Eva Christabel Williams

Controlled release niosome embedded chitosan system: Effect of crosslink mesh dimensions on drug release†

We report on a model chemotherapy drug delivery system comprising nonionic surfactant vesicles (niosomes) packaged within a temperature-sensitive chitosan network. This smart packaging, or package-within-a package system, provides two distinct advantages. First, the gel prevents circulation of the niosomes and maintains delivery in the vicinity of a tumor. Second, the chitosan network protects the niosomes against fluctuations in tonicity, which affects delivery rates. Tonicity is the sum of the concentrations of the solutes which have the capacity to exert an osmotic force across the membrane. All release rate experiments were conducted with 5,6-carboxyfluorescein, a fluorescent dye. Release rates were monitored from both bare niosomes alone and niosome-embedded, chitosan networks. It was observed that chitosan networks prolonged delivery from 100 h to 55 days in low ionic strength environment and pH conditions similar to a tumor site. The primary effect of chitosan is to add control on release time and dosage, and stabilize the niosomes through a high ionic strength surrounding that prevents uncontrolled bursting of the niosomes. Secondary factors include crosslink density of the chitosan network, molecular weight of the individual chitosan polymers, dye concentration within the niosomes, and the number density of niosomes packaged within the chitosan network. Each of these factors can be altered to fine-tune release rates.

Abstract 3677: Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system

We report a development of a novel localized nanodelivery system featuring an enhanced targeted and controlled paclitaxel (PTX) delivery to epithelial and neuroectodermal origin tumor cells. The presented drug delivery system consists of a chitosan hydrogel embedded with PTX loaded non-ionic surfactant vesicles (PTX-niosomes) and chlorotoxin (CTX). We found a higher accumulation of chitosan on the surface of ovarian epithelial carcinoma cells known to overexpress MUC1 antigen as compared to normal ovarian epithelial cells. We therefore hypothesized that besides serving as a localized drug delivery platform, chitosan hydrogel can actively target MUC1 overexpressing tumor cells. To further improve the tumor-specific delivery of PTX, we have incorporated CTX, a 36-amino acid peptide, which binds to tumor cells of neuroectodermal origin, but not to non-transformed cells. Using a High Performance Liquid Chromatography (HPLC) assay, we have measured the in-vitro release profiles of PTX from chitosan hydrogel embedded with PTX-niosomes and CTX. We have assessed the morphology of CTX and PTX-nisomes as well as chitosan hydrogel embedded with PTX-niosomes and CTX using Transmission Electron Microscopy (TEM). We have used Attenuated Total Reflectance-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy to investigate the possible molecular interactions between chitosan hydrogel and various tumor cells. To better understand the mechanism involved in interactions between chitosan and specific tumor cells, we have developed a new Cell based Enzyme Linked Immunosorbent Assay (Cell ELISA) allowing us to quantify the expression level of MUC1 in epithelial and neuroectodermal origin tumor cells. Our Cell ELISA results revealed higher expression level of MUC1 (around 1.5 fold) in ovarian epithelial carcinoma cell line (OV2008) when compared to normal ovarian epithelial cell line (MCC3). The ATR-FTIR results showed a specific interaction of chitosan with OV2008 cells implying the mucoadhesive property of chitosan and its potential in targeting of MUC1 overexpressing tumor cells. We observed a sustained PTX release from chitosan hydrogel embedded with PTX-niosomes ( Citation Format: Rana Falahat, Eva Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Enhanced targeted delivery of paclitaxel to tumor cells of epithelial and neuroectodermal origin using chlorotoxin-chitosan nanodelivery system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3677. doi:10.1158/1538-7445.AM2015-3677

Abstract 5410: Enhanced targeting delivery to tumor cells using mucoadhesive chitosan and chlorotoxin

Specific drug delivery to tumor cells without affecting normal cells remains a major challenge in cancer treatment. We present a localized drug delivery system with enhanced targeting ability consisting of non-ionic surfactant vesicles (niosomes) with chlorotoxin (CTX) embedded in a chitosan hydrogel. This system represents a novel approach in cancer therapy through the controlled and targeted delivery of drugs to tumor cells. We have found the specific accumulation of chitosan on the surface of ovarian epithelial carcinoma cells known to have high expression of mucin antigen MUC1, but not on the normal ovarian epithelial cells. This finding demonstrates the capability of chitosan to target tumor cells expressing MUC1. Similarly, the incorporation of CTX (a 36-amino acid peptide capable of binding preferentially to tumor cells of neuroectodermal origin but not to normal cells) along with niosomes in the chitosan hydrogel has been used as the second targeting strategy to further improve the specific delivery of drugs to tumor cells such as glioma. To investigate the possible molecular interaction between the chitosan hydrogel and various cell lines, Attenuated Total Reflectance-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy has been used. The expression level of MUC1 in different cell lines was quantitatively evaluated using Cell based Enzyme Linked Immunosorbent Assay (Cell-ELISA) to better understand the mechanism involved in interactions between chitosan and specific tumor cells. In vitro release studies were performed to examine the effect of CTX on the release rates. Transmission Electron Microscopy (TEM) is being used for morphology assessments of CTX and nisomes as well as chitosan hydrogel embedded with niosomes and CTX. Cell-ELISA data revealed high expression level of MUC1 in ovarian epithelial carcinoma cell line (OV2008) and a moderate expression level in normal ovarian epithelial cell line (MCC3) implicating higher affinity of chitosan for OV2008 than MCC3 cell lines. This is consistent with ATR-FTIR results as well as the previously observed higher chitosan accumulation on the surface of OV2008 compared to MCC3, confirming the mucoadhesive property of chitosan and indicating its specificity in targeting MUC1 overexpressing tumor cells. In vitro release studies show that embedding CTX along with the niosomes does not disturb the controlled release from the chitosan network. In fact, attachment of CTX to the surface of niosomes shown by TEM imaging improves the stability of niosomes resulting in extended release rates. Based on these findings, we anticipate that the drug delivery system described here would improve tumor cell uptake of drugs due to the enhanced tumor targeting and its controlled, sustained and localized drug delivery to cancer cells. Citation Format: Rana Falahat, Eva Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Enhanced targeting delivery to tumor cells using mucoadhesive chitosan and chlorotoxin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5410. doi:10.1158/1538-7445.AM2014-5410

Abstract 4523: Targeted delivery to tumor cells by using tunable nano-delivery system with chlorotoxin.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The aim of this research is to develop the Tunable Nano-Delivery System (TNDS) for specific targeting of the tumor cells using Chlorotoxin (CTX). We present preliminary findings for the TNDS localized drug delivery system comprising of non-ionic surfactant vesicles (niosomes) with CTX embedded in a biodegradable and temperature sensitive chitosan network. The system demonstrates a new approach in the treatment of cancers, through the controlled and targeted delivery to tumor cells (the release time and dosage can be accurately controlled) while sparing normal cells. CTX is a scorpion-derived peptide that binds specifically to neuroectodermal tumors, e.g., glioma, but not to normal tissues. Previously, we have shown that there is a specific interaction between chitosan and Mucin1 (MUC1) receptors over expressed in epithelial ovarian carcinoma resulting in higher chitosan accumulation on the cancer cell surface than on normal ovarian epithelial cells. The current research addresses the incorporation of CTX embedded in Chitosan network to enhance specific targeting, which already benefits from the intrinsic interaction of MUC1 with Chitosan. Our current studies incorporating CTX in chitsoan resulting in TNDS-CTX complex are preformed in cell-free system. First, the release rates and release kinetics of TNDS-CTX are being measured in cell-free system using High Performance Liquid Chromatography (HPLC) and Attenuated Total Reflectance- Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. Results are being compared to our established TNDS delivery system to better determine CTX specific chemical binding sites along with the sites already determined to be specific for MUC1. Next, we will be testing TNDS-CTX in vitro in glioma, epithelial ovarian carcinoma, and normal ovarian epithelial and astrocyte cells. The level of fluorescence of normal cells and tumor cells exposed to the TNDS-CTX-fluorescently labeled are evaluated in in vitro by confocal microscopy. The studies in cell-free system showed that the release rates could be finely controlled depending on the specific design of the TNDS-CTX e.g., ratio of niosomes to chitosan and amount of crosslinker for the chitosan network. We are currently evaluating TNDS-CTX containing benchmark chemotherapeutics such as Paclitaxel, and it is expected that the presence of CTX will enhance cellular uptake of chemotherapeutics when compared to the noisome-chitosan delivery system without CTX. Based on our preliminary data, it is anticipated that CTX will perform as a targeting ligand improving the TNDS-CTXs anti-tumor efficacy owing to enhanced tumor targeting and its tunable and localized delivery. Citation Format: Rana Falahat, Eva Christabel Williams, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Targeted delivery to tumor cells by using tunable nano-delivery system with chlorotoxin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4523. doi:10.1158/1538-7445.AM2013-4523

Abstract 2185: Cross-disciplinary optimization of nano-drug delivery to ovarian carcinoma and glioma cells

This study reports on the quantitative analysis of the diffusion and localization of a targeted drug delivery system (DDS) consisting of fluorescent labeled 0.01 µM paclitaxel-BODIPY 564/570 encapsulated in non-ionic surfactant vesicles embedded in a thermosensitive cross-linked chitosan hydrogel. This is a multi-tiered DDS that allows for enhanced stability, sustained and controlled delivery of embedded drugs to tumor sites, and reduced toxicity. We showed that cancer cells of epithelial origin, such as human ovarian epithelial carcinoma OV2008 and highly migratory mouse glioma G-26, overexpress MUC1, a mucin surface antigen, which effectively enhances the specific targeting capacity of chitosan. Utilizing this DDS we found that OV2008 carcinoma cells had ~2 times higher fluorescence intensity level than normal ovarian epithelial IMCC3 cells with a statistical significance at 5 min and 24 h incubation times suggesting that this DDS had a higher affinity for tumor cells. Therefore, we hypothesized that there is a difference in PTX diffusion towards epithelial origin tumor cells when compared to normal cells in both distance/location and time. Diffusion and localization of fluorescent labeled PTX was evaluated in vitro using confocal microscopy. The fluorescence intensity captured on the images was quantified with ImageJ software in OV2008 carcinoma cells and compared to IMCC3 normal cells at time intervals 5 min, 1 h, 24 h, 48 h, and 72 h. The fluorescence intensity image data was analyzed in multiple radial line segments separated into three different zones of 24 µm each to represent multiple diffusion distances. Our results showed that fluorescence intensity levels in the zones around the IMCC3 normal cells at 5 min and 1 h were significantly higher (pl0.05) than around OV2008 carcinoma cells correlating reciprocally with our finding of intracellular fluorescence intensity in these two cell lines. Therefore, the data evaluating fluorescence levels in the radial zones outside the cells indicated that the migrating DDS-PTX had already been taken up by the tumor cells. Furthermore, the normal cells which showed significantly lower intracellular levels of fluorescence had higher levels of fluorescence in the measured radial zones outside the cells. This PTX-BODIPY 564/570 diffusion data from in vitro studies is currently being used for computational modeling in the in vivo intracerebral model of G-26 glioma. It will be integrated with mathematical model simulations that describe the pharmacokinetic and pharmacodynamic (PK/PD) properties of this drug delivery system for the evaluation of its efficacy and to optimize drug delivery in vivo. Computational modelling is being done in Matlab using the treatment scenario of the post-surgical late-stage glioma. The simulation studies will be used to determine optimal drug concentrations, chitosan density, and localization. Citation Format: Rupin Singh, Rana Falahat, Eva Williams, Joseph O. Johnson, Norma Alcantar, Aleksandra Karolak, Katarzyna Rejniak, Marzenna Wiranowska. Cross-disciplinary optimization of nano-drug delivery to ovarian carcinoma and glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2185. doi:10.1158/1538-7445.AM2017-2185

Abstract 2897: Preferential drug delivery to cancer cells than to normal cells by using the Niosome-Chitosan Thermo-responsive Double Package System (NCTR-DPS)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The main challenge of this research is to develop a localized, cost effective drug delivery system that can provide control over the release of drugs in ovarian cancer or brain tumor patients, in a manner that also reduces chemotherapeutic toxicity. We have designed a drug delivery system consisting of non-ionic surfactant vesicles (niosomes) packaged within a biodegradable, temperature sensitive hydrogel (chitosan) network (NCTR-DPS). Two chemotherapeutics, Paclitaxel and Carboplatin, conjugated to BODIPY 564/570 (RED fluorescence dye) were encapsulated in the NCTR-DPS. Their release was evaluated either in cell free system or following incubation with human cell lines: glioma (U373), ovarian carcinoma (OV2008), and two normal ovarian epithelial (MCC3 and I-Low) cell lines. Cellular localization of Paclitaxel-RED in live cells was evaluated using confocal microscopy and the level of direct fluorescence quantified with LAS AF software. Confocal imaging of the cells verified that the activity of the drugs remained intact within the drug delivery system. The cellular drug delivery was confirmed by cells toxicity observed within two hours of incubation. The control study showed no toxicity of the niosome-chitosan alone (without the chemotherapeutics). Chitosan accumulation was greater on OV2008 cell surface than on MCC3 or I-Low cells. Also, significantly higher Paclitaxel-RED fluorescence was found in OV2008 compared to normal MCC3 cells, indicating a specific interaction between chitosan and Mucin1 (MUC1) receptors over expressed in ovarian carcinoma. These data were further confirmed by ATR-FTIR (Attenuated Total Reflectance- Fourier Transform Infra-Red) spectroscopy showing higher MUC1 levels in OV2008 than in normal epithelial cells. In addition, in-vivo examinations of the NCTR-DPS were carried out in 8-week old female mice (Strain: FVB/NJ) using Xenogen IVIS Spectrum to quantitatively monitor release of tracer molecules.In-vivo examinations revealed the release dependence on the packaging density; 25 - 40% of release occurred within 24 hours, followed by a steady release for over 14 days. By the 14th day, 98% of dye was released. Higher percentage release was observed for greater niosome chitosan ratios. Our results showed that the time release of the drugs could be finely controlled by varying packaging and crosslinking ratio in the chitosan network from 3:1 (loose network which translates into high release) to 5:1 (tight network which translates into low release). This NCTS-DPS drug delivery system can be injected directly to tumor sites preventing the need for surgical implantation. It may have positive implications in the treatment of ovarian cancer preferentially targeting tumor cells or brain tumors because of its controlled and localized release. 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 2897. doi:1538-7445.AM2012-2897

Abstract 3229: Thermo-responsive double packaged system for localized drug delivery for cancer treatment

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL This research aims at developing a drug delivery system that will provide a plethora of benefits including cost effectiveness, reduction of toxicity and a control over the release of chemotherapeutics in Ovarian Cancer and Brain Tumor patients. We have designed a drug delivery system consisting of non-ionic surfactant vesicles (niosomes) packaged within a biodegradable, temperature and pH sensitive hydrogel (chitosan) network. Optimization of the release rates were accomplished by altering the molecular weight and cross-link density of the chitosan network and the size of the niosomes. Two chemotherapeutics Paclitaxel and Carboplatin were used for encapsulation. The interaction between niosome and chitosan were observed using Attenuated Total Reflectance- Fourier Transform Infrared (ATR-FTIR) spectroscopy. The drug delivery system was further investigated in human ovarian (OV2008) and human glioma (U373) cell lines. The concentration of the therapeutics used ranged from 0.04-0.4 µg/ml. Confocal imaging of the cells verified that the activity of the drugs remained intact within the niosome-chitosan drug delivery system and cells toxicity was observed within two hours confirming cellular delivery of the drug. The effect of the niosome-chitosan without the chemotherapeutic drugs on normal cells was also examined indicating that this system is non-toxic. Our results showed that this route of drug delivery to the cancer cells was effective since microtubule stabilization, fragmentation of the nucleus, and condense morphology were noted. Moreover, the time release of the drugs can be finely controlled by varying the crosslinking ratio in the chitosan network from 3:1 (loose network which translates into high release) to 5:1 (tight network which translates into low release). In addition, this system is a liquid solution at room temperature and undergoes a change of phase to a soft, self-containing gel at physiological conditions. The system therefore, can be injected into a tumor site. The materials used to construct this system are low cost and biocompatible and provides a flexible method for drug delivery with applications to intracavitary ovarian cancer and glioma treatment. 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 3229. doi:10.1158/1538-7445.AM2011-3229

Abstract 3707: Design and characterization of a double packaged system for localized drug delivery for the treatment of cancers

This research aims at developing a drug delivery system that will provide a plethora of benefits such as cost effectiveness, reduction of toxicity and a control over the release of chemotherapeutic drugs in brain and ovarian cancer patients. We have designed a model drug delivery system consisting of non-ionic surfactant vesicles (niosomes) packaged within a biodegradable, temperature and pH sensitive hydrogel (chitosan) network. Optimization of the release rates were accomplished by altering the condition of its two components, chitosan and niosomes. Two main cancer drugs, Paclitaxel and Carboplatin, were used for encapsulation. It was found that medium molecular weight chitosan with a crosslinker: chitosan ratio of 4:1, which corresponded to a pH of 7.4, resulted in the finest controlled release. Surface characteristics, such as the interaction between the niosomes and chitosan were determined using Surface Forces Apparatus. The system was also tested in-vivo in mice models, where the success of the drug delivery has been measured by measuring the drug concentrations of the mice blood with respect to time and by analyzing shrinking effects in induced cancer tumors in mice. Xenogen was used to visualize the release of drugs in-vivo. Release rates of this system have been compared with other delivery methods. Our results will help in the development of low cost and improved methods for drug delivery with applications to brain tumors and intracavitary ovarian cancer treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3707.

Double Packaged System for Localized Drug Delivery for Ovarian Cancer

This research aims at developing a drug delivery system that will provide a plethora of benefits such as cost effectiveness, reduction of toxicity and a control over the release of chemotherapeutic drugs in Ovarian Cancer patients. We have designed a model drug delivery system consisting of non-ionic surfactant vesicles (niosomes) packaged within a biodegradable, temperature and pH sensitive hydrogel (chitosan) network. Optimization of the release rates were accomplished by altering the condition of its two components, chitosan and niosome. Two ovarian cancer drugs Paclitaxel and Carboplatin were used for encapsulation. It was found that medium molecular weight chitosan with a crosslinker:chitosan ratio of 4:1 which corresponded to a pH of 7.4 resulted in the finest controlled release. Surface characteristics, such as the interaction between the niosomes and chitosan were determined using Surface Forces Apparatus. The system was also tested in-vivo in mice models. Xenogen was used to study the release of drugs in-vivo. Our results will help in the development of a low cost and improved method for drug delivery with application to intracavitary ovarian cancer treatment and other cancer types.