Cale D. Fahrenholtz

Large-Pore Functionalized Mesoporous Silica Nanoparticles as Drug Delivery Vector for a Highly Cytotoxic Hybrid Platinum-Acridine Anticancer Agent

Large-pore mesoporous silica nanoparticles (MSN) were prepared and functionalized to serve as a highly robust and biocompatible delivery platform for platinum-acridine (PA) anticancer agents. The material showed a high loading capacity for the dicationic, hydrophilic hybrid agent [PtCl(en)(N-[acridin-9-ylaminoethyl]-N-methylpropionamidine)] dinitrate salt (P1A1) and virtually complete retention of payload at neutral pH in a high-chloride buffer. In acidic media mimicking the pH inside the cell lysosomes, rapid, burst-like release of P1A1 from the nanoparticles is observed. Coating of the materials in phospholipid bilayers resulted in nanoparticles with greatly improved colloidal stability. The lipid and carboxylate-modified nanoparticles containing 40 wt % drug caused S-phase arrest and inhibited cell proliferation in pancreatic cancer cells at submicromolar concentrations similar to carrier-free P1A1. The most striking feature of nanoparticle-delivered P1A1 was that the payload did not escape from the acidified lysosomal vesicles into the cytoplasm, but was shuttled to the nuclear membrane and released into the nucleus.

Design and cellular studies of a carbon nanotube-based delivery system for a hybrid platinum-acridine anticancer agent.

A three-component drug-delivery system has been developed consisting of multi-walled carbon nanotubes (MWCNTs) coated with a non-classical platinum chemotherapeutic agent ([PtCl(NH3)2(L)]Cl (P3A1; L=N-(2-(acridin-9-ylamino)ethyl)-N-methylproprionimidamide) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-mPEG). The optimized P3A1-MWCNTs are colloidally stable in physiological solution and deliver more P3A1 into breast cancer cells than treatment with the free drug. Furthermore, P3A1-MWCNTs are cytotoxic to several cell models of breast cancer and induce S-phase cell cycle arrest and non-apoptotic cell death in breast cancer cells. By contrast, free P3A1 induces apoptosis and allows progression to G2/M phase. Photothermal activation of P3A1-MWCNTs to generate mild hyperthermia potentiates their cytotoxicity. These findings suggest that delivery of P3A1 to cancer cells using MWCNTs as a drug carrier may be beneficial for combination cancer chemotherapy and photothermal therapy.

Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane

There is a growing interest in the use of multiwalled carbon nanotubes (MWCNTs) to treat diseases of the brain. Little is known about the effects of MWCNTs on human brain microvascular endothelial cells (HBMECs), which make up the blood vessels in the brain. In our studies, we evaluate the cytotoxicity of MWCNTs and acid oxidized MWNCTs, with or without a phospholipid-polyethylene glycol coating. We determined the cytotoxic effects of MWCNTs on both tissue-mimicking cultures of HBMECs grown on basement membrane and on monolayer cultures of HBMECs grown on plastic. We also evaluated the effects of MWCNT exposure on the capacity of HBMECs to form rings after plating on basement membrane, a commonly used assay to evaluate angiogenesis. We show that tissue-mimicking cultures of HBMECs are less sensitive to all types of MWCNTs than monolayer cultures of HBMECs. Furthermore, we found that MWCNTs have little impact on the capacity of HBMECs to form rings. Our results indicate that relative cytotoxicity of MWCNTs is significantly affected by the type of cell culture model used for testing, and supports further research into the use of tissue-mimicking endothelial cell culture models to help bridge the gap between in vitro and in vivo toxicology.

Heterogeneous Responses of Ovarian Cancer Cells to Silver Nanoparticles as a Single Agent and in Combination with Cisplatin

We investigated the effects of silver nanoparticle (AgNP) exposure in three ovarian cancer cell lines (A2780, SKOV3, and OVCAR3). We found that AgNPs were highly cytotoxic toward A2780 and SKOV3 cells but OVCAR3 cells were less sensitive to AgNPs. In agreement with the cytotoxicity data, AgNPs caused DNA damage in A2780 and SKOV3 cells, but not in OVCAR3 cells. A2780 and SKOV3 showed higher levels of basal reactive oxygen species (ROS) relative to OVCAR3 cells. AgNP exposure increased ROS levels in both A2780 and SKOV3 cells, but not in OVCAR3 cells. We found that the heterogeneous cytotoxicity was specific to the uptake of intact particles and was not due to differences in sensitivity to silver ions. Furthermore, the combination of AgNPs and standard-of-care platinum therapy, cisplatin (cis-diamminedichloroplatinum(II), CDDP), was synergistic for treatment of A2780 andOVCAR3 cells and the combination of AgNPs and CDDP showed a favorable dose reduction in all cell lines tested. These results provide insight into potential applications of AgNPs for treatment of ovarian cancer.

Abstract B04: Systemic delivery of silver nanoparticles and targeting of the folate receptor alpha for the treatment of triple-negative breast cancer

Triple-negative breast cancer (TNBC) lacks expression of the estrogen and progesterone receptors and does not overexpress the HER-2 receptor. Thus, current targeted therapies are rendered ineffective against this subtype of breast cancer, leaving a gap in treatment options for these patients. The use of nanotechnology has the potential to dramatically enhance the way cancer is diagnosed and treated. However, concerns persist about the toxicity of nanomaterials, which may not degrade, or are only slowly degraded and excreted after long body residence time. For clinical translation to become a reality, the potential risk–benefit balance for these materials must be resolved. We recently identified that silver nanoparticles (AgNPs) have the capacity to act as a single, self-therapeutic agent with a desirable combination of selective cytotoxicity and radiation dose enhancement effects in TNBC cells in vitro and in vivo at doses that that are non-toxic to non-cancerous breast and other cells. This is the first time any nanomaterial has been demonstrated to possess a TNBC specific toxicity profile and provides evidence that a therapeutic window exists for the safe use of AgNPs. Our current studies focus on determining the properties of the nanomaterial that are important to retain or enhance this TNBC selective response. Nanoparticle size and aggregation can have a dramatic effect on the cytotoxic and tumor targeting properties of nanomaterials. We used AgNP preparations with varying diameters (5 nm, 25 nm, 50 nm, 75 nm) to determine the influence of AgNP size on the increased sensitivity of TNBC cell lines to AgNPs. Nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, and nanoparticle tracking analysis. Using this combinatorial analysis, we identify spherical AgNPs preparations that resisted aggregation or dissolution in physiologic solutions for up to one month. We find that the increased sensitivity of TNBC cells as compared to non-cancerous cells was independent of nanoparticle size, and TNBC cell lines (MDA-MB-231, BT-549, SUM-159) are more sensitive to AgNP exposure than luminal A (MCF-7) or non-cancerous breast (MCF-10A, 184B5). We then performed tumor treatment studies in nude mice bearing MDA-MB-231 TNBC tumors in the 4th mammary fat pad. Mice were intravenously injected three times per week with AgNPs (6 mg/kg) or phosphate buffered saline (PBS). Remarkably, AgNP treatment significantly slows tumor TNBC tumor growth in vivo with no apparent systemic toxicity. Targeting has the potential to increase selectivity and uptake of AgNPs leading to increased therapeutic efficacy. Therefore, we functionalized our AgNPs with folic acid (FA) to exploit the folate receptor α (FRA), which is overexpressed in approximately 80% of TNBC. AgNPs were functionalized with a SH-PEG-FA targeting moiety to form FRA targeted AgNPs (FA-AgNPs). Using a modified dot blot probed with an anti-FA antibody, we demonstrate that FA-AgNPs were free of unbound FA and that AgNP bound- FA was available for binding with the FRA. In comparison to non-targeted AgNPs, FA-AgNPs significantly increase the cytotoxic effects of our AgNPs against TNBC in vitro. Furthermore, excess FA decreased the cytotoxicity of FA-AgNPs but had no effect on the cytotoxicity of the non-targeted AgNPs, indicating that the increased cytotoxicity of FA-AgNPs was FRA mediated. In summary, we identified that AgNPs exert significant anti-cancer activity toward TNBC cells in vitro and in vivo, and point to potential vulnerabilities in TNBC cells that could be exploited for the development of new therapeutic agents. Additionally, these AgNPs are suitable for systemic delivery and can be modified with FA to increase the specificity which further decreases the potential for off-target toxicity. Citation Format: Jessica L. Swanner, Cale D. Fahrenholtz, Ravi N. Singh. Systemic delivery of silver nanoparticles and targeting of the folate receptor alpha for the treatment of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B04.

Abstract B47: Silver nanoparticles exhibit subtype specific cytotoxic and therapeutic effects in claudin low breast cancer in vitro and in vivo

Triple negative breast cancers (TNBC) are characterized by loss of expression of hormone receptors and decreased expression of the human epidermal growth factor receptor 2 (HER2). TNBC patients do not benefit from current targeted breast cancer (BC) treatments. Molecular profiling of breast cancer has found that TNBC is largely comprised of basal-like and claudin-low intrinsic molecular subtypes. Claudin-low breast cancer (CLBC) accounts for approximately one third of TNBCs, and early evidence suggests CLBC tumors may be more resistant to neoadjuvant anthracycline/taxane-based chemotherapy compared to basal-like tumors. For the development of novel breast cancer therapeutics, attention must be paid to therapeutic efficacy in specific sub-types of the disease. We discovered a type of silver nanoparticle (AgNP) that is selectively cytotoxic for treatment of CLBC. We find that the increased sensitivity of CLBC cells as compared to non-cancerous cells was independent of nanoparticle size, and CLBC cell lines (MDA-MB-231, BT-549, SUM-159) are more sensitive to AgNP exposure than luminal A BC (MCF-7), HER2 positive BC (SKBR3), and basal-like BC (MDA-MB-468, BT-20), or non-cancerous breast cells (MCF-10A, 184B5, HMT-3522 S1) via MTT assay. By treating CLBC cells and non-cancer breast cells with AgNPs or silver ions, in the form of silver nitrate, we demonstrated that intact AgNPs are necessary for selective cytotoxicity in CLBC. To determine the mechanism of cell death caused by AgNPs, annexin V (AnnV) and propidium iodide (PI) co-staining was performed on non-cancerous MCF-10A breast cells and MDA-MB-231 CLBC cells treated with AgNPs for 48 hours. AgNPs induced a dose-dependent increase in late-stage apoptosis and an increase in necrosis in MDA-MB-231 compared to vehicle control. Conversely, AgNPs had a minimal effect on late-stage apoptosis and necrosis in non-cancerous MCF-10A. Utilizing western blots, we showed that AgNPs induce oxidative damage to protein thiols and activate the unfolded protein response (UPR) in CLBC, but not in non-cancerous breast cells. To better recapitulate the tumor volume in an in vitro setting, multicellular tumor nodules from MDA-MB-231 TNBC cells or HMT-3522 S1 non-transformed mammary epithelial cells were formed by culturing the cells on basement membrane. When grown using 3D culture techniques, HMT-3522 S1 cells can develop growth arrested, polarized spherical structures containing lumens resembling a normal breast acinar structure characterized by a central lumen, cell-cell junctional complexes (including apical tight junctions, TJ), and basal expression of hemidesmosomal α6/β4 integrins ligating an endogenous basement membrane. 48h treatment with AgNPs did not prevent apical localization of the TJ marker ZO-1, alter the basal deposition of collagen-IV, induce proliferation of the growth arrested acini, or induce DNA damage. Conversely, significant amounts of DNA damage were observed in the MDA-MB-231 tumor nodules following 48h AgNP treatment. We conducted a 3 month in vivo safety/efficacy study in CLBC tumor-bearing mice where we showed that AgNPs can be delivered repeatedly at substantial doses intravenously and are effective for treatment of tumors without systemic toxicity. This is the first time any nanomaterial has been demonstrated subtype-specific therapeutic efficacy. This work demonstrates that AgNPs may provide a significant benefit for the CLBC patient population. Citation Format: Jessica L. Swanner, Iliana Tenvooren, Brian W. Bernish, Cale D. Fahrenholtz, Pierre A. Vidi, Katherine L. Cook, Ravi N. Singh. Silver nanoparticles exhibit subtype specific cytotoxic and therapeutic effects in claudin low breast cancer in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B47.

Abstract B37: Photothermal therapy of glioblastoma multiforme using multiwalled carbon nanotubes optimized for diffusion in extracellular space

Glioblastoma multiforme (GBM) is the most common and most lethal primary brain tumor with a 5 year overall survival rate of approximately 5%. Currently, no therapy is curative and all have significant side effects. Focal thermal ablative therapies are being investigated as a new therapeutic approach. Such therapies can be enhanced using nanotechnology. Carbon nanotube mediated thermal therapy (CNMTT) uses lasers that emit near infrared radiation to excite carbon nanotubes (CNTs) localized to the tumor to generate heat needed for thermal ablation. Clinical translation of CNMTT for GBM will require development of effective strategies to deliver CNTs to tumors, clear structure-activity and structure-toxicity evaluation, and an understanding of the effects of inherent and acquired thermotolerance on the efficacy of treatment. In our studies, we show that a dense coating of phospholipid-poly(ethylene glycol) on multiwalled CNTs (MWCNTS) allows for better diffusion through brain phantoms, while maintaining the ability to achieve ablative temperatures after laser exposure. Phospholipid-poly(ethylene glycol) coated MWCNTs do not induce a heat shock response (HSR) in GBM cell lines. Activation of the HSR in GBM cells via exposure to sub-ablative temperatures or short term treatment with an inhibitor of heat shock protein 90 (17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)), induces a protective heat shock response that results in thermotolerance and protects against CNMTT. Finally, we evaluate the potential for CNMTT to treat GBM multicellular spheroids. These data provide pre-clinical insight into key parameters needed for translation of CNMTT including nanoparticle delivery, cytotoxicity, and efficacy for treatment of thermotolerant GBM.

Abstract B05: Self-assembling platinum-acridine loaded carbon nanotubes for triple-negative breast cancer chemotherapy

Triple-negative breast cancer (TNBC) accounts for 10-15% of breast cancers, has the highest levels of recurrence, and the lowest five-year survival of all breast cancer subtypes. TNBC does not express estrogen or progesterone receptors and does not overexpress HER2 receptors. Therefore, TNBC does not benefit from current FDA-approved targeted therapies against HER2 or hormone-positive cancers. Early clinical trials show that TNBC is susceptible to platinum chemotherapy, but dose-limiting toxicities and cross-resistance amongst current FDA-approved platinum agents may limit clinical efficacy. To address this issue, we have developed a novel drug delivery system consisting of a potent, non-classical platinum chemotherapeutic that self-assembles onto the surface of carbon nanotubes. Our pharmacophore, termed platinum-acridines (PA), is an anticancer agent composed of a platinum group modified with an acridine. The platinum-group of the PA functions to bind and platinate DNA, while the acridine group functions as a classical DNA intercalator. This coordination allows for platination of DNA near the intercalation site, leading to a more severe form of damage than the crosslinks induced by cisplatin which may increase potency and limit cross-resistance. Platinum-acridines show efficacy against breast cancer in vitro, but preclinical studies show a possibility of dose-limiting toxicities; thus PA may be most beneficial specifically delivered to the tumor using a nanocarrier such as carbon nanotubes (CNT). Carbon nanotubes have a large surface area to volume ratio for high capacity drug loading, can be made safe for systemic administration, and selectively accumulate in tumors due the enhanced permeability and retention (EPR) effect. Platinum-acridines readily adsorb onto biocompatible carbon nanotubes (CNTs) through non-covalent pi stacking. Therefore, CNTs can be used for controlled delivery of high dose platinum chemotherapy to the tumor and may decrease dose-limiting toxicities. Transmission electron microscopy confirmed that PA loads onto biocompatible CNTs. We found that these platinum-acridine loaded carbon nanotubes (PA-CNTs) are stable in physiological solution for extended periods of time. PA-CNTs were found intracellularly and successfully delivered PA chemotherapy to MDA-MB-231 TNBC cells. Furthermore, PA-CNTs were cytotoxic to several models of TNBC (MDA-MB-231, MDA-MB-468, SUM159, BT20); whereas, control CNTs were not appreciably cytotoxic. PA-CNTs induced non-apoptotic cell death in MDA-MB-231 breast cancer cells, whereas free PA favored apoptosis. Our nanotube-mediated delivery system is also readily adaptable to load a variety of cargo such as imaging agents or additional chemotherapeutics for multi-modal therapy and diagnostic applications. These findings indicate our self-assembling carbon nanotube delivery system loaded with platinum-acridines may be beneficial for the treatment of triple-negative breast cancer and warrants further preclinical evaluation. Citation Format: Cale D. Fahrenholtz, Song Ding, Brian W. Bernish, Mariah Wright, Ulrich Bierbach, Ravi N. Singh. Self-assembling platinum-acridine loaded carbon nanotubes for triple-negative breast cancer chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B05.