reactive oxygen species within the cell and cytotoxicity that was comparable to free hydrogen peroxide.

PMID: 17275091 [PubMed - indexed for MEDLINE]

Department of Pharmaceutical Sciences, College of Pharmacy, University of
Kentucky, Lexington, KY 40536-0082, USA.

Serum and tumor copper levels are significantly elevated in a variety of alignancies including breast, ovarian, gastric, lung, and leukemia. D-Penicillamine (D-pen), a copper-chelating agent, at low concentrations in the
presence of copper generates concentration-dependent cytotoxic hydrogen peroxide (H(2)O(2)). The purpose of these studies was to investigate the in vitro cytotoxicity, intracellular reactive oxygen species (ROS) generation, and the eduction in intracellular thiol levels due to H(2)O(2) and other ROS generated from copper-catalyzed D-pen oxidation in human breast cancer cells (BT474, MCF-7) and human leukemia cells (HL-60, HL-60/VCR, HL-60/ADR). D-pen (</=400 muM) in the resence of cupric sulfate (10 muM) resulted in concentration-dependent cytotoxicity. Catalase was able to completely protect the cells, substantiating
the involvement of H(2)O(2) in cancer cell cytotoxicity. A linear correlation between the D-pen concentration and the intracellular ROS generated was shown in both breast cancer and leukemia cells. D-pen in the presence of copper also resulted in a reduction in intracellular reduced thiol levels. The H(2)O(2)-mediated cytotoxicity was greater in leukemia cells compared to breast cancer cells. These results support the hypothesis that D-pen can be employed as a cytotoxic copper-chelating agent based on its ROS-generating ability.

PMID: 17893040 [PubMed - in process]

Pharm. Res. (2007) 24:728-737.

Department of Oral Maxillofacial Surgery & Pathology, College of Dentistry, The Ohio State University, Columbus, OH 43210-1241, USA. mallery.1@osu.edu

PURPOSE: The purpose of these studies was to formulate mucoadhesive gels containing freeze dried black raspberries (FBR) and to determine optimum parameters for a subset of FBR bioactive compounds including anthocyanin stability, absorption and penetration in-vitro and in-vivo. MATERIALS AND METHODS: Berry gels were prepared having FBR at 5% and 10% w/w and final pHs ranging from 3.5 to 7.5. A HPLC assay was developed to quantify and determine the stability of the anthocyanins in the gels. A single time-point study was performed to determine anthocyanin uptake when the gels were applied to oral mucosa. Penetration of anthocyanins into human oral tissue explants was determined as a function of gel pH and FBR content. A HPLC-mass spectroscopy assay was utilized to quantify the anthocyanin levels in human oral tissue explants, saliva, and blood. RESULTS: The stability of anthocyanins in the gel was directly related to gel pH and storage temperature. Maximum stability of anthocyanins was found at lower pH (pH 3.5) and storage temperature (4 degrees C). Anthocyanins contained in mucoadhesive berry gel formulations were readily absorbed into human oral mucosa tissue as evidenced by detectable blood levels within 5 min after gel application. There was a trend for greater penetration of  anthocyanins into tissue explants for berry gels with a final pH of 6.5 versus pH 3.5. CONCLUSIONS: Formulation and characterization of a novel gel formulation for local delivery of chemopreventive compounds to human oral mucosal tissues has been described. The results show anthocyanin stability was dependent upon gel pH and storage temperature and also demonstrate that the gel composition is well-suited for absorption and penetration into the target oral mucosal tissue site.

Publication Types:
Clinical Trial
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

PMID: 17372698 [PubMed - indexed for MEDLINE]

Pharm Res. 2007 Feb;24(2):343-52. Epub 2006 Dec 19.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

PURPOSE: The purpose of these studies was to prepare nanoparticles (NPs) with a small amount of surface-chelated nickel for obtaining enhanced binding of histidine-tagged (his-tag) proteins compared to non-histidine-tagged protein binding to charged nanoparticles. MATERIALS AND METHODS: NPs were prepared from oil-in-water microemulsion precursors using emulsifying wax, 3 mM Brij 78 and 0.1 mM DOGS-NTA-Ni lipid (referred to as Ni-NPs). The amount of lipid entrapped in the NPs was quantitated by atomic emission spectroscopy (AES). The Ni-NPs were investigated for binding to two his-tag proteins, green fluorescent protein (GFP) and his-tag HIV-1 Gag p24. In vivo studies in mice were carried out to evaluate the immune responses obtained to his-tag Gag p24 bound to Ni-NPs. RESULTS: AES studies demonstrated that approximately 5% of the DOGS-NTA-Ni lipid used was entrapped in the NPs. The optimal binding ratio his-tag GFP and his-tag Gag p24 to Ni-NPs was found to be 1:33.7 and 1:35.4 w/w, respectively. This interaction was stable at 37 degrees C in PBS, pH 7.4 over 4 h and the interaction of his-tag GFP with the Ni-NPs was enhanced compared to control NPs prepared with no Ni on the surface (NTA-NPs). The in vivo studies demonstrated enhanced serum IgG and IgG2a responses to his-tag Gag p24 bound to Ni-NPs compared to protein adjuvanted with Alum or adsorbed on the surface of control NTA-NPs. CONCLUSIONS: Ni-NPs can be used to bind strongly to his-tag proteins. This system was demonstrated to have potential applications in vaccine delivery for enhancing immune responses to protein-based vaccines.

Publication Types:
&amp;nbsp;&amp;nbsp;&amp;nbsp; Research Support, N.I.H., Extramural
&amp;nbsp;&amp;nbsp;&amp;nbsp; Research Support, Non-U.S. Gov&amp;amp;#39;t

PMID: 17180725 [PubMed - indexed for MEDLINE]

Drug Dev. Ind. Pharm. (2006) 32:973-980.

Fatty alcohols are commonly used in lipid-based drug delivery systems including parenteral emulsions and solid lipid nanoparticles (NPs). The purpose of these studies was to determine whether horse liver alcohol dehydrogenase (HLADH), a NAD-dependent enzyme, could metabolize the fatty alcohols within the NPs and thus serve as a mechanism to degrade these NPs in the body. Solid nanoparticles (<100 nm) were engineered from oil-in-water microemulsion precursors using emulsifying wax NF as the oil phase and polyoxyethylene 20-stearyl ether (Brij 78) as the surfactant. Emulsifying wax contains both cetyl and stearyl alcohols. NPs were incubated with the enzyme and NAD+ at 37 degrees C for up to 48 h, and the concentrations of fatty alcohols were quantitatively determined over time by gas chromatography (GC). The concentrations of cetyl alcohol and stearyl alcohol within the NPs decreased to only 10-20% remaining after 15-24 h of incubation. In parallel, NP size, turbidity and the fluorescence intensity of NADH all increased over time. It was concluded that horse liver alcohol dehydrogenase/NAD+ was able to metabolize the fatty alcohols within the NPs, suggesting that NPs made of fatty alcohols may be metabolized in the body via endogenous alcohol dehydrogenase enzyme systems.

PMID: 16954110 [PubMed - indexed for MEDLINE]

J Pharm. Innovation. (2006) 1:76-82 .

Read Article Here.

J Nanosci Nanotech, (2006) 6:996-1003.

Department of Pharmaceutical Sciences, University of Kentucky, Lexington 40536-0082, USA.

In this article, we use a nanotemplate engineering approach to prepare biodegradable nanoparticles composed of FDA-approved materials and possessing accessible gadolinium (Gd) atoms and demonstrate their potential as a Magnetic Resonance Imaging (MRI) contrast agent. Nanoparticles containing dimyristoyl phosphoethanolamine diethylene triamine penta acetate (PE-DTPA) were prepared using 3.5 mg of Brij 78, 2.0 mg of emulsifying wax and 0.5 mg of PE-DTPA/ml from a microemulsion precursor. After the addition of GdCl3, the presence of Gd on the surface of nanoparticles was characterized using inductively coupled plasma atomic emission spectroscopy and Scanning Transmission Electron Microscopy (STEM). The in vitro relaxivities of the PE-DTPA-Gd nanoparticles in different media were assessed at different field strengths. The conditional stability constant of Gd binding to the nanoparticles was determined using competitive spectrophotometric titration. Transmetallation kinetics of the gadolinium ion from PE-DTPA-Gd nanoparticles with zinc as the competing ionic was measured using the relaxivity evolution method. Nanoparticles with a diameter of approximately 130 nm possessing surface chelating functions were made from GRAS (Generally Regarded As Safe) materials. STEM demonstrated the uniform distribution of Gd3+ on the surface of the nanoparticles. The thermodynamic binding constant for Gd3+ to the nanoparticles was approximately 10(18) M(-1) and transmetallation studies with Zn2+ yielded kinetic constants K1 and K(-1) of 0.033 and 0.022 1/h, respectively, with an equilibrium constant of 1.5. A payload of approximately 10(5) Gd/nanoparticle was achieved; enhanced relaxivities were observed, including a pH dependence of the transverse relaxivity (r2). Nanoparticles composed of materials that have been demonstrated to be hemocompatible and enzymatically metabolized and possessing accessible Gd ions on their surface induce relaxivities in the bulk water signal that make them potentially useful as next-generation MRI tumor contrast enhancement agents.

PMID: 16736756 [PubMed - indexed for MEDLINE]

J. Controlled Rel. (2006) May 30;112(3):312-9. Epub 2006 Apr 19.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

Colloidal carriers have been shown to improve tumor therapy by increased drug delivery into tumor sites resulting directly from the enhanced permeability and retention effect (EPR). However, the clinical outcome of tumor therapy is often limited due to multidrug resistance. Several different types of resistance exist with expression of p-glycoprotein being the most commonly described. Paclitaxel entrapped in emulsifying wax nanoparticles (PX NPs) was shown to overcome drug resistance in a human colon adenocarcinoma cell line (HCT-15). In the present studies, the in-vivo efficacy of PX NPs in a HCT-15 mouse xenograft model was demonstrated. Significant inhibition in tumor growth was observed in mice receiving PX NPs treatment. Additionally, mice dosed with Taxol also demonstrated slower tumor growth; however, the efficacy of the Taxol treatment was shown in the in-vitro HUVEC model to be due to the antiangiogenic effect of paclitaxel. It was concluded that the enhanced efficacy of PX NPs over Taxol in the xenograft model was due to both overcoming paclitaxel resistance and an antiangiogenic effect.

Publication Types:Comparative Study

PMID: 16626835 [PubMed - indexed for MEDLINE]

Vaccine. 2006 Apr 24;24(17):3564-73. Epub 2006 Feb 17.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

HIV-1 Tat has been identified as an attractive target for vaccine development and is currently under investigation in clinical trials as both a therapeutic and preventative vaccine for HIV-1. It is well known that protein based vaccines produce poor immune responses by themselves and therefore require adjuvants to
enhance immune responses. We have previously reported on the use of anionic nanoparticles (NPs) for enhancing cellular and humoral immune responses to Tat (1-72). The purpose of this study was to further evaluate the immune response of HIV-1 Tat (1-72) coated on anionic nanoparticles compared to alum using various doses of Tat (1-72). Nanoparticles were effective at generating comparable antibody titers at both 1 and 5 microg doses of Tat (1-72), whereas the antibody titers significantly decreased at the lower dose of Tat (1-72) using alum. Anti-sera from Tat (1-72) immunized mice reacted greatest to the N-terminal and
basic regions of Tat, with the NP groups showing stronger reactivity to these regions compared to alum. Moreover, the anti-sera from all Tat (1-72) immunized groups contained Tat-neutralizing antibodies and were able to significantly inhibit Tat-mediated long terminal repeat (LTR) transactivation.

Publication Types:
Comparative Study
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

PMID: 16516358 [PubMed - indexed for MEDLINE]

Pharm Res. 2005 Nov;22(11):1821-8. Epub 2005 Aug 26.

Department of Pharmaceutical Sciences, Center of Pharmaceutical Science and Technology, College of Pharmacy, University of Kentucky, 725 Rose Street, Lexington, KY 40536-0082, USA.

PURPOSE: Pegylated and nonpegylated cetyl alcohol/polysorbate nanoparticles (E78 NPs) are being tested as drug carriers for specific tumor and brain targeting. Because these nanoparticle formulations are designed for systemic administration, it is important to test the compatibility of these lipid-based NPs with blood and blood cells. METHODS: The hemocompatibility of E78 NPs was evaluated with a particular focus on hemolytic activity, platelet function, and blood coagulation. Human red blood cell lysis was determined by measuring hemoglobin release. Activation and aggregation of human platelets were determined using flow cytometry and aggregometry, respectively. Finally, the whole blood clotting time was measured using human blood. RESULTS: E78 NPs did not cause in vitro red blood cell lysis at concentrations up to 1 mg/mL. In addition, under conditions tested, E78 and polyethylene glycol (PEG)-coated E78 NPs (PEG-E78 NPs) did not activate platelets. In fact, both NP formulations very rapidly inhibited agonist-induced platelet activation and aggregation in a dose-dependent manner. Additionally, E78 NPs significantly prolonged in vitro whole blood clotting time at a concentration of 500 microg/mL or greater. CONCLUSIONS: It was concluded that PEG-coated and nonpegylated E78 NPs have potential blood compatibility at clinically relevant doses. Based on the calculated nanoparticle-to-platelet ratio, the concentration at which E78 NPs could potentially affect platelet function in vivo was approximately 1 mg/mL.

PMID: 16132346 [PubMed - indexed for MEDLINE]

Vaccine. (2004) 22:2631-2640.

Center for Pharmaceutical Science and Technology, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

A significant emphasis has been placed on the development of adjuvants and/or delivery systems to improve both antibody production and cell-mediated immune responses. We previously reported on a novel anionic nanoparticle, which led to enhanced humoral and T helper type-1 (Th1) biased immune responses in mice when coated with cationized model antigen. Tat (1-72) is a conserved regulatory HIV-1 protein. It was hypothesized that HIV vaccine strategies employing Tat (1-72) may be a promising approach. Although previous reports have suggested that Tat (1-86) may be immunosuppressive, it was demonstrated in this present study that Tat (1-72) was not immunosuppressive when co-administered to mice with ovalbumin (OVA). Tat (1-72) was coated on novel anionic nanoparticles. BALB/c mice were immunized with Tat (5 microg)-coated nanoparticles (15 microg) by subcutaneous injection on days 0 and 14. Antibody and cytokine release were determined on day 28 and compared to Tat (5 microg) adjuvanted with Alum (15 microg) as a Th2 control, Tat (5 microg) adjuvanted with Lipid A (50 microg) as a Th1 control. Immunization of BALB/c mice with Tat-coated nanoparticles resulted in antibody levels (IgG and IgM) comparable to those elicited from Tat and Alum. However, Tat-coated nanoparticles led to a Th1 biased immune response. The IFN-gamma release from splenocytes with Tat-coated nanoparticles was comparable to that from mice immunized with Tat and Lipid A, and 3.3-fold greater than that from mice immunized with Tat and Alum. These studies warrant further investigation of these nanoparticles to enhance both antibody and cellular-based immune responses.

PMID: 15193389 [PubMed - indexed for MEDLINE]

Journal of Drug Targeting. (2004) 12:635-41

Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106-1712, USA.

PURPOSE: The blood-brain barrier (BBB) presents both a physical and electrostatic barrier to limit brain permeation of therapeutics. Previous work has demonstrated that nanoparticles (NPs) overcome the physical barrier, but there is little known regarding the effect of NP surface charge on BBB function. Therefore, this work evaluated: (1) effect of neutral, anionic and cationic charged NPs on BBB integrity and (2) NP brain permeability. Methods: Emulsifying wax NPs were prepared from warm oil-in-water microemulsion precursors using neutral, anionic or cationic surfactants to provide the corresponding NP surface charge. NPs were characterized by particle size and zeta potential. BBB integrity and NP brain permeability were evaluated by in situ rat brain perfusion. RESULTS: Neutral NPs and low concentrations of anionic NPs were found to have no effect on BBB integrity, whereas, high concentrations of anionic NPs and cationic NPs disrupted the BBB. The brain uptake rates of anionic NPs at lower concentrations were superior to neutral or cationic formulations at the same concentrations. CONCLUSIONS: (1) Neutral NPs and low concentration anionic NPs can be utilized as colloidal drug carriers to brain, (2) cationic NPs have an immediate toxic effect at the BBB and (3) NP surface charges must be considered for toxicity and brain distribution profiles.

PMID: 15621689 [PubMed - indexed for MEDLINE]

J. Controlled Rel. (2004) 99:259-269.

Division of Pharmaceutical Sciences, Center for Pharmaceutical Science and Technology, College of Pharmacy, University of Kentucky, 725 Rose Street, Lexington, KY 40536-0082, USA.

Despite the advances in tumor therapy, patients with primary brain tumors and brain metastases have a very poor prognosis. Low responses to chemotherapy are mainly attributed to impermeability of the blood-brain barrier to cytotoxic agents. Paclitaxel has been shown to be active against gliomas and various brain metastases. However, its use in treatment of brain tumors is limited due to low blood-brain barrier permeability and serious side effects associated with administration of the paclitaxel solvent, Cremophor EL. Lack of paclitaxel brain uptake is thought to be associated with the p-glycoprotein (p-gp) efflux
transporter. In this work, paclitaxel (PX) was entrapped in novel cetyl alcohol/polysorbate nanoparticles. Paclitaxel nanoparticles (PX NPs) were characterized by means of size, short-term stability, drug entrapment efficiency, and release profile. The PX NP cytotoxicity profile was monitored using two
different cell lines, U-118 and HCT-15. Brain uptake of PX NPs was evaluated using an in situ rat brain perfusion model. The results suggest that entrapment of paclitaxel in nanoparticles significantly increases the drug brain uptake and its toxicity toward p-glycoprotein expressing tumor cells. It was hypothesized
that PX NPs could mask paclitaxel characteristics and thus limit its binding to p-gp, which consequently would lead to higher brain and tumor cell uptake of the otherwise effluxed drug.

Publication Types:
Comparative Study

PMID: 15380635 [PubMed - indexed for MEDLINE]

Appl. Rad. Isotop. (2004) 60:887-891.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

A microemulsion comprised of water, Brij 78, pentanol and styrene into which PPO and bis-MSB had been dissolved was prepared. Polymerization of the styrene resulted in a suspension of fluor-containing polystyrene nanoparticles (<100 nm). After a concentration step, the aqueous nanosuspension was able to detect (14)C with counting efficiencies over 50% of those of a commercially available scintillation cocktail. Monte Carlo calculations demonstrated that the size and concentration of the nanoparticles were appropriate for optimum detection efficiency.

PMID: 15110354 [PubMed - indexed for MEDLINE]

J.Controlled Rel. (2004) 95:613-626.

Center for Pharmaceutical Science and Technology, Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 907 Rose Street, Lexington, KY 40536-0082, USA.

The purpose of these studies was to compare the cell uptake, biodistribution and tumor retention of folate-coated and PEG-coated gadolinium (Gd) nanoparticles. Gd is a potential agent for neutron capture therapy (NCT) of tumors. Gd nanoparticles were engineered from oil-in-water microemulsion templates. To obtain folate-coated nanoparticles, a folate ligand [folic acid chemically linked to distearoylphosphatidylethanolamine (DSPE) via a PEG spacer MW 3350] was included in nanoparticle preparations. Similarly, control nanoparticles were coated with DSPE-PEG-MW 3350 (PEG-coated). Nanoparticles were characterized based on size, size distribution, morphology, biocompatibility and tumor cell uptake. In vivo studies were carried out in KB (human nasopharyngeal carcinoma) tumor-bearing athymic mice. Biodistribution and tumor retention studies were carried out at pre-determined time intervals after injection of nanoparticles (10 mg/kg). Gd nanoparticles did not aggregate platelets or activate neutrophils. The retention of nanoparticles in the blood 8, 16 and 24 h post-injection was 60%, 13% and 11% of the injected dose (ID), respectively. A maximum Gd tumor localization of 33+/-7 microg Gd/g was achieved. Both folate-coated and PEG-coated nanoparticles had comparable tumor accumulation. However, the cell uptake and tumor retention of folate-coated nanoparticles was significantly enhanced over PEG-coated nanoparticles. Thus, the benefits of folate ligand coating were to facilitate tumor cell internalization and retention of Gd-nanoparticles in the tumor tissue. The engineered nanoparticles may have potential in tumor-targeted delivery of Gd thereby enhancing the therapeutic success of NCT.

PMID: 15023471 [PubMed - indexed for MEDLINE]

Drug Dev. Ind. Pharm. (2003) 29:689-700.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082, USA.

Previously, we reported on a cationic nanoparticle-based DNA vaccine delivery system engineered from warm oil-in-water microemulsion precursors. In these present studies, the feasibility of lyophilizing the nanoparticles and their thermal properties were investigated. Also, the binding and uptake of the nanoparticles by a macrophage cell line were studied. The nanoparticles (prior to pDNA coating) were freeze-dried with lactose or sucrose as cryoprotectants. The stability of lyophilized nanoparticles at room temperature was monitored and compared to that of the aqueous nanoparticle suspension. The thermal properties of the nanoparticles were investigated using differential scanning calorimetry (DSC). The nanoparticles, coated or uncoated with mannan as a ligand, were incubated with a mannose receptor positive (MR+) mouse macrophage cell line (J774E), at either 4 degrees C or 37 degrees C to study the binding and uptake of the nanoparticles by the cells. It was found that lactose or sucrose (1-5%, w/v) was required for successful lyophilization of the nanoparticles. After 4 months of storage, the size of lyophilized nanoparticles did not significantly increase while those in aqueous suspension grew by over 900%. Unlike its individual components, emulsifying wax (m.p., approximately 55 degrees C) and hexadecyltrimethyl ammonium bromide, the nanoparticles showed a melting point of approximately 90 degrees C. Moreover, the DSC profile of the nanoparticles was different from that of the physical mixture of emulsifying wax and CTAB. After 1 hour incubation at 37 degrees C, the uptake of mannan-coated nanoparticles was 50% higher than that of the uncoated nanoparticles. At 4 degrees C and after one hour, the binding of the mannan-coated nanoparticles by J774E was over 2-fold higher than that of the uncoated nanoparticles. This increase in J774E binding could be abolished by preincubating the cells with free mannan, suggesting that the binding and uptake were receptor-mediated. In conclusion, the nanoparticles were lyophilizable, and lyophilization was shown to enhance the stability of the nanoparticles. DSC provided evidence that the nanoparticles were not a physical mixture of their individual components. Finally, cell binding and uptake studies demonstrated that the nanoparticles have potential application for cell-specific targeting.

PMID: 12889787 [PubMed - indexed for MEDLINE]

J Pharm Biomed Anal. (2003) Sep 19;33(2):181-9.

Division of Pharmaceutical Sciences, Center for Pharmaceutical Science and Technology, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

More rapid, reproducible, and cost-effective methods to control product quality in the pharmaceutical industry continue to be a major emphasis, particularly with the FDA through its recent process analytical technologies (PAT) initiative. Many different methods have been used to determine the stability and content uniformity of a drug in various dosage forms; however, most of these methods include the destruction of the sample. Therefore, the development of nondestructive methods that allow the analysis of each individual dosage form has become the basis of much research. A new assay for the nondestructive determination of testosterone content in mucoadhesive bi-layer thin-film composites (TFCs) using near-infrared spectroscopy (NIR) was developed. Five sets of the circular films (n=5) with theoretical testosterone content of 0, 1, 2, 3, and 4 mg per 3/8th in. diameter disks were scanned in the near-infrared region of 1100-2500 nm to determine testosterone content. The NIR results were directly compared with those obtained using a previously developed ultraviolet assay for testosterone at 240 nm. Principal component regression (PCR) was performed to calibrate the NIR assay. This correlation produced r2=0.99 with a standard error of estimate (SEE)=0.18 mg, and a standard error of performance (SEP)=0.18 on cross validation with an equal number of samples (F test passed at P=0.05). Though the UV assay showed a slightly better r2 value, the NIR assay was much quicker, easier, and nondestructive. Therefore, the NIR assay may have significant potential for use in the quality control of pharmaceutical films containing drugs.

PMID: 12972083 [PubMed - indexed for MEDLINE]

Inter. J. Pharm. (2003) 251:85-97.

Division of Pharmaceutical Sciences, Center for Pharmaceutical Science and Technology, College of Pharmacy, University of Kentucky, 907 Rose Street, Lexington, KY 40536-0082, USA.

Emulsifying wax and polyoxyl 2 stearyl ether (Brij 72) nanoparticles (2 mg/ml) containing high concentrations of gadolinium hexanedione (GdH) (0-3 mg) have been engineered from oil-in-water microemulsion templates. Solid nanoparticles were cured by cooling warm microemulsion templates (prepared at 55 degrees C) to room temperature in one vessel. Nanoparticles were characterized by transmission electron microscopy (TEM), photon correlation spectroscopy (PCS) and gel permeation chromatography (GPC). To obtain folate-coated nanoparticles, a folate ligand was added to either the microemulsion templates or nanoparticle suspensions at 25 degrees C. Since the concentration of Gd in the tumor is critical to the success of Gd-neutron capture therapy (NCT), the effects of various formulation factors on GdH entrapment in nanoparticles as well as tumor-targeting were studied. GdH entrapment in nanoparticles was affected mostly by the method of GdH incorporation and surfactant concentration used in preparing the microemulsion templates. Cell uptake studies were carried out in KB cells (human nasopharyngeal epidermal carcinoma cell line). The method of adding folate ligand to the formulations did not significantly affect nanoparticle cell uptake (P>0.11; t-test). However, the concentration of folate ligand added to nanoparticles had the greatest influence on nanoparticle uptake (P<0.01; t-test). The results showed that GdH entrapment and cell uptake were optimized and suggested that engineered folate-coated nanoparticles may serve as effective carrier systems for Gd-NCT of tumors.

PMID: 12527178 [PubMed - indexed for MEDLINE]

J. Controlled Rel. (2003) 93:271-282.

Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University HSC, Amarillo, TX 79106-1712, USA.

Recently, a novel nanoparticle (NP) comprised of emulsifying wax and Brij 78 was shown to have significant brain uptake using the in-situ rat brain perfusion technique. To further these studies and to specifically target brain, we have incorporated thiamine as a surface ligand on the nanoparticles. Solid nanoparticles were prepared from oil-in-water microemulsion precursors. Nanoparticles were radiolabeled and a thiamine ligand (thiamine linked to distearoylphosphatidylethanolamine via a polyethylene glycol spacer) was coated on the surface of the nanoparticles. Initial experiments focused on assessing uptake of [3H]nanoparticles with and without thiamine surface ligands. Biodistribution nanoparticle studies were also carried out in BALB/c mice. The results showed: (1) the effectiveness of using microemulsions as precursors to engineer nanoparticles, (2) kinetic modeling for brain uptake of nanoparticles with and without the thiamine surface ligands, and (3) initial data suggesting mechanisms for nanoparticle brain entry. Comparison of NP brain uptake demonstrated that the thiamine-coated nanoparticle associated with the blood-brain barrier (BBB) thiamine transporter and had an increased K(in) between 45 and 120 s (thiamine coated NP 9.8 +/- 1.1 x 10(-3) ml/s/g versus uncoated NPs; 7.0 +/- 0.3 x 10(-3) ml/s/g). It was concluded that the thiamine ligand facilitated binding and/or association with blood-brain barrier thiamine transporters, which may be a viable mechanism for nanoparticle mediated brain drug delivery.

PMID: 14644577 [PubMed - indexed for MEDLINE]

Pharm. Res. (2003) 20:1772-1778.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

PURPOSE: Two novel types of nanoparticles were evaluated as poten tial carriers for drugs across the blood-brain barrier (BBB). METHODS: Nanoparticles were composed of biocompatible materials including emulsifying wax (E. Wax) or Brij 72. Brij 78 and Tween 80 were used as surfactants for E. Wax nanoparticles (E78 NPs) and Brij 72 nanoparticles (E72 NPs), respectively. Both nanoparticle formulations were prepared from warm microemulsion precursors usin melted E. Wax or Brij 72 as the oil phase. Nanoparticles were radio-labeled by entrapment of [3H]cetyl alcohol, and entrapment efficiency and release of radiolabel were evaluated. The transport of E78 and E72 NPs across the BBB was measured by an in situ rat brai perfusion method. RESULTS: Both formulations were successfully radiolabeled by entrapment of [3H]cetyl alcohol; -98% of radiolabel remained associated with nanoparticles at experimental conditions. The transfer rate (Kin) of E78 NPs from perfusion fluid into the brain was 4.1 +/- 0.5 x 10(-3) ml/s/g, and the permeability-surface area product (PA) was 4.3 +/- 0.7 x 10(-3) ml/s/g. The values for Kin and PA for E72 NPs were 5.7 +/- 1.1 x 10(-3) ml/s/g and 6.1 +/- 1.4 x 10(-3) ml/s/g, respectively. CONCLUSIONS: For both nanoparticle types, statistically significant uptake was observed compared to [14C]sucrose, suggesting central nervous system uptake of nanoparticles. The mechanism underlying th nanoparticle brain uptake has yet to be fully understood.

PMID: 14661921 [PubMed - indexed for MEDLINE]