Investigation on nanoparticle based combination therapy for targeted cancer treatment

“The current treatment methods in cancer are associated with toxicity in healthy tissues, partial therapeutic response, drug resistance and finally recurrence of the disease. The cancer drugs are challenged by non-specific binding, undesired toxicity in healthy cells, low therapeutic index and finally poor therapeutic outcome. In this work, a targeted nanoscale therapeutic system Antibody Drug Nanoparticle (ADN) was engineered to selectively inhibit the breast cancer cell growth with reduced toxicity in healthy cells. The ADNs were designed by synthesizing rod shaped anoparticles using pure chemotherapeutic drug and covalently conjugating a therapeutic monoclonal antibody (mAb) on the surface of the drug nanorods. The rod shaped nanosized formulation of ADNs significantly enhanced the aqueous phase stability and therapeutic payload of the system while the conjugated mAb was utilized for specific targeting of breast cancer cells. The designed ADN was effective for active targeting and synergistic inhibition of breast cancer cells. The mechanisms of actions of ADN was investigated at the cellular, molecular and genetic levels in cancer cells. The engineered AND synergistically inhibited the growth of \u3e 80% of the human epidermal growth factor receptor 2 (HER2) - positive breast cancer cells in vitro. The cell cycle and protein expression analysis showed that ADN arrested the cellular growth for a prolonged time and induced a programmed cell death mechanism in HER2-positive breast cancer cells in vitro. Finally, the gene regulatory analysis showed the genetic mechanisms of programmed cell death regulation induced by ADN in breast cancer cell lines”--Abstract, page iv

Antibody-Drug Nanoparticle Induces Synergistic Treatment Efficacies in HER2 Positive Breast Cancer Cells

Chemotherapeutic drugs suffer from non-specific binding, undesired toxicity, and poor blood circulation which contribute to poor therapeutic efficacy. In this study, antibody–drug nanoparticles (ADNs) are engineered by synthesizing pure anti-cancer drug nanorods (NRs) in the core of nanoparticles with a therapeutic monoclonal antibody, Trastuzumab on the surface of NRs for specific targeting and synergistic treatments of human epidermal growth factor receptor 2 (HER2) positive breast cancer cells. ADNs were designed by first synthesizing ~ 95 nm diameter × ~ 500 nm long paclitaxel (PTX) NRs using the nanoprecipitation method. The surface of PTXNRs was functionalized at 2′ OH nucleophilic site using carbonyldiimidazole and conjugated to TTZ through the lysine residue interaction forming PTXNR-TTZ conjugates (ADNs). The size, shape, and surface charge of ADNs were characterized using scanning electron microscopy (SEM), SEM, and zeta potential, respectively. Using fluorophore labeling and response surface analysis, the percentage conjugation efficiency was found \u3e 95% with a PTX to TTZ mass ratio of 4 (molar ratio ≈ 682). In vitro therapeutic efficiency of PTXNR-TTZ was evaluated in two HER2 positive breast cancer cell lines: BT-474 and SK-BR-3, and a HER2 negative MDA-MB-231 breast cancer cell using MTT assay. PTXNR-TTZ inhibited \u3e 80% of BT-474 and SK-BR-3 cells at a higher efficiency than individual PTX and TTZ treatments alone after 72 h. A combination index analysis indicated a synergistic combination of PTXNR-TTZ compared with the doses of single-drug treatment. Relatively lower cytotoxicity was observed in MCF-10A human breast epithelial cell control. The molecular mechanisms of PTXNR-TTZ were investigated using cell cycle and Western blot analyses. The cell cycle analysis showed PTXNR-TTZ arrested \u3e 80% of BT-474 breast cancer cells in the G2/M phase, while \u3e 70% of untreated cells were found in the G0/G1 phase indicating that G2/M arrest induced apoptosis. A similar percentage of G2/M arrested cells was found to induce caspase-dependent apoptosis in PTXNR-TTZ treated BT-474 cells as revealed using Western blot analysis. PTXNR-TTZ treated BT-474 cells showed ~ 1.3, 1.4, and 1.6-fold higher expressions of cleaved caspase-9, cytochrome C, and cleaved caspase-3, respectively than untreated cells, indicating up-regulation of caspase-dependent activation of apoptotic pathways. The PTXNR-TTZ ADN represents a novel nanoparticle design that holds promise for targeted and efficient anti-cancer therapy by selective targeting and cancer cell death via apoptosis and mitotic cell cycle arrest

PolyRad -- Protection Against Free Radical Damage

The effects of elevated levels of radiation contribute to the instability of pharmaceutical formulations in space compared to those on earth. Existing technologies are ineffective at maintaining the therapeutic efficacies of drugs in space. Thus, there is an urgent need to develop novel space-hardy formulations for preserving the stability and efficacy of drug formulations. This work aims to develop a novel approach for the protection of space pharmaceutical drug molecules from the radiation-induced damage to help extend or at least preserve their structural integrity and potency. To achieve this, free radical scavenging antioxidant, Trolox was conjugated on the surface of poly-lactic-co-glycolic acid (PLGA) nanoparticles for the protection of a candidate drug, melatonin that is used as a sleep aid medication in International Space Station (ISS). Melatonin-PLGA-PLL-Trolox nanoparticle as named as PolyRad was synthesized employing single oil in water (o/w) emulsion solvent evaporation method. PolyRad is spherical in shape and has an average diameter of ~600 nm with a low polydispersity index of 0.2. PolyRad and free melatonin (control) were irradiated by UV light after being exposed to a strong oxidant, hydrogen peroxide (H2O2). Bare melatonin lost ~80% of the active structure of the drug following irradiation with UV light or treatment with H2O2. In contrast, PolyRad protected \u3e 80% of the active structure of melatonin. The ability of PolyRad to protect melatonin structure was also carried out using 0, 1, 5 and 10 Gy gamma radiation. Gamma irradiation showed \u3e 98% active structures of melatonin encapsulated in PolyRads. Drug release and effectiveness of melatonin using PolyRad were evaluated on human umbilical vein endothelial cells (HUVEC) in vitro. Non-irradiated PolyRad demonstrated maximum drug release of ~70% after 72 h, while UV-irradiated and H2O2-treated PolyRad showed a maximum drug release of ~85%. Cytotoxicity of melatonin was carried out using both live/dead and MTT assays. Melatonin, non-radiated PolyRad and irradiated PolyRad inhibited the viability of HUVEC in a dose-dependent manner. Cell viability of melatonin, PolyRad alone without melatonin (PolyRad carrier control), non-radiated PolyRad, and irradiated PolyRad were ~98, 87, 75 and 70%, respectively at a concentration ~ 0.01 mg/ ml (10 μg/ ml). Taken together, PolyRad nanoparticle provides an attractive formulation platform for preventing damage to pharmaceutical drugs in potential space mission applications

Isolation and Purification of Glycoglycerolipids to Induce Apoptosis in Breast Cancer Cells

Monogalactosyldiacylglycerol (MGDG) is the most abundant type of glycoglycerolipid found in the plant cell membrane and mostly in the chloroplast thylakoid membrane. The amphiphilic nature of MGDG is attractive in pharmaceutical fields for interaction with other biological molecules and hence exerting therapeutic anti-cancer, anti-viral, and anti-inflammatory activities. In this study, we investigated the therapeutic efficacy of cyanobacteria derived MGDG to inhibit breast cancer cell growth. MGDG was extracted from a cyanobacteria Synechocystis sp. PCC 6803 followed by a subsequent fractionation by column chromatographic technique. The purity and molecular structure of MGDG were analyzed by nuclear magnetic resonance (NMR) spectroscopy analysis. The presence of MGDG in the extracted fraction was further confirmed and quantified by high-performance liquid chromatography (HPLC). The anti-proliferation activity of the extracted MGDG molecule was tested against BT-474 and MDA-MB-231 breast cancer cell lines. The in vitro study showed that MGDG extracted from Synechocystis sp. PCC 6803 induced apoptosis in (70 ± 8) % of BT-474 (p \u3c 0.001) and (58 ± 5) % of MDA-MB-231 cells (p \u3c 0.001) using ~ 60 and 200 ng/ml of concentrations, respectively. The half-maximal inhibitory concentration, IC50 of MGDG extracted from Synechocystis sp. PCC 6803 were (27.2 ± 7.6) and (150 ± 70) ng/ml in BT-474 and MDA-MB-231 cell lines, respectively. Quantification of caspase-3/7 activity using flow cytometry showed (3.0 ± 0.4) and (2.1 ± 0.04)-fold (p \u3c 0.001) higher protein expressions in the MGDG treated BT-474 and MDA-MB-231 cells, respectively than untreated controls conferring to the caspase-dependent apoptosis. The MGDG did not show any significant cytotoxic side effects in human dermal fibroblasts cells. A commercially available MGDG control did not induce any apoptotic cell death in cancer cells substantiating the potential of the MGDG extracted from Synechocystis sp. PCC 6803 for the treatment of breast cancer cells through the apoptosis-mediated pathway

Microparticles for Suspension Culture of Mammalian Cells

The focus of this work is to develop a technology for the synthesis of polymer microcarriers that demonstrate mammalian cell culture adhesion on the surface of the microcarriers. Most mammalian cells are adherent in nature that requires multilayer vessels, large volume, expensive cell culture media, high manufacturing time, and high costs of cell culture supplies for the commercial-scale manufacturing of cells. The development of an efficient, scalable technology for producing large volumes of cells is a need in bioprocess industries to improve product potency. We developed a method of synthesizing soft biocompatible US FDA approved polymer based microparticle carrier system of approximately 260 ± 27 µm in diameter that serves as an adherent platform for human umbilical vein endothelial cells (HUVEC) to grow in suspension. Our preliminary experimental results showed that using the polymeric microcarrier system cell adhesion to the surface of the microcarriers was 2-3-fold higher than conventional cell culture flasks while using 10-fold lower cell culture media in a bioreactor than a tissue-culture treated flask. The survival of HUVEC on microparticles was confirmed by live cell staining (green fluorescent calcein AM), dead cell staining (ethidium homodimer-1), nuclear DAPI staining, actin cytoskeleton staining, confocal microscopy, and flow cytometry analysis. This technology will provide high cell culture productivity while reducing the costs of growing adherent cells

Polymer Coated Gold-Ferric Oxide Superparamagnetic Nanoparticles for Theranostic Applications

Background: Engineered inorganic nanoparticles (NPs) are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. We show that by changing the coating of magnetic iron oxide NPs using poly-l-lysine (PLL) polymer the colloidal stability of the dispersion is improved in aqueous solutions including water, phosphate buffered saline (PBS), PBS with 10% fetal bovine serum (FBS) and cell culture medium, and the internalization of the NPs toward living mammalian cells is profoundly affected. Methods: A multifunctional magnetic NP is designed to perform a near-infrared (NIR)-responsive remote control photothermal ablation for the treatment of breast cancer. In contrast to the previously reported studies of gold (Au) magnetic (Fe3O4) core-shell NPs, a Janus-like nanostructure is synthesized with Fe3O4 NPs decorated with Au resulting in an approximate size of 60 nm mean diameter. The surface of trisoctahedral Au-Fe3O4 NPs was coated with a positively charged polymer, PLL to deliver the NPs inside cells. The PLL-Au-Fe3O4 NPs were characterized by transmission electron microscopy (TEM), XRD, FT-IR and dynamic light scattering (DLS). The unique properties of both Au surface plasmon resonance and superparamagnetic moment result in a multimodal platform for use as a nanothermal ablator and also as a magnetic resonance imaging (MRI) contrast agent, respectively. Taking advantage of the photothermal therapy, PLL-Au-Fe3O4 NPs were incubated with BT-474 and MDA-MB-231 breast cancer cells, investigated for the cytotoxicity and intracellular uptake, and remotely triggered by a NIR laser of ~ 808 nm (1 W/cm2 for 10 min). Results: The PLL coating increased the colloidal stability and robustness of Au-Fe3O4 NPs (PLL-Au-Fe3O4) in biological media including cell culture medium, PBS and PBS with 10% fetal bovine serum. It is revealed that no significant ( \u3c 10%) cytotoxicity was induced by PLL-Au-Fe3O4 NPs itself in BT-474 and MDA-MB-231 cells at concentrations up to 100 µg/ml. Brightfield microscopy, fluorescence microscopy and TEM showed significant uptake of PLL-Au-Fe3O4 NPs by BT-474 and MDA-MB-231 cells. The cells exhibited 40 and 60% inhibition in BT-474 and MDA-MB-231 cell growth, respectively following the internalized NPs were triggered by a photothermal laser using 100 µg/ml PLL-Au-Fe3O4 NPs. The control cells treated with NPs but without laser showed \u3c 10% cell death compared to no laser treatment control. Conclusion: Combined together, the results demonstrate a new polymer gold superparamagnetic nanostructure that integrates both diagnostics function and photothermal ablation of tumors into a single multimodal nanoplatform exhibiting a significant cancer cell death

Polymer coated gold-ferric oxide superparamagnetic nanoparticles for theranostic applications

Abstract Background Engineered inorganic nanoparticles (NPs) are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. We show that by changing the coating of magnetic iron oxide NPs using poly-l-lysine (PLL) polymer the colloidal stability of the dispersion is improved in aqueous solutions including water, phosphate buffered saline (PBS), PBS with 10% fetal bovine serum (FBS) and cell culture medium, and the internalization of the NPs toward living mammalian cells is profoundly affected. Methods A multifunctional magnetic NP is designed to perform a near-infrared (NIR)-responsive remote control photothermal ablation for the treatment of breast cancer. In contrast to the previously reported studies of gold (Au) magnetic (Fe3O4) core–shell NPs, a Janus-like nanostructure is synthesized with Fe3O4 NPs decorated with Au resulting in an approximate size of 60 nm mean diameter. The surface of trisoctahedral Au–Fe3O4 NPs was coated with a positively charged polymer, PLL to deliver the NPs inside cells. The PLL–Au–Fe3O4 NPs were characterized by transmission electron microscopy (TEM), XRD, FT-IR and dynamic light scattering (DLS). The unique properties of both Au surface plasmon resonance and superparamagnetic moment result in a multimodal platform for use as a nanothermal ablator and also as a magnetic resonance imaging (MRI) contrast agent, respectively. Taking advantage of the photothermal therapy, PLL–Au–Fe3O4 NPs were incubated with BT-474 and MDA-MB-231 breast cancer cells, investigated for the cytotoxicity and intracellular uptake, and remotely triggered by a NIR laser of ~ 808 nm (1 W/cm2 for 10 min). Results The PLL coating increased the colloidal stability and robustness of Au–Fe3O4 NPs (PLL–Au–Fe3O4) in biological media including cell culture medium, PBS and PBS with 10% fetal bovine serum. It is revealed that no significant (< 10%) cytotoxicity was induced by PLL–Au–Fe3O4 NPs itself in BT-474 and MDA-MB-231 cells at concentrations up to 100 μg/ml. Brightfield microscopy, fluorescence microscopy and TEM showed significant uptake of PLL–Au–Fe3O4 NPs by BT-474 and MDA-MB-231 cells. The cells exhibited 40 and 60% inhibition in BT-474 and MDA-MB-231 cell growth, respectively following the internalized NPs were triggered by a photothermal laser using 100 μg/ml PLL–Au–Fe3O4 NPs. The control cells treated with NPs but without laser showed < 10% cell death compared to no laser treatment control Conclusion Combined together, the results demonstrate a new polymer gold superparamagnetic nanostructure that integrates both diagnostics function and photothermal ablation of tumors into a single multimodal nanoplatform exhibiting a significant cancer cell death