Supriya Srinivasan

Comparing cellular uptake and cytotoxicity of targeted drug carriers in cancer cell lines with different drug resistance mechanisms

UNLABELLED The purpose of this study was to compare the cellular uptake and cytotoxicity of targeted and nontargeted doxorubicin (DOX)-loaded poly(d,l-lactide co-glycolide) (PLGA) nanoparticle (NP) drug delivery systems in drug-resistant ovarian (SKOV-3) and uterine (MES-SA/Dx5) cancer cell lines. The cellular uptakes of DOX from nonconjugated DOX-loaded NPs (DNPs) and from HER-2 antibody-conjugated DOX-loaded NPs (ADNPs) in MES-SA/Dx5 cancer cells were higher compared to free DOX. Results also showed higher uptake of DOX from ADNPs in SKOV-3 cells compared with both free DOX and DNPs treatment. Cytotoxicity results at 10 μM extracellular DOX concentration were consistent with the cellular uptake results. Our study concludes that cellular uptake and cytotoxicity of DOX can be improved in MES-SA/Dx5 cells by loading DOX into PLGA NPs. DNPs targeted to membrane receptors may enhance cellular uptake and cytotoxicity in SKOV-3 cells. FROM THE CLINICAL EDITOR The authors of this study compare the cellular uptake and cytotoxicity of targeted and nontargeted doxorubicin loaded PLGA nanoparticle delivery systems in drug-resistant ovarian and uterine cancer cell lines, concluding that cellular uptake and cytotoxicity of doxorubicin can be improved by the proposed methods.

Simultaneous Delivery of Chemotherapeutic and Thermal-Optical Agents to Cancer Cells by a Polymeric (PLGA) Nanocarrier: An In Vitro Study

ABSTRACTPurposeTo test the effectiveness of a dual–agent-loaded PLGA nanoparticulate drug delivery system containing doxorubicin (DOX) and indocyanine green (ICG) in a DOX-sensitive cell line and two resistant cell lines that have different resistance mechanisms.MethodsThe DOX-sensitive MES-SA uterine sarcoma cell line was used as a negative control. The two resistant cell lines were uterine sarcoma MES-SA/Dx5, which overexpresses the multidrug resistance exporter P-glycoprotein, and ovarian carcinoma SKOV-3, which is less sensitive to doxorubicin due to a p53 gene mutation. The cellular uptake, subcellular localization and cytotoxicity of the two agents when delivered via nanoparticles (NPs) were compared to their free-form administration.ResultsThe cellular uptake and cytotoxicity of DOX delivered by NPs were comparable to the free form in MES-SA and SKOV-3, but much higher in MES-SA/Dx5, indicating the capability of the NPs to overcome P-glycoprotein resistance mechanisms. NP-encapsulated ICG showed slightly different subcellular localization, but similar fluorescence intensity when compared to free ICG, and retained the ability to generate heat for hyperthermia delivery.ConclusionThe dual-agent-loaded system allowed for the simultaneous delivery of hyperthermia and chemotherapy, and this combinational treatment greatly improved cytotoxicity in MES-SA/Dx5 cells and to a lesser extent in SKOV-3 cells.

Near-infrared fluorescing IR820-chitosan conjugate for multifunctional cancer theranostic applications

This study reports the preparation and characterization of IR820-chitosan conjugates that have potential multifunctional imaging-hyperthermia applications in cancer. The conjugates were formulated by covalentcouplingofchitosan to a carboxyl derivatized IR820, and studied for optical imaging and hyperthermia applications. IR820-chitosan conjugates were able to generate heat upon exposure to 808nm laser and produce hyperthermic cell growth inhibition in cancer cell lines MES-SA, SKOV-3 and Dx5. The level of cell growth inhibition caused by hyperthermia was significantly higher for IR820-chitosan compared to IR820 in MES-SA and Dx5 cells. Fluorescent microscope images of these cancer cell lines after 3-h exposure to 5μM IR820-chitosan showed that the conjugates can be used for in vitro near-infrared imaging. In an in vivo rat model, the conjugates accumulated in the liver after i.v. injection and were excreted through the gastrointestinal tract, demonstrating a different biodistribution when compared to the free dye. The accumulation of these conjugates in bile with subsequent gastrointestinal excretion allows for potential applications as gastrointestinal contrast agents and delivery vehicles. This formulation can potentially be used in multifunctional cancer theranostics.

Covalent IR820-PEG-diamine nanoconjugates for theranostic applications in cancer.

Near-infrared dyes can be used as theranostic agents in cancer management, based on their optical imaging and localized hyperthermia capabilities. However, their clinical translatability is limited by issues such as photobleaching, short circulation times, and nonspecific biodistribution. Nanoconjugate formulations of cyanine dyes, such as IR820, may be able to overcome some of these limitations. We covalently conjugated IR820 with 6 kDa polyethylene glycol (PEG)-diamine to create a nanoconjugate (IRPDcov) with potential for in vivo applications. The conjugation process resulted in nearly spherical, uniformly distributed nanoparticles of approximately 150 nm diameter and zeta potential −0.4±0.3 mV. The IRPDcov formulation retained the ability to fluoresce and to cause hyperthermia-mediated cell-growth inhibition, with enhanced internalization and significantly enhanced cytotoxic hyperthermia effects in cancer cells compared with free dye. Additionally, IRPDcov demonstrated a significantly longer (P<0.05) plasma half-life, elimination half-life, and area under the curve (AUC) value compared with IR820, indicating larger overall exposure to the theranostic agent in mice. The IRPDcov conjugate had different organ localization than did free IR820, with potential reduced accumulation in the kidneys and significantly lower (P<0.05) accumulation in the lungs. Some potential advantages of IR820-PEG-diamine nanoconjugates may include passive targeting of tumor tissue through the enhanced permeability and retention effect, prolonged circulation times resulting in increased windows for combined diagnosis and therapy, and further opportunities for functionalization, targeting, and customization. The conjugation of PEG-diamine with a near-infrared dye provides a multifunctional delivery vector whose localization can be monitored with noninvasive techniques and that may also serve for guided hyperthermia cancer treatments.

Targeted nanoparticles for simultaneous delivery of chemotherapeutic and hyperthermia agents--an in vitro study.

The purpose of this study was to prepare targeted Poly lactide-co-glycolide (PLGA) nanoparticles with simultaneous entrapment of indocyanine green (ICG) and doxorubicin (DOX) by surface decorating them with tumor specific monoclonal antibodies in order to achieve simultaneous therapy and imaging. ICG was chosen as an imaging and hyperthermia agent and DOX was used as a chemotherapeutic agent. ICG and DOX were incorporated into PLGA nanoparticles using the oil-in-water emulsion solvent evaporation technique. These nanoparticles were further surface decorated with antibodies against Human Epithelial Receptor-2 (HER-2) using carbodiimide chemistry. The uptake of antibody conjugated ICG-DOX-PLGA nanoparticles (AIDNP) was enhanced in SKOV-3 (HER-2 overexpressing cell lines) compared to their non-conjugated counterparts (ICG-DOX-PLGA nanoparticles (IDNP)). The uptake of antibody conjugated ICG-DOX-PLGA nanoparticles, however, was similar in MES-SA and MES-SA/Dx5 cancer cells (HER-2 negative cell lines), which were used as negative controls. The cytotoxicity results after laser treatment (808 nm, 6.7 W/cm(2)) showed an enhanced toxicity in treatment of SKOV-3. The negative controls exhibited comparable cytotoxicity with or without exposure to the laser. Thus, this study showed that these antibody conjugated ICG-DOX-PLGA nanoparticles have potential for combinatorial chemotherapy and hyperthermia.

AgNPs-based Label-Free Colloidal SERS Nanosensor for the Rapidand Sensitive Detection of Stress-Proteins Expressed in Response toEnvironmental-Toxins

Among several physical, chemical and immunoassay-based methods for the detection of biomolecules, the Enzyme-Linked Immuno-Sorbent Assay (ELISA) is the standard technique that is routinely used for quantification of known proteins. However, it is a label-based, end-point sensor technique that is time-consuming, labor-intensive and fairly costly. This sandwich assay typically involves a series of peptide binding and washing steps. Here, we report a Surface-Enhanced Raman Spectroscopy (SERS) immuno-nanosensor technique that allows rapid and label-free extracellular detection of proteins compared to ELISA, and can potentially be used for intracellular detection. Our study shows that the silver nanoparticles (AgNPs) based SERS sensor can detect the stress-proteins, HSP70 and RAD54 expressed by yeast in response to environmental-toxins, in a dose dependent manner. As compared to the multi-step sandwich ELISA technique, the detection of stress-proteins using the SERS sensor is a simple two-step process. The simplicity of the SERS nanosensor design allowed the rapid detection of proteins within two hours in a fairly cost-effective and user-friendly approach. The SERS sensor we reported has an edge over ELISA as it directly quantifies the proteins without using any label (label-free) and also gives qualitative information about the antigen-antibody interaction. The SERS sensor showed good correlation and comparable sensitivity with ELISA. However, SERS was found to be less reproducible. Compared to previous reports on SERS-based protein detection techniques, our colloidal SERS sensor is easy to fabricate, offers improved biocompatibility, and allows rapid detection of the proteins in a cellular environment at picogram-levels. As a result, the SERS sensor demonstrates great potential for biomedical and environmental sensor technology (BEST) allowing label-free, rapid and sensitive detection, and could possibly replace ELISA.

Polyethylene glycol modified ORMOSIL theranostic nanoparticles for triggered doxorubicin release and deep drug delivery into ovarian cancer spheroids

A novel pegylated multifunctional probe of Ormosil nanoparticles (PEGCDSIR820) loaded with Near Infrared dye (NIR; IR820) and a chemotherapeutic drug, Doxorubicin (DOX) was developed for cancer theranostic applications. PEGCDSIR820 nanoparticles had an average diameter of 58.2±3.1nm, zeta potential of -6.9±0.1mV in cell culture media and stability against aggregation in physiological buffers. The encapsulation efficiency of DOX was 65.0±3.0%, and that of IR820 was 76.0±2.1%. PEGCDSIR820 showed no cytotoxicity in ovarian cancer cells (Skov-3). The cytotoxicity markedly increased when Skov-3 cells incubated with PEGCDSIR820 particles were exposed to 808nm laser due to the combination of adjuvant hyperthermia (43°C) and enhanced DOX release. Exposure to laser enhanced the release of DOX, 45% of DOX release was observed in 3h compared to 23% without laser exposure. Confocal imaging in Skov-3 cells showed that the combination of hyperthermia due to NIR exposure and release of DOX caused cell necrosis. Furthermore, in spheroids exposed to NIR laser penetration of DOX was deeper compared to the absence of laser exposure. Skov-3 spheroids incubated with pegylated nanoparticles for 24h and exposed to laser showed 94% reduction in cell viability. Encapsulation of IR820 in PEGCDSIR820 increased the in-vivo elimination half-life to 41.0±7.2h from 30.5±0.5h of free IR820.

IR820 Conjugates for Image-Guided Cancer Therapy

The development of novel agents for multifunctional approaches to cancer diagnosis and therapy is a growing area of research with great promise for clinical translation. Our group has created two conjugate formulations of the near-infrared dye IR820 that can be used as image-guided therapy agents. The first formulation combined IR820 with PEG-diamine to create ionic IR820-PEG diamine nanoplexes (IR820-PDNCs), and the second formulation was a covalent conjugate of IR820 and chitosan (IR820-chitosan). Both formulations retained the ability to fluoresce and generate heat upon laser exposure. IR820-PDNCs, approximately 50-nm in diameter, were characterized and studied in vitro using the cancer cell lines MES-SA, Dx5, and SKOV-3. Fluorescent imaging after 2.5-hour incubation with IR820-PDNCs showed enhanced cell uptake compared to free IR820 dye. Cytotoxicity studies showed an enhanced cytotoxic effect of IR820-PDNCs after exposure to 808-nm when compared to the use of free IR820 for all three cell lines. This enhancement was statistically significant in MES-SA and Dx5 (p<0.05). IR820-chitosan conjugates were characterized for their absorption and fluorescence properties and studied in vitro using the same cell lines as for IR820-PDNCs. The IR820-conjugate formulation was also studied in vivo using a Fisher-344 rat model. Our results showed that IR820-chitosan conjugates can be used in cell imaging and have an enhanced hyperthermia-mediated cytotoxic effect in MES-SA and Dx5 compared to free dye (p<0.05). During in vivo studies, IR-820 conjugates accumulated in the liver after an i.v. injection, and were subsequently excreted in bile. The conjugates traveled through the intestines and retained the ability to fluoresce for at least 24 hours. This indicates that IR820-chitosan conjugates may have applications as gastrointestinal contrast agents or delivery vehicles. Both IR820 conjugate formulations have the potential to be further developed as theranostic agents.

Targeted Delivery of Doxorubicin by PLGA Nanoparticles Increases Drug Uptake in Cancer Cell Lines

Doxorubicin (DOX) is an anthracycline drug widely used in the treatment of a large spectrum of solid tumors. Its application is limited by its efflux through the multidrug resistance (MDR) protein. In this study, we explored the potential of DOX - PLGA nanoparticles (DNPs) and antibody decorated nanoparticles (ADNPs) to overcome MDR in cancer cells. DNPs were prepared by the O/W emulsion solvent evaporation method. The surface decoration of nanoparticles with antibody was performed via carbodiimide reaction. The particles were characterized for their size and zeta potential, and their in vitro uptake was compared with their unconjugated counterparts. Entrapment efficiency of DNPs and ADNPs was measured by fluorescence using the DMSO burst release procedure. Cytotoxicity was measured using the SRB assay. The DNP and ADNP nanoparticles had average diameter of 162.6 ± 2.0 nm and 213.0 ± 3.5 nm respectively. Their corresponding surface charges were -13.2 ± 2.3 mV and -1.3 ± 3.8 mV respectively. Our results showed that cellular uptake of DOX from DNPs in DOX-resistant MES-SA/Dx5 cancer cell was higher compared to free DOX. However, the uptake of DOX from DNPs in MES-SA and SKOV-3 cancer cell lines was comparable to free DOX treatment. Next, we conjugated the DNPs with HER-2 antibody to specifically target to SKOV-3 cancer cell line and MES-SA and MES-SA/Dx5 were used as negative controls. Results showed higher uptake of DOX from ADNPs compared to free DOX and DNPs in SKOV-3. However, the cellular uptake of DOX from ADNPs was comparable to DNPs in MES-SA and MES-SA/Dx5. Cytotoxicity results were consistent with the cellular uptake data. Our study concludes that the targeted DNPs may enhance the cellular uptake and cytotoxicity in SKOV-3.

Abstract 5259: Urolithin A prevents pancreatic tumor growth and increases survival by inhibiting PI3K/PDK1 and STAT3 signaling

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related mortality in the United States. Most patients present with an advanced disease and the majority die within five years, many surviving less than six months. Cytotoxic chemotherapy including Gemcitabine (Gem), FOLFIRINOX, nab-paclitaxel offer modest improvement in survival, albeit at the cost of increased side effects and unwanted toxicities. Therefore, developing novel chemotherapeutic agents for PDAC treatment is critical to improve survival. Ellagic acid/ellatitannins are abundantly present in the pomegranate and berries, are actively metabolized by the intestinal microflora to Urolithin A (UA). Oral administration of UA has shown to be highly bioavailable and non-toxic. UA inhibits multiple kinases that are known to be involved in PDAC progression and metastasis. Therefore, we hypothesized that UA would elicit potent anti-cancer therapeutic potential in PDAC. The effect of UA on kinase activity was assessed . Inhibition of AKT (downstream of PI3K/PDK1), p70 S6 Kinase (PS6K) and STAT3 activation was quantified in PDAC cells treated with UA in dose-dependent manner. The mechanism of action was validated for UA’s activity on PI3K/PDK1, PS6K and STAT3 activation using immunoblot analysis. MiaPaCa2 cells were treated with specific inhibitors for either AKT (MK2206) or STAT3 (AZD1480) and analyzed for tumorigenicity. UA treated PDAC cells were analyzed for cell proliferation, cell invasion and colony formation. Cell cycle analysis and cell apoptosis were measured by flow cytometry. To test the efficacy of UA in vivo, cells were implanted subcutaneously in athymic nude mice. The animals received UA daily and tumor volume was measured for 5 weeks. Next, we assessed tumor growth and overall survival (OS) in PKT (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl) mice, an aggressive genetically engineered PDAC mouse model, in response to UA and/or Gem treatment. Tissues from the xenografts and PKT mice treated with vehicle or UA were analyzed for cell proliferation (Ki67) and apoptosis (cleaved Caspase 3) by immunohistochemistry. High expression levels of activated STAT3 or AKT correlate with decreased survival in PDAC. UA treated MiaPaCa2 cells showed significant dose-dependent increase in apoptosis and decrease in anchorage-independent growth. UA inhibited AKT, PS6K and STAT3 signaling. As a single agent, UA effectively reduced in vivo PDAC tumor growth. Immunohistochemistry of UA treated tissues from tumor xenografts and PKT mice showed inhibition of Ki67 positive tumor cells and increased cleaved caspase 3 staining. PKT mice treated with UA showed a decrease in tumor size and an increased OS when compared to vehicle or Gem treated mice alone. These findings show that UA is a novel inhibitor/modulator/regulator for multiple signaling pathways in PDAC. These results suggest UA has potential for pre-clinical development in pancreatic cancer. Citation Format: Supriya Srinivasan, Venkatakrishna Jala, Kumar Honnenahally, Jason Castellanos, Praveen Kumar Vermula, Michael VanSaun, Nipun Merchant, Nagaraj Nagathihalli. Urolithin A prevents pancreatic tumor growth and increases survival by inhibiting PI3K/PDK1 and STAT3 signaling [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 5259. doi:10.1158/1538-7445.AM2017-5259