Alex E. Grill

Folic Acid Functionalized Nanoparticles for Enhanced Oral Drug Delivery

The oral absorption of drugs that have poor bioavailability can be enhanced by encapsulation in polymeric nanoparticles. Transcellular transport of nanoparticle-encapsulated drug, possibly through transcytosis, is likely the major mechanism through which nanoparticles improve drug absorption. We hypothesized that the cellular uptake and transport of nanoparticles can be further increased by targeting the folate receptors expressed on the intestinal epithelial cells. The objective of this research was to study the effect of folic acid functionalization on transcellular transport of nanoparticle-encapsulated paclitaxel, a chemotherapeutic with poor oral bioavailability. Surface-functionalized poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles loaded with paclitaxel were prepared by the interfacial activity assisted surface functionalization technique. Transport of paclitaxel-loaded nanoparticles was investigated using Caco-2 cell monolayers as an in vitro model. Caco-2 cells were found to express folate receptor and the drug efflux protein, p-glycoprotein, to high levels. Encapsulation of paclitaxel in PLGA nanoparticles resulted in a 5-fold increase in apparent permeability (Papp) across Caco-2 cells. Functionalization of nanoparticles with folic acid further increased the transport (8-fold higher transport compared to free paclitaxel). Confocal microscopic studies showed that folic acid functionalized nanoparticles were internalized by the cells and that nanoparticles did not have any gross effects on tight junction integrity. In conclusion, our studies indicate that folic acid functionalized nanoparticles have the potential to enhance the oral absorption of drugs with poor oral bioavailability.

Interfacial Activity Assisted Surface Functionalization: A Novel Approach to Incorporate Maleimide Functional Groups and cRGD Peptide on Polymeric Nanoparticles for Targeted Drug Delivery

Nanoparticles formulated using poly(d,l-lactide-co-glycolide) (PLGA) copolymer have emerged as promising carriers for targeted delivery of a wide variety of payloads. However, an important drawback with PLGA nanoparticles is the limited types of functional groups available on the surface for conjugation to targeting ligands. In the current report, we demonstrate that the interfacial activity assisted surface functionalization (IAASF) technique can be used to incorporate reactive functional groups such as maleimide onto the surface of PLGA nanoparticles. The surface maleimide groups were used to conjugate cRGD peptide to nanoparticles. The cRGD peptide targets alpha(v)beta(3) integrins overexpressed on tumor vasculature and some tumor cells, and was used as model targeting ligand in this study. Incorporation of biologically active cRGD peptide on the surface of nanoparticles was confirmed by in vitro cell uptake studies and in vivo tumor accumulation studies. Functionalization of nanoparticles with cRGD peptide increased the cellular uptake of nanoparticles 2-3-fold, and this enhancement in uptake was substantially reduced by the presence of excess cRGD molecules. In a syngeneic mouse 4T1 tumor model, cRGD functionalization resulted in increased accumulation and retention of nanoparticles in the tumor tissue (nearly 2-fold greater area under the curve), confirming the in vivo activity of cRGD functionalized nanoparticles. In conclusion, the IAASF technique enabled the incorporation of reactive maleimide groups on PLGA nanoparticles, which in turn permitted efficient conjugation of biologically active cRGD peptide to the surface of PLGA nanoparticles.

Mutagenicity of furan in female Big Blue B6C3F1 mice.

Furan is an abundant food and environmental contaminant that is a potent liver carcinogen in rodent models. To determine if furan is genotoxic in vivo, female B6C3F1 Big Blue transgenic mice were treated with 15 mg/kg bw furan by gavage 5 days a week for 6 weeks, or once weekly for 3 weeks. Liver cII transgene mutation-frequency and mutation spectra were determined. Furan did not increase the mutation frequency under either treatment condition. In the 6-week treatment regimen, there was a change in the cII transgene mutation-spectrum, with the fraction of GC to AT transitions significantly reduced. The only other significant change was an increase in GC to CG transversions; these represented a minor contribution to the overall mutation spectrum. A much larger furan-dependent shift was observed in the 3-week study. There was a significant increase in transversion mutations, predominantly GC to TA transversions as well as smaller non-significant changes in GC to CG and AT to TA transversions. To determine if these mutations were caused by cis-2-butene-1,4-dial (BDA), a reactive metabolite of furan, the mutagenic activity and the mutation spectrum of BDA was determined in vitro, in Big Blue mouse embryonic fibroblasts. This compound did not increase the cII gene mutation-frequency but caused a substantial increase in AT to CG transversions. This increase, however, lost statistical significance when adjusted for multiple comparisons. Together, these findings suggest that BDA may not be directly responsible for the in-vivo effects of furan on mutational spectra. Histopathological analysis of livers from furan-treated mice revealed that furan induced multifocal, hepatocellular necrosis admixed with reactive leukocytes and pigment-laden Kupffer cells, enhanced oval-cell hyperplasia, and increased hepatocyte mitoses, some of which were atypical. An indirect mechanism of genotoxicity is proposed in which chronic toxicity followed by inflammation and secondary cell proliferation triggers cancer development in furan-exposed rodents.

Epithelial proinflammatory response and curcumin-mediated protection from staphylococcal toxic shock syndrome toxin-1.

Staphylococcus aureus initiates infections and produces virulence factors, including superantigens (SAgs), at mucosal surfaces. The SAg, Toxic Shock Syndrome Toxin-1 (TSST-1) induces cytokine secretion from epithelial cells, antigen presenting cells (APCs) and T lymphocytes, and causes toxic shock syndrome (TSS). This study investigated the mechanism of TSST-1-induced secretion of proinflammatory cytokines from human vaginal epithelial cells (HVECs) and determined if curcumin, an anti-inflammatory agent, could reduce TSST-1-mediated pathology in a rabbit vaginal model of TSS. TSST-1 caused a significant increase in NF-κB-dependent transcription in HVECs that was associated with increased expression of TNF- α, MIP-3α, IL-6 and IL-8. Curcumin, an antagonist of NF-κB-dependent transcription, inhibited IL-8 production from ex vivo porcine vaginal explants at nontoxic doses. In a rabbit model of TSS, co-administration of curcumin with TSST-1 intravaginally reduced lethality by 60% relative to 100% lethality in rabbits receiving TSST-1 alone. In addition, TNF-α was undetectable from serum or vaginal tissue of curcumin treated rabbits that survived. These data suggest that the inflammatory response induced at the mucosal surface by TSST-1 is NF-κB dependent. In addition, the ability of curcumin to prevent TSS in vivo by co-administration with TSST-1 intravaginally suggests that the vaginal mucosal proinflammatory response to TSST-1 is important in the progression of mTSS.

Abundant Rodent Furan-Derived Urinary Metabolites Are Associated with Tobacco Smoke Exposure in Humans

Furan, a possible human carcinogen, is found in heat treated foods and tobacco smoke. Previous studies have shown that humans are capable of converting furan to its reactive metabolite, cis-2-butene-1,4-dial (BDA), and therefore may be susceptible to furan toxicity. Human risk assessment of furan exposure has been stymied because of the lack of mechanism-based exposure biomarkers. Therefore, a sensitive LC-MS/MS assay for six furan metabolites was applied to measure their levels in urine from furan-exposed rodents as well as in human urine from smokers and nonsmokers. The metabolites that result from direct reaction of BDA with lysine (BDA-N(α)-acetyllysine) and from cysteine-BDA-lysine cross-links (N-acetylcysteine-BDA-lysine, N-acetylcysteine-BDA-N(α)-acetyllysine, and their sulfoxides) were targeted in this study. Five of the six metabolites were identified in urine from rodents treated with furan by gavage. BDA-N(α)-acetyllysine, N-acetylcysteine-BDA-lysine, and its sulfoxide were detected in most human urine samples from three different groups. The levels of N-acetylcysteine-BDA-lysine sulfoxide were more than 10 times higher than that of the corresponding sulfide in many samples. The amount of this metabolite was higher in smokers relative to that in nonsmokers and was significantly reduced following smoking cessation. Our results indicate a strong relationship between BDA-derived metabolites and smoking. Future studies will determine if levels of these biomarkers are associated with adverse health effects in humans.

Dihydromethysticin from kava blocks tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis and differentially reduces DNA damage in A/J mice

We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice with a preferential reduction in NNK-induced O (6)-methylguanine (O (6)-mG). In this study, we first identified natural (+)-dihydromethysticin (DHM) as a lead compound through evaluating the in vivo efficacy of five major compounds in Fraction B on reducing O (6)-mG in lung tissues. (+)-DHM demonstrated outstanding chemopreventive activity against NNK-induced lung tumorigenesis in A/J mice with 97% reduction of adenoma multiplicity at a dose of 0.05mg/g of diet (50 ppm). Synthetic (±)-DHM was equally effective as the natural (+)-DHM in these bioassays while a structurally similar analog, (+)-dihydrokavain (DHK), was completely inactive, revealing a sharp in vivo structure-activity relationship. Analyses of an expanded panel of NNK-induced DNA adducts revealed that DHM reduced a subset of DNA adducts in lung tissues derived from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the active metabolite of NNK). Preliminary 17-week safety studies of DHM in A/J mice at a dose of 0.5mg/g of diet (at least 10× its minimum effective dose) revealed no adverse effects, suggesting that DHM is likely free of kavas hepatotoxic risk. These results demonstrate the outstanding efficacy and promising safety margin of DHM in preventing NNK-induced lung tumorigenesis in A/J mice, with a unique mechanism of action and high target specificity.

Abstract 602: Chemopreventive efficacy of sustained release curcumin microparticles depends on tumorigenesis stage during initial treatment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Our lab has developed novel curcumin loaded PLGA microparticles for effectively maintaining curcumin blood and tissue levels for at least 45 days. Efficacy of this system was tested in Balb-neuT mice, a transgenic model of HER2 driven breast cancer. This model mimics human tumorigenesis and follows a well predicted timeline: hyperplasia begins at 3 weeks of age followed by carcinoma in situ at 8 weeks, palpable tumors appearing at 17 weeks, and all mammary glands developing tumors by 33 weeks of age. Our previous studies showed that starting curcumin microparticle treatment at 4 weeks of age resulted in significant chemopreventive efficacy. The goal of the current work was to examine the chemopreventive efficacy of curcumin microparticle treatment as a function of tumorigenesis stage at which the treatment was started. Mice were injected subcutaneously with curcumin loaded or empty microparticles at 2, 4, 7 or 12 weeks of age and then once a month thereafter until all mammary pads developed palpable tumors. Mice that started receiving curcumin microparticle treatment at 2 weeks of age showed similar delays in tumor multiplicity compared to mice initially treated at 4 weeks. Times to first tumor (14 vs 15.5 weeks) and 100% tumor multiplicity (18.5 vs 21 weeks) were significantly delayed compared to blank microparticle controls. Additionally, tumor growth was slower in curcumin treated animals. At 8 weeks of age, mammary tissue showed significantly decreased abnormal mammary tissue (3 vs 7%) and decreased CD-31 staining (16 vs 20 counts/field) compared to age-matched controls. Mammary vasculature was also visually smaller and less developed. Based on these results, decreased angiogenesis appears to be an important chemopreventive mechanism for curcumin before the onset of hyperplasia. In mice receiving curcumin microparticle treatment initially at 4 weeks of age showed a difference in age to 100% tumor multiplicity (19 vs 21 weeks) compared to mice that received control microparticles. At 12 weeks, histology studies showed that curcumin treatment resulted in a significant (p<0.05) decrease in Ki-67 expression and overall percent area of abnormal mammary tissue (lobular hyperplasia, carcinoma in situ and invasive carcinoma). Western blotting showed decreased HER2 protein levels in curcumin-treated mice. Decreased cellular proliferation is a key chemopreventive mechanism of action in this case. No difference in tumor multiplicity was seen when the treatments were started at 7 or 12 weeks of age. In summary, sustained release curcumin microparticles show promise as a chemopreventive against HER-2 positive breast cancer. These results indicate that curcumin treatment should be started before the appearance of carcinoma in situ. Future studies will further examine the different mechanisms of action of curcumin in HER2 driven breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 602. doi:1538-7445.AM2012-602

Abstract 1856: Sustained release curcumin microparticles delay mammary tumorigenesis in BALB-neuT transgenic mice

This work examined the chemopreventive efficacy of curcumin delivered by poly(lactide-co-glycolide) (PLGA) microparticles in a HER-2 transgenic mouse mammary tumor model. HER-2 positive breast cancers have a high incidence of metastasis to the brain and are not well treated by conventional means. Curcumin, a dietary polyphenol derived from turmeric, is a powerful antioxidant and has shown chemopreventive activity in vitro. Curcumin suffers from poor oral bioavailability, requiring the need for a controlled release system that could maintain steady plasma levels over a long period of time. Our lab has formulated a curcumin loaded microparticle formulation using the FDA approved polymer, PLGA. Previously published data using this system showed steady curcumin plasma concentrations for a month and efficacy against MDA-MB-231 tumor xenografts (Shahani et al, Cancer Res. 70(11):4443-52). In the present study, Balb-neuT +/- mice were injected subcutaneously with curcumin loaded or empty microparticles at 4, 7 or 12 weeks of age and then once a month thereafter until all mammary pads developed palpable tumors. Mice that started receiving curcumin microparticles at 4 weeks of age showed a difference in age to 50% tumor multiplicity (16 vs 18.5 weeks) and 100% tumor multiplicity (20 vs 22 weeks) compared to those that received control microparticles. No difference was seen in tumor multiplicity when the treatments were started at 7 or 12 weeks of age. Mammary pads from mice that started receiving curcumin treatment at 4 weeks of age showed a significant decrease in Ki-67 expression compared to those from control mice. Curcumin treated mice also showed a decrease in overall percent area of tissue showing abnormal morphology (lobular hyperplasia and carcinoma in situ). These data suggest that curcumin delays tumorigenesis in this model through inhibition of cell proliferation. In summary, sustained release curcumin microparticles show promise as a chemopreventive against Her-2 positive breast cancer. Future studies will examine the effect of starting curcumin treatment at 2 weeks of age (prior to induction of hyperplasia) and determine other potential mechanisms of action of curcumin including inhibition of angiogenesis and induction of apoptosis in this transgenic model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1856. doi:10.1158/1538-7445.AM2011-1856