Hillevi Bauer

Size and surface charge significantly influence the toxicity of silica and dendritic nanoparticles

Abstract The influence of size, surface charge and surface functionality of poly(amido amine) dendrimers and silica nanoparticles (SNPs) on their toxicity was studied in immunocompetent mice. After systematic characterization of nanoparticles, they were administered to CD-1 (caesarean derived-1) mice to evaluate acute toxicity. A distinct trend in nanotoxicity based on surface charge and functional group was observed with dendrimers regardless of their size. Amine-terminated dendrimers were fatal at doses >10 mg/kg causing haematological complications such as disseminated intravascular coagulation-like manifestations whereas carboxyl- and hydroxyl-terminated dendrimers of similar sizes were tolerated at 50-fold higher doses. In contrast, larger SNPs were less tolerated than smaller SNPs irrespective of their surface functionality. These findings have important implications in the use of these nanoparticles for various biomedical applications.

Biorecognition and subcellular trafficking of HPMA copolymer-anti-PSMA antibody conjugates by prostate cancer cells.

A new generation of antibodies against the prostate specific membrane antigen (PSMA) has been proven to bind specifically to PSMA molecules on the surface of living prostate cancer cells. To explore the potential of anti-PSMA antibodies as targeting moieties for macromolecular therapeutics for prostate cancer, fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymer-anti-PSMA antibody conjugates (P-anti-PSMA) were synthesized and the mechanisms of their endocytosis and subcellular trafficking in C4-2 prostate cancer cells were studied. Radioimmunoassays showed the dissociation constants of P-anti-PSMA for C4-2 prostate cancer cells in the low nanomolar range, close to values for free anti-PSMA. It indicated that conjugation of anti-PSMA to HPMA copolymers did not compromise their binding affinity. The rate of endocytosis of P-anti-PSMA was much faster than that of control HPMA copolymer conjugates containing nonspecific IgG. Selective pathway inhibitors of clathrin-mediated endocytosis and of macropinocytosis inhibited the internalization of P-anti-PSMA. Inhibition of clathrin-mediated endocytosis was further evidenced by down-regulation of clathrin heavy chain expression by siRNA. Using a dominant-negative mutant of dynamin (Dyn K44A) to abolish the clathrin-, caveolae-independent endocytic pathway, we found that some of P-anti-PSMA adopted this pathway to be endocytosed into C4-2 cells. Thus multiple receptor-mediated endocytic pathways, including clathrin-mediated endocytosis, macropinocytosis, and clathrin-, caveolae-independent endocytosis, were involved in the internalization of P-anti-PSMA. The extent of the participation of each pathway in P-anti-PSMA endocytosis was estimated. Membrane vesicles containing P-anti-PSMA rapidly colocalized with membrane vesicles overexpressing Rab7, a late endosome localized protein, demonstrating that a part of P-anti-PSMA was transported to late endosomes.

Endocytic uptake of a large array of HPMA copolymers: Elucidation into the dependence on the physicochemical characteristics

Endocytic uptake and subcellular trafficking of a large array of HPMA (N-(2-hydroxypropyl)methacrylamide) based copolymers possessing positively or negatively charged residues, or hydrophobic groups were evaluated by flow cytometry and living cell confocal microscopy in cultured prostate cancer cells. The degrees of cellular uptake of various copolymer fractions with narrow polydispersities were quantified. The copolymer charge was the predominant physicochemical feature in terms of cellular uptake. Fast and efficient uptake occurred in positively charged copolymers due to non-specific adsorptive endocytosis, whereas slow uptake of negatively charged copolymers was observed. The uptake of copolymers was also molecular weight dependent. The copolymers were internalized into the cells through multiple endocytic pathways: positively charged copolymers robustly engaged clathrin-mediated endocytosis, macropinocytosis and dynamin-dependent endocytosis, while weakly negatively charged copolymers weakly employed these pathways; strongly negatively charged copolymers only mobilized macropinocytosis. HPMA copolymer possessing 4 mol% of moderately hydrophobic functional groups did not show preferential uptake. All copolymers ultimately localized in late endosomes/lysosomes via early endosomes; with varying kinetics among the copolymers. This study indicates that cell entry and subsequent intracellular trafficking of polymeric drug carriers are strongly dependent on the physicochemical characteristics of the nanocarrier, such as charge and molecular weight.

Comparison of active and passive targeting of docetaxel for prostate cancer therapy by HPMA copolymer-RGDfK conjugates.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel-RGDfK conjugate was synthesized, characterized, and evaluated in vitro and in vivo in comparison with untargeted low and high molecular weight HPMA copolymer-docetaxel conjugates. The targeted conjugate was designed to have a hydrodynamic diameter below renal threshold to allow elimination post treatment. All conjugates demonstrated the ability to inhibit the growth of DU145 and PC3 human prostate cancer cells and the HUVEC at low nanomolar concentrations. The targeted conjugate showed active binding to α(v)β(3) integrins in both HUVEC and DU145 cells, whereas the untargeted conjugate demonstrated no evidence of specific binding. Efficacy at two concentrations (20 mg/kg and 40 mg/kg) was evaluated in nu/nu mice bearing DU145 tumor xenografts treated with a single dose of conjugates and compared with controls. RGDfK targeted and high molecular weight nontargeted conjugates exhibited the highest antitumor efficacy as evaluated by tumor regression. These results demonstrate that α(v)β(3) integrin targeted polymeric conjugates with improved water solubility, reduced toxicity and ease of elimination post treatment in vivo are promising candidates for prostate cancer therapy.

Anticancer and antiangiogenic activity of HPMA copolymer-aminohexylgeldanamycin-RGDfK conjugates for prostate cancer therapy.

Tumor progression is dependent on neoangiogenesis for blood supply. Neovasculature over-express α(v)β(3) integrins which recognize the Arg-Gly-Asp (RGD) sequence in the extracellular matrix. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing side chains terminated in cyclic RGD analogs such as RGDfK show increased accumulation in prostate tumors. Geldanamycin and their derivatives (e.g., aminohexylgeldanamycin (AH-GDM)) are benzoquinone ansamycins that have both antiangiogenic and antitumor activity. In this work the antiangiogenic and antitumor activities of targetable HPMA copolymer-RGDfK-AH-GDM conjugates were compared with non-targetable systems in vitro and in vivo. Copolymer-drug conjugates containing RGDfK in the side chains showed superior activity against endothelial and prostate cancer cell lines in vitro, as well as higher inhibition of angiogenesis in vivo. At equimolar doses of the drug, the RGDfK containing conjugates showed significantly higher antitumor activity in nude mice bearing DU-145 human prostate cancer xenografts. These findings suggest the utility of HPMA copolymer-RGDfK conjugates for targeted delivery of geldanamycin analogs with a dual mode of action.

Synthesis and evaluation of poly(styrene-co-maleic acid) micellar nanocarriers for the delivery of tanespimycin

Polymeric micelles carrying the heat shock protein 90 inhibitor tanespimycin (17-N-allylamino-17-demethoxygeldanamycin) were synthesized using poly(styrene-co-maleic acid) (SMA) copolymers and evaluated in vitro and in vivo. SMA-tanespimycin micelles were prepared with a loading efficiency of 93%. The micelles incorporated 25.6% tanespimycin by weight, exhibited a mean diameter of 74 ± 7 nm by dynamic light scattering and a zeta potential of -35 ± 3 mV. Tanespimycin was released from the micelles in a controlled manner in vitro, with 62% released in 24h from a pH 7.4 buffer containing bovine serum albumin. The micellar drug delivery systems for tanespimycin showed potent activity against DU145 human prostate cancer cells, with an IC(50) of 230 nM. They further exhibited potent anti-cancer activity in vivo in nu/nu mice bearing subcutaneous DU145 human prostate cancer tumor xenografts, with significantly higher anticancer efficacy as measured by tumor regression when compared to free tanespimycin at an equivalent single dose of 10mg/kg. These data suggest further investigation of SMA-tanespimycin as a promising agent in the treatment of prostate cancer.

Assessment of HER2 testing patterns, HER2+ disease, and the utilization of HER2-directed therapy in early breast cancer

Context Determining human epidermal growth factor receptor 2 (HER2) status is critical for the management of early-stage breast cancer (ESBC). An understanding of HER2 testing practices can provide insight into how test results influence the use of HER2-directed therapy. Objective To assess HER2 testing, HER2+ disease, and HER2-directed therapy in ESBC at the Huntsman Cancer Institute before and after the 2007 American Society of Clinical Oncology and College of American Pathologist (ASCO/CAP) guidelines on HER2 testing were published. Methods Patients were identified from an institutional tumor registry. HER2 testing patterns and results were examined using a chart review of pathology and clinical notes. Patient characteristics, HER2+ rate, and trastuzumab use were evaluated descriptively. Discordance rate with reflex testing (immunohistochemistry [IHC]2+ retested by fluorescence in situ hybridization [FISH]) was also evaluated. Results A total of 1,459 women were included (mean age: 57 years). The rate of HER2+ disease was 17% (number [N] =245). The discordance rate between IHC2+ and FISH was 10%. After the 2007 ASCO/CAP guidelines, fewer tumors were classified as IHC3+ (16% post- versus 21.9% pre-2007), more tumors were characterized as IHC2+ (26.4% post- versus 20.7% pre-2007), and the overall HER2+ rate was decreased (18.7% versus 21.9%), but this was not statistically significant (P=0.519). Most patients with HER2+ ESBC received HER2-targeted therapy (N=185). Conclusion The HER2+ rate was 17% and within the range of the reported rates in the literature. Reflex testing identified additional HER2+ tumors by approximately 10%, and should be considered a potential quality indicator. ASCO/CAP HER2 testing guidelines in 2007 appeared to impact the interpretation and classification of HER2+ tumors.

Impact of a genetic counseling requirement prior to genetic testing

BackgroundGenetic counseling by a Genetic Counselor (GC) is a requirement prior to genetic testing for cancer susceptibility genes (GC-mandate policy) for some insurers. This study evaluated the impact of this policy from the patient perspective.MethodsSurveys were sent to individuals for whom their insurer ordered genetic testing for the cancer susceptibility genes BCRA1 and BRCA2 over a 1 year time period that spanned the introduction of a GC-mandate policy. Responses were assessed by time period (before/after policy introduction) and genetic test completion.ResultsThe surveys were completed by 1247/4950 (25.7%) eligible individuals. After policy introduction, there was no change in the proportion of respondents who completed genetic testing (p = 0.13) or had a mutation (p = 0.55). Overall decisional conflict (uncertainty or feeling uninformed) around genetic testing did not change after policy introduction (p = 0.16), but was significantly higher among respondents who did not complete genetic testing (p < 0.01). Although a larger proportion of respondents saw a GC after policy introduction (p < 0.01), fewer did so to better understand their test results (p < 0.01). The proportion of respondents who did not see a GC due to insurance issues/requirements and time restraints was higher among those tested after policy introduction or who did not complete genetic testing (p < 0.01). In multivariate analysis, respondents with a household income of

Response Monitoring, Tolerability, and Effectiveness of Imatinib Treatment for Chronic Myeloid Leukemia in a Retrospective Research Database

25,000 or greater were 3-times more likely to complete testing.ConclusionsA GC-mandate policy did not improve decisional conflict or increase the number of deleterious mutations identified and low-income respondents were less likely to complete testing. On the contrary, insurance requirements and time constraints may be preventing individuals at risk from receiving appropriate testing.