Perlecan-targeted nanoparticles for drug delivery to triple-negative breast cancer.

AIM: We previously developed two antibodies that bind to a cell surface protein, perlecan, overexpressed in triple-negative breast cancer (TNBC). The goal of this study was to investigate these antibodies as targeting ligands for nanoparticle-mediated drug delivery. METHODS: Paclitaxel-loaded poly(D,L-lactide-co-glycolide) nanoparticles were functionalized with antibodies using thiol-maleimide chemistry. Effect of antibody functionalization on therapeutic efficacy of drug-loaded nanoparticles was investigated using in vitro and in vivo models of TNBC. RESULTS: The antibodies were covalently conjugated to nanoparticles without affecting antibody binding affinity or nanoparticle properties. Perlecan-targeted nanoparticles showed improved cell uptake, retention, cytotoxicity in vitro and enhanced tumor growth inhibition in vivo. CONCLUSION: The data presented here indicates that perlecan-targeted nanoparticles can improve tumor drug delivery to TNBC.

Discovery of HSPG2 (Perlecan) as a Therapeutic Target in Triple Negative Breast Cancer.

In recent years, there have been significant advances in the treatment of breast cancer resulting in remarkably high survival rates. However, treatment options for metastatic triple negative breast cancer (TNBC) are quite limited due to a lack of identifiable, unique markers. Using a phage display-based whole cell biopanning procedure, we developed two human antibodies that bind to tumor cells with a metastatic TNBC phenotype. Our studies further identified domain 1 of HSPG2 (perlecan) protein as the cognate cell surface antigen bound by the antibody. Immunohistochemistry studies utilizing patient tissue samples revealed significant cell surface expression of HSPG2 in both primary tumors and metastatic lesions. Further, higher HSPG2 expression correlated with poor survival in TNBC. The affinity-matured antibody inhibited the growth of triple negative MDA-MB-231 tumors to a greater extent in nude mice than in NSG mice, pointing to the potential role of natural killer cell-mediated antibody-dependent cell cytotoxicity. This mechanism of action was confirmed through in vitro assays using mouse splenocytes and human peripheral blood mononuclear cells (PBMCs). These results suggest that HSPG2 is a promising target in metastatic TNBC and HSPG2-targeted antibodies could represent a potentially novel class of targeted therapeutics for TNBC.

Nanoparticles Containing High Loads of Paclitaxel-Silicate Prodrugs: Formulation, Drug Release, and Anticancer Efficacy.

We have investigated particle size, interior structure, drug release kinetics, and anticancer efficacy of PEG-b-PLGA-based nanoparticles loaded with a series of paclitaxel (PTX)-silicate prodrugs [PTX-Si(OR)3]. Silicate derivatization enabled us to adjust the hydrophobicity and hydrolytic lability of the prodrugs by the choice of the alkyl group (R) in the silicate derivatives. The greater hydrophobicity of these prodrugs allows for the preparation of nanoparticles that are stable in aqueous dispersion even when loaded with up to ca. 75 wt % of the prodrug. The hydrolytic lability of silicates allows for facile conversion of prodrugs back to the parent drug, PTX. A suite of eight PTX-silicate prodrugs was investigated; nanoparticles were made by flash nanoprecipitation (FNP) using a confined impingement jet mixer with a dilution step (CIJ-D). The resulting nanoparticles were 80-150 nm in size with a loading level of 47-74 wt % (wt %) of a PTX-silicate, which corresponds to 36-59 effective wt % of free PTX. Cryogenic transmission electron microscopy images show that particles are typically spherical with a core-shell structure. Prodrug/drug release profiles were measured. Release tended to be slower for prodrugs having greater hydrophobicity and slower hydrolysis rate. Nanoparticles loaded with PTX-silicate prodrugs that hydrolyze most rapidly showed in vitro cytotoxicity similar to that of the parent PTX. Nanoparticles loaded with more labile silicates also tended to show greater in vivo efficacy.

Silicate esters of paclitaxel and docetaxel: synthesis, hydrophobicity, hydrolytic stability, cytotoxicity, and prodrug potential.

We report here the synthesis and selected properties of various silicate ester derivatives (tetraalkoxysilanes) of the taxanes paclitaxel (PTX) and docetaxel (DTX) [i.e., PTX-OSi(OR)3 and DTX-OSi(OR)3]. Both the hydrophobicity and hydrolytic lability of these silicates can be (independently) controlled by choice of the alkyl group (R). The synthesis, structural characterization, hydrolytic reactivity, and in vitro cytotoxicity against the MDA-MB-231 breast cancer cell line of most of these derivatives are described. We envision that the greater hydrophobicity of these silicates (vis-à-vis PTX or DTX itself) should be advantageous from the perspective of preparation of stable aqueous dispersions of amphiphilic block-copolymer-based nanoparticle formulations.

Exploiting nanotechnology to overcome tumor drug resistance: Challenges and opportunities.

Tumor cells develop resistance to chemotherapeutic drugs through multiple mechanisms. Overexpression of efflux transporters is an important source of drug resistance. Efflux transporters such as P-glycoprotein reduce intracellular drug accumulation and compromise drug efficacy. Various nanoparticle-based approaches have been investigated to overcome efflux-mediated resistance. These include the use of formulation excipients that inhibit transporter activity and co-delivery of the anticancer drug with a specific inhibitor of transporter function or expression. However, the effectiveness of nanoparticles can be diminished by poor transport in the tumor tissue. Hence, adjunct therapies that improve the intratumoral distribution of nanoparticles may be vital to the successful application of nanotechnology to overcome tumor drug resistance. This review discusses the mechanisms of tumor drug resistance and highlights the opportunities and challenges in the use of nanoparticles to improve the efficacy of anticancer drugs against resistant tumors.

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.

Inhibition of vinyl carbamate-induced pulmonary adenocarcinoma by indole-3-carbinol and myo-inositol in A/J mice.

In previous studies, we reported that indole-3-carbinol (I3C) and myo-inositol (MI) inhibit lung adenoma induced by tobacco smoke carcinogens in A/J mice. In this paper, we extended our work and examined the effects of I3C (70 or 30 micromol/g diet) and MI (56 micromol/g diet) against vinyl carbamate (VC)-induced lung adenocarcinoma by administering the agents from 1 week after the second of two injections of VC until termination of the study at week 18. The higher dose of I3C decreased multiplicities of tumors on the surface of the lung (26%, P = 0.0005), carcinoma incidence (38%), multiplicity (67%, P < 0.0001) and size (complete abolition of carcinoma with an area of >1.0 cm(2)) as well as adenoma with cellular pleomorphism (46%, P < 0.0001). The lower dose of I3C was less effective. MI decreased multiplicities of pulmonary surface tumors (20%, P = 0.0005), adenoma with cellular pleomorphism (40%, P < 0.0001) and lung adenoma (52%, P < 0.0001) and the proportion of the biggest carcinoma (carcinoma with an area of >1.0 cm(2), P < 0.05). Immunoblot analyses of lung tissues for potential target identification showed that I3C (70 micromol/g diet) inhibits IkappaBalpha degradation, nuclear factor-kappaB activation, expression of cyclooxygenase-2, phospho-Akt and fatty acid synthase (FAS) and activates caspase-3 and poly ADP ribose polymerase cleavage. The effect of MI was limited to inhibition of phospho-Akt and FAS expression. Our data show that I3C and MI inhibit lung carcinoma and provide a basis for future evaluation of these compounds in clinical trials as chemopreventive agents for current and former smokers.

Differential expression of microRNAs in early-stage neoplastic transformation in the lungs of F344 rats chronically treated with the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

While numerous microRNAs (miRNAs) have been reported to alter their expression levels in human lung cancer tissues compared with normal tissues, the function of these miRNAs and their contribution to the long process of lung cancer development remains largely unknown. We applied a tobacco-specific carcinogen-induced cancer model to investigate the involvement of miRNAs in early lung cancer development, which could also provide information on potential, early biomarkers of lung cancers. Male F344 rats were first chronically treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogen present in tobacco products, for up to 20 weeks. The expression profiles of miRNAs in rat lungs were then determined. As measured by miRNA microarrays and confirmed by Northern blot and real-time polymerase chain reaction analyses, NNK treatment reduced the expression of a number of miRNAs, such as miR-101, miR-126*, miR-199 and miR-34. Significantly, these miRNAs overlap with previously published reports on altered miRNA expression in human lung cancer samples. These miRNAs might, therefore, represent early-response miRNAs that signify the molecular changes associated with pulmonary tumorigenesis. Moreover, we identified cytochrome P450 (CYP) 2A3, a critical enzyme in rat lungs that activates NNK to render it carcinogenic, as a potential target of miR-126*. NNK treatment in rats repressed miR-126* but induced CYP2A3 expression, a mechanism that may potentiate the oncogenic effects of NNK.

Quantitation of pyridylhydroxybutyl-DNA adducts in liver and lung of F-344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in rats and is believed to be one cause of lung cancer in smokers. NNK is metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a strong lung carcinogen in rats and has a chiral center at its 1-carbon. Previous studies have demonstrated that cytochrome P450-catalyzed alpha-hydroxylation of NNK in the lung leading to the formation of methyl and pyridyloxobutyl (POB)-DNA adducts is critical for its carcinogenicity. alpha-Hydroxylation of NNAL would similarly produce pyridylhydroxybutyl (PHB)-DNA adducts, but these have not been previously investigated in vivo. POB- and PHB-DNA adduct levels can indicate the amounts of pyridyloxobutylating and pyridylhydroxybutylating agents present in tissues of NNK- or NNAL-treated rats at any given point. Therefore, in this study, we developed a sensitive and quantitative liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring method to determine levels of the PHB-DNA adducts O(6)-[4-(3-pyridyl)-4-hydroxybut-1-yl]-2'-deoxyguanosine (O(6)-PHB-dGuo, 10b), O(2)-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (O(2)-PHB-dThd, 11b), and 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]-2'-deoxyguanosine (7-PHB-dGuo, 12b), the latter as the corresponding base 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]-Gua (7-PHB-Gua, 14b) in DNA isolated from liver and lung of rats treated with 10 ppm NNK, (S)-NNAL, or (R)-NNAL in the drinking water for 20 weeks and sacrificed at 1, 2, 5, 10, 16, and 20 weeks. PHB-DNA adduct levels were higher in lung than in liver at each time point, consistent with previous studies of POB-DNA adducts in rats treated with NNK and NNAL in the drinking water. The results showed that NNK and (S)-NNAL behaved in a similar fashion, while (R)-NNAL was strikingly different. In the rats treated with NNK or (S)-NNAL, levels of each adduct at each time point were remarkably similar in lung, and levels of O(2)-PHB-dThd were generally greater than 7-PHB-Gua > O(6)-PHB-dGuo. The highest PHB-DNA adduct levels were found in lung and liver of rats treated with (R)-NNAL, suggesting that there are cytochrome P450s that efficiently catalyze the alpha-methyl hydroxylation of this compound. The results of this study provide further support for our hypothesis that (S)-NNAL is rapidly formed from NNK, sequestered at an unknown site in the lung, and then released and reoxidized to NNK with consequent DNA adduct formation resulting in lung carcinogenicity.