Cancer Nanotechnology: Opportunities for Prevention, Diagnosis, and Therapy.

Nanotechnological innovations over the last 16 years have brought about the potential to revolutionize specific therapeutic drug delivery to cancer tissue without affecting normal tissues. In addition, there are new nanotechnology-based platforms for diagnosis of cancers and for theranostics, i.e., integrating diagnosis with therapy and follow-up of effectiveness of therapy. This chapter presents an overview of these nanotechnology-based advancements in the areas of prevention, diagnosis, therapy, and theranostics for cancer. In addition, we stress the need to educate bio- and medical students in the field of nanotechnology.

Novel nanotechnology approaches to diagnosis and therapy of ovarian cancer.

OBJECTIVE: Ovarian carcinoma is the leading cause of death from gynecologic malignancies, which is a direct outcome of missing its diagnosis at an early stage. Approximately 75% of ovarian cancer patients are initially diagnosed with disseminated intra-abdominal disease (stages III-IV) when ~30% of patients have a 5-year survival rate. In addition to the challenge of early detection of ovarian cancer, its therapy presents several challenges including the route of therapy, resistance to therapy with recurrence of cancer, and specific targeting of ovarian cancer to reduce cytotoxic side effects. METHODS: We reviewed recent literature employing nanotechnology approaches to diagnosis and therapy of ovarian cancer. RESULTS: Recent innovations in nanotechnology with applications in cancer diagnostics and therapy help circumvent many pre-existing problems with conventional chemotherapy and present new ways of diagnosis and therapy. CONCLUSIONS: Nanotechnology has promising potential in enhancing early detection of ovarian cancer and treatment of recurrent disease.

Biomimetic silica microspheres in biosensing.

Lipid vesicles spontaneously fuse and assemble into a lipid bilayer on planar or spherical silica surfaces and other substrates. The supported lipid bilayers (SLBs) maintain characteristics of biological membranes, and are thus considered to be biomembrane mimetic systems that are stable because of the underlying substrate. Examples of their shared characteristics with biomembranes include lateral fluidity, barrier formation to ions and molecules, and their ability to incorporate membrane proteins into them. Biomimetic silica microspheres consisting of SLBs on solid or porous silica microspheres have been utilized for different biosensing applications. The advantages of such biomimetic microspheres for biosensing include their increased surface area to volume ratio which improves the detection limits of analytes, and their amenability for miniaturization, multiplexing and high throughput screening. This review presents examples and formats of using such biomimetic solid or porous silica microspheres in biosensing.

The constitutive activity of epidermal growth factor receptor vIII leads to activation and differential trafficking of wild-type epidermal growth factor receptor and erbB2.

A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII's activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII's activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis.

Targeting the EGF receptor for ovarian cancer therapy.

Ovarian carcinoma is the leading cause of death from gynecologic malignancy in the US. Factors such as the molecular heterogeneity of ovarian tumors and frequent diagnosis at advanced stages hamper effective disease treatment. There is growing emphasis on the identification and development of targeted therapies to disrupt molecular pathways in cancer. The epidermal growth factor (EGF) receptor is one such protein target with potential utility in the management of ovarian cancer. This paper will discuss contributions of EGF receptor activation to ovarian cancer pathogenesis and the status of EGF receptor inhibitors and EGF receptor targeted therapies in ovarian cancer treatment.

Role of polyethylene glycol integrity in specific receptor targeting of carbon nanotubes to cancer cells.

We demonstrate that dispersion of single walled carbon nanotubes (SWNTs) by ultrasonication with phospholipid-polyethylene glycol (PL-PEG) fragments it, thus interfering with its ability to block nonspecific uptake by cells. However, unfragmented PL-PEG promoted specific cellular uptake of targeted SWNTs to two distinct classes of receptors expressed by cancer cells. Since fragmentation is a likely consequence of ultrasonication, a technique commonly used to disperse SWNTs, this maybe a concern for certain applications such as drug delivery.

Phenotypic plasticity of neoplastic ovarian epithelium: unique cadherin profiles in tumor progression.

The mesodermally derived normal ovarian surface epithelium (OSE) displays both epithelial and mesenchymal characteristics and exhibits remarkable phenotypic plasticity during post-ovulatory repair. The majority of epithelial ovarian carcinomas (EOC) are derived from the OSE and represent the most lethal of all gynecological malignancies, as most patients (approximately 70%) present at diagnosis with disseminated intra-abdominal metastasis. The predominant pattern of EOC metastasis involves pelvic dissemination rather than lymphatic or hematologic spread, distinguishing EOC from other solid tumors. Acquisition of the metastatic phenotype involves a complex series of interrelated cellular events leading to dissociation (shedding) and dispersal of malignant cells. A key event in this process is disruption of cell-cell contacts via modulation of intercellular junctional components. In contrast to most carcinomas that downregulate E-cadherin expression during tumor progression, a unique feature of primary well-differentiated ovarian cancers is a gain of epithelial features, characterized by an increase in expression of E-cadherin. Subsequent reacquisition of mesenchymal features is observed in more advanced tumors with concomitant loss of E-cadherin expression and/or function during progression to metastasis. The functional consequences of this remarkable phenotypic plasticity are not fully understood, but may play a role in modulation of cell survival in suspension (ascites), chemoresistance, and intraperitoneal anchoring of metastatic lesions.

Detection of membrane biointeractions based on fluorescence superquenching.

Assays for biointeractions of molecules with supported lipid bilayers using fluorescence superquenching are described. A conjugated cationic polymer was adsorbed on to silica microspheres, which were then coated with an anionic lipid bilayer. The lipid bilayer attenuated superquenching by acting as a barrier between the conjugated polymer and its quencher. Biointeractions of the lipid bilayer with a membrane lytic peptide, melittin, were detected and quantitated by superquenching of the conjugated polyelectrolyte in flow cytometric and microfluidic bioassays. A higher sensitivity for detecting melittin lysis of the lipid bilayer at lower concentrations and shorter times for melittin action was found using flow cytometry in this study in comparison to other existing methods. This study combined the sensitivity of superquenching and flow cytometry to detect biointeractions with a lipid bilayer, which serves as a platform for developing functional assays for sensor applications, lipid enzymology, and investigations of molecular interactions. In addition, this study demonstrated proof-of-concept for using superquenching detected as a result of lipid bilayer disruption in a microfluidic format.

Biosensors based on release of compounds upon disruption of lipid bilayers supported on porous microspheres.

The authors describe a biosensing concept based on the release of compounds, which are encapsulated within lipid-coated porous silica microspheres, by detergents and toxins that disrupt supported lipid bilayers (SLBs) on the microspheres. Suspension and microfluidic based methods have been developed to monitor the release of the encapsulated compounds in response to membrane disruption. The authors established that the SLBs on porous microspheres can endure experimental conditions necessary for their incorporation into packed microchannels while maintaining the bilayer integrity and functionality. Model compounds including a nonionic detergent (Triton X-100), a membrane active protein (alpha-hemolysin), and a membrane lytic antimicrobial peptide (melittin) were successfully utilized to interact with different formulations of SLBs on porous silica microspheres. The results demonstrate the stability of the SLBs on the microspheres for several weeks, and the feasibility of using this system to detect the release of fluorescent dyes as well as other molecular reporters. The latter were detected by their involvement in subsequent biospecific interactions that were detected by fluorescence. This study exemplifies proof of concept for developing new chemical and biochemical sensors and drug delivery systems based on the disruption of lipid membranes coating porous silica microspheres that encapsulate dyes or bioactive compounds.

PEA3 is necessary for optimal epidermal growth factor receptor-stimulated matrix metalloproteinase expression and invasion of ovarian tumor cells.

Elevated expression of the epidermal growth factor (EGF) receptor (EGFR) is detected in human ovarian tumors and is associated with decreased recurrence-free and overall survival. EGFR activation affects tumor progression in part by promoting tumor invasion through the induction of prometastatic matrix metalloproteinases (MMP). PEA3, an ETS family transcription factor, is elevated in advanced and metastatic ovarian cancer and regulates MMPs in various cell types, therefore, we investigated whether PEA3 is required for the EGFR-dependent induction of MMP mRNA. MMP-9 and MMP-14 mRNA levels were selectively increased in response to EGFR activity in ovarian tumor cells. EGFR activation resulted in nuclear accumulation of PEA3 and direct binding of PEA3, but not the related protein ETS-1, to the endogenous MMP-9 and MMP-14 promoters. Furthermore, PEA3 overexpression was sufficient to induce MMP-9 and MMP-14 mRNA, tumor cell migration, and invasion, suggesting that PEA3 is an important contributor to the metastatic phenotype. Additionally, inhibition of PEA3 expression via short interfering RNA reduced the EGF induction of MMP-9 and MMP-14 gene expression by 92% and 50%, respectively, and impaired EGF-stimulated tumor cell invasion. These results suggest that PEA3 is regulated by EGFR and that the elevated PEA3 expression detected in human ovarian cancer may divert cells to a more invasive phenotype by regulating MMP-9 and MMP-14.

Using bicellar mixtures to form supported and suspended lipid bilayers on silicon chips.

Bicellar mixtures, planar lipid bilayer assemblies comprising long- and short-chain phosphatidylcholine lipids in suspension, were used to form supported lipid bilayers on flat silicon substrate and on nanotextured silicon substrates containing arrays of parallel troughs (170 nm wide, 380 nm deep, and 300 nm apart). Confocal fluorescence and atomic force microscopies were used to characterize the resulting lipid bilayer. Formation of a continuous biphasic undulating lipid bilayer membrane, where the crests and troughs corresponded to supported and suspended lipid bilayer regions, is demonstrated. The use of interferometric lithography to fabricate nanotexured substrates provides an advantage over other nanotextured substrates such as nanoporous alumina by offering flexibility in designing different geometries for suspending lipid bilayers.

Mesenchymal transformation in epithelial ovarian tumor cells expressing epidermal growth factor receptor variant III.

Overexpression of the epidermal growth factor (EGF) receptor occurs frequently in ovarian cancer and is associated with poor patient prognosis. A constitutively active mutant EGF receptor termed variant III (EGFRvIII) has been detected at a high frequency in many human tumors, including those of the ovary. To identify the consequences of EGFRvIII expression in ovarian tumor cells, we introduced EGFRvIII into the epithelial ovarian cancer cell line (OVCA 433). The EGFRvIII-transfected cells displayed a dissociated, motile phenotype and fibroblastic morphology. The EGFRvIII-dependent phenotype was comparable to that observed in EGF-stimulated parental OVCA 433 cultures and required the catalytic activity of the mutant receptor. Disruption of adherens and desmosomal junctions in EGFRvIII expressing cells was evident by immunofluorescent detection of specific junctional components. In addition, Western blot analysis confirmed decreased levels of cellular plakoglobin and beta-catenin in EGFRvIII-expressing cells, and E-cadherin protein and mRNA were nearly absent. The loss of E-cadherin was accompanied by decreased expression of additional ovarian epithelial markers, including keratins 7, 8, and 18 and mucins 1 and 4. In contrast, the mesenchymal markers N-cadherin and vimentin were elevated in EGFRvIII expressing cells. Overall, the switch in cadherins from E-cadherin to N-cadherin, coupled with gain of vimentin expression and loss of the epithelial keratins and mucins typically expressed in well-differentiated epithelial ovarian carcinomas, are consistent with transition to a mesenchymal phenotype as an outcome of EGFRvIII expression. These findings suggest that EGFRvIII expression may regulate phenotypic plasticity in ovarian cancer and thereby contribute to more aggressive disease.

Epithelial cell migration in response to epidermal growth factor.

Epidermal growth factor (EGF) is a ligand for the EGF receptor, a member of the erbB family of receptor tyrosine kinases. Activation of EGF receptor by EGF or other high-affinity ligands often results in increased migration of cells in physiological and pathological situations. There are numerous approaches for evaluating cell migratory response following EGF stimulation. Both qualitative and quantitative techniques will be presented in this chapter.

Superquenching as a detector for microsphere-based flow cytometric assays.

BACKGROUND: Fluorescent conjugated polymers display high fluorescence quantum yields and enhanced sensitivity to quenching (superquenching) by oppositely charged quenchers through energy or electron transfer. Fluorescent polymers and their quenchers are used in bead-based biosensor applications where the polymers are coated on particles. In this work, we investigate a detection method that utilizes superquenching on microspheres, which can be used for flow cytometric assays. METHODS: Microspheres were coated with the fluorescent cationic polyelectrolyte poly(p-phenylene-ethynylene) (PPE), and its superquenching by 9,10-anthraquinone-2,6-disulfonic acid (AQS) was examined by fluorometric methods in presence and in absence of a barrier to superquenching in the form of an anionic lipid bilayer. RESULTS: Flow cytometry detected superquenching of PPE on microspheres (MS-PPE) by AQS where high levels of reduction in fluorescence were observed. Adding different concentrations of AQS to MS-PPE yielded a Stern-Volmer quenching constant of 0.8x10(6) M-1. While forming an anionic lipid bilayer around the MS-PPE acted as a barrier to superquenching by AQS, disrupting the lipid bilayer allowed superquenching to take place. CONCLUSIONS: The sensitivity of flow cytometry in detecting fluorescence of microspheres and the amplified quenching sensitivity of fluorescent conjugated polymers both offer advantages over other fluorometric methods and conventional quenching detection. This study used superquenching of fluorescent polymers as a new tool in flow cytometry, thus combining the advantages offered by both method and detector. In addition, we employed the formation and the disruption of a supported lipid bilayer in mediating superquenching to offer new biosensing applications.

Down-regulation of integrin alpha2 surface expression by mutant epidermal growth factor receptor (EGFRvIII) induces aberrant cell spreading and focal adhesion formation.

Elevated expression or activity of the epidermal growth factor receptor (EGFR) is common in ovarian cancer and is associated with poor patient prognosis. A naturally occurring EGFR mutation termed variant III (EGFRvIII) has been detected in many human tumors, including those of the ovary. This mutant receptor does not bind EGF; however, it is constitutively active as detected by receptor dimerization, autophosphorylation, and stimulation of signal transduction cascades. To identify the consequences of EGFRvIII expression in ovarian tumor cells, we introduced EGFRvIII into the epithelial ovarian cancer cell line OVCA 433. The EGFRvIII-transfected cells displayed a motile phenotype, defects in cell spreading, and decreased integrin alpha2 protein expression as detected by Western blot analysis and flow cytometry. Inhibition of EGFRvIII catalytic activity using the EGFR-selective tyrphostin AG1478 restored integrin alpha2 expression within 4 to 8 hours after treatment. The modulation of integrin alpha2 expression corresponded to marked changes in the actin cytoskeleton as detected by redistribution of filamentous-actin. Furthermore, focal adhesions were evident only when EGFRvIII activity was inhibited. Together, these findings suggest that expression of the constitutively active mutant EGFRvIII promotes changes in cell shape and focal adhesion formation, mediated in part through specific modulation of integrin alpha2 expression and function. We conclude that EGFR-activating mutations, such as EGFRvIII, in ovarian cancer may contribute to a more aggressive disease.