Hitesh Handa

Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance in vitro

Drug resistance limits the success of many anticancer drugs. Reduced accumulation of the drug at its intracellular site of action because of overexpression of efflux transporters such as P-glycoprotein (P-gp) is a major mechanism of drug resistance. In this study, we investigated whether photodynamic therapy (PDT) using methylene blue, also a P-gp inhibitor, can be used to enhance doxorubicin-induced cytotoxicity in drug-resistant tumor cells. Aerosol OT (AOT)-alginate nanoparticles were used as a carrier for the simultaneous cellular delivery of doxorubicin and methylene blue. Methylene blue was photoactivated using light of 665 nm wavelength. Induction of apoptosis and necrosis following treatment with combination chemotherapy and PDT was investigated in drug-resistant NCI/ADR-RES cells using flow cytometry and fluorescence microscopy. Effect of encapsulation in nanoparticles on the intracellular accumulation of doxorubicin and methylene blue was investigated qualitatively using fluorescence microscopy and was quantitated using HPLC. Encapsulation in AOT-alginate nanoparticles significantly enhanced the cytotoxicity of combination therapy in resistant tumor cells. Nanoparticle-mediated combination therapy resulted in a significant induction of both apoptosis and necrosis. Improvement in cytotoxicity could be correlated with enhanced intracellular and nuclear delivery of the two drugs. Further, nanoparticle-mediated combination therapy resulted in significantly elevated reactive oxygen species (ROS) production compared to single drug treatment. In conclusion, nanoparticle-mediated combination chemotherapy and PDT using doxorubicin and methylene blue was able to overcome resistance mechanisms and resulted in improved cytotoxicity in drug-resistant tumor cells.

Surfactant−Polymer Nanoparticles Enhance the Effectiveness of Anticancer Photodynamic Therapy

Photodynamic therapy (PDT) is a promising treatment modality for cancer. PDT is based on the concept that photosensitizers, when exposed to light of specific wavelength, generate cytotoxic reactive oxygen species (ROS) capable of killing tumor cells. The effectiveness of PDT has been limited in part by the lack of photosensitizers that accumulate sufficiently in tumor cells and poor yield of ROS from existing photosensitizers. In this report, we investigated whether aerosol OT-alginate nanoparticles can be used as a carrier to enhance the therapeutic efficacy of a model photosensitizer, methylene blue. Methylene blue loaded nanoparticles were evaluated for PDT effectiveness in two cancer cell lines, MCF-7 and 4T1. Encapsulation of methylene blue in nanoparticles significantly enhanced intracellular ROS production, and the overall cytotoxicity following PDT. It also resulted in higher incidence of necrosis. Greater effectiveness of nanoparticles could be correlated with higher yield of ROS with nanoparticle-encapsulated methylene blue. Further, treatment of tumor cells with nanoparticle-encapsulated methylene blue resulted in significant nuclear localization of methylene blue while free drug treatment resulted in its accumulation mainly in the endolysosomal vesicles. In conclusion, encapsulation of methylene blue in aerosol OT-alginate nanoparticles enhanced its anticancer photodynamic efficacy in vitro. Increased ROS production and favorable alteration in the subcellular distribution contribute to the enhanced PDT efficacy of nanoparticle-encapsulated photosensitizer.

The attenuation of platelet and monocyte activation in a rabbit model of extracorporeal circulation by a nitric oxide releasing polymer

Nitric oxide (NO) has been shown to reduce thrombogenicity by decreasing platelet and monocyte activation by the surface glycoprotein, P-selectin and the integrin, CD11b, respectively. In order to prevent platelet and monocyte activation with exposure to an extracorporeal circulation (ECC), a nitric oxide releasing (NORel) polymeric coating composed of plasticized polyvinyl chloride (PVC) blended with a lipophilic N-diazeniumdiolate was evaluated in a 4 h rabbit thrombogenicity model using flow cytometry. The NORel polymer significantly reduced ECC thrombus formation compared to polymer control after 4 h blood exposure (2.8 +/- 0.7 NORel vs 6.7 +/- 0.4 pixels/cm(2) control). Platelet count (3.4 +/- 0.3 NORel vs 2.3 +/- 0.3 x 10(8)/ml control) and function as measured by aggregometry (71 +/- 3 NORel vs 17 +/- 6% control) were preserved after 4 h exposure in NORel versus control ECC. Plasma fibrinogen levels significantly decreased in both NORel and control groups. Platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry was attenuated after 4 h on ECC to ex vivo collagen stimulation (27 +/- 1 NORel vs 40 +/- 2 MFI control). Monocyte CD11b expression was reduced after 4 h on ECC with NORel polymer (87 +/- 14 NORel vs 162 +/- 30 MFI control). These results suggest that the NORel polymer coatings attenuate the increase in both platelet P-selectin and monocytic CD11b integrin expression in blood exposure to ECCs. These NO-mediated platelet and monocytic changes were shown to improve thromboresistance of these NORel-polymer-coated ECCs for biomedical devices.

Long-term nitric oxide release and elevated temperature stability with S-nitroso-N-acetylpenicillamine (SNAP)-doped Elast-eon E2As polymer

Nitric oxide (NO) is known to be a potent inhibitor of platelet activation and adhesion. Healthy endothelial cells that line the inner walls of all blood vessels exhibit a NO flux of 0.5-4 × 10(-10) mol cm(-2) min(-1) that helps prevent thrombosis. Materials with a NO flux that is equivalent to this level are expected to exhibit similar anti-thrombotic properties. In this study, five biomedical grade polymers doped with S-nitroso-N-acetylpenicillamine (SNAP) were investigated for their potential to control the release of NO from the SNAP within the polymers, and further control the release of SNAP itself. SNAP in the Elast-eon E2As polymer creates an inexpensive, homogeneous coating that can locally deliver NO (via thermal and photochemical reactions) as well slowly release SNAP. Furthermore, SNAP is surprisingly stable in the E2As polymer, retaining 82% of the initial SNAP after 2 months storage at 37 °C. The E2As polymer containing SNAP was coated on the walls of extracorporeal circulation (ECC) circuits and exposed to 4 h blood flow in a rabbit model of extracorporeal circulation to examine the effects on platelet count, platelet function, clot area, and fibrinogen adsorption. After 4 h, platelet count was preserved at 100 ± 7% of baseline for the SNAP/E2As coated loops, compared to 60 ± 6% for E2As control circuits (n = 4). The SNAP/E2As coating also reduced the thrombus area when compared to the control (2.3 ± 0.6 and 3.4 ± 1.1 pixels/cm(2), respectively). The results suggest that the new SNAP/E2As coating has potential to improve the thromboresistance of intravascular catheters, grafts, and other blood-contacting medical devices, and exhibits excellent storage stability compared to previously reported NO release polymeric materials.

Optimized polymeric film-based nitric oxide delivery inhibits bacterial growth in a mouse burn wound model.

Nitric oxide (NO) has many biological roles (e.g. antimicrobial agent, promoter of angiogenesis, prevention of platelet activation) that make NO releasing materials desirable for a variety of biomedical applications. Localized NO release can be achieved from biomedical grade polymers doped with diazeniumdiolated dibutylhexanediamine (DBHD/N2O2) and poly(lactic-co-glycolic acid) (PLGA). In this study, the optimization of this chemistry to create film/patches that can be used to decrease microbial infection at wound sites is examined. Two polyurethanes with different water uptakes (Tecoflex SG-80A (6.2±0.7wt.%) and Tecophilic SP-60D-20 (22.5±1.1wt.%)) were doped with 25wt.% DBHD/N2O2 and 10wt.% of PLGA with various hydrolysis rates. Films prepared with the polymer that has the higher water uptake (SP-60D-20) were found to have higher NO release and for a longer duration than the polyurethane with the lower water uptake (SG-80A). The more hydrophilic polymer enhances the hydrolysis rate of the PLGA additive, thereby providing a more acidic environment that increases the rate of NO release from the NO donor. The optimal NO releasing and control SG-80A patches were then applied to scald burn wounds that were infected with Acinetobacter baumannii. The NO released from these patches applied to the wounds is shown to significantly reduce the A. baumannii infection after 24h (∼4 log reduction). The NO release patches are also able to reduce the level of transforming growth factor-β in comparison to controls, which can enhance re-epithelialization, decrease scarring and reduce migration of bacteria. The combined DBHD/N2O2 and PLGA-doped polymer patches, which could be replaced periodically throughout the wound healing process, demonstrate the potential to reduce risk of bacterial infection and promote the overall wound healing process.

Cross-Linked Bioreducible Layer-by-Layer Films for Increased Cell Adhesion and Transgene Expression

The effect of cross-linking layer-by-layer (LbL) films consisting of bioreducible poly(2-dimethylaminoethyl methacrylate) (rPDMAEMA) and DNA is examined with regard to rigidity, biodegradability, cell adhesion, and transfection activity using 1,5-diiodopentane (DIP) cross-linker. DIP chemically reacts with the tertiary amines of rPDMAEMA, altering the chemical composition of these LbL films. The result is a change in surface morphology, film swelling behavior, and film rigidity, measured with AFM and ellipsometry. It is found that the apparent Youngs modulus is increased more than 4 times its original value upon cross-linking. Cross-linking mass is additionally confirmed with a quartz crystal microbalance with dissipation (QCM-D). Comprehensive analyses of these experimental values were investigated to calculate the degree of cross-linking using the rubber elasticity theory and the Flory-Rehner theory. Additionally, the Flory-Huggins parameter, chi, was calculated. Good agreement in the two methods yields a cross-linking density of approximately 0.82 mmol/cm(3). The Flory-Huggins parameter increased upon cross-linking from 1.07 to 1.2, indicating increased hydrophobicity of the network and formation of bulk water droplets within the films. In addition, the effects of cross-linking on film disassembly by 1,4-dithiothreitol (DTT) are found to be insignificant despite the alteration in film rigidity. Mouse fibroblast cells and smooth muscle cells are used to study the effect of cross-linking on cell adhesion and cell transfection activity. In vitro transfection activity up to seven days is quantified using secreted alkaline phosphatase (SEAP) DNA. Film cross-linking is found to enhance cell adhesion and prolong the duration of cellular transfection. These results contribute to the development of bioreducible polymer coatings for localized gene delivery.

Hemocompatibility comparison of biomedical grade polymers using rabbit thrombogenicity model for preparing nonthrombogenic nitric oxide releasing surfaces

Nitric oxide (NO) is an endogenous vasodilator as well as natural inhibitor of platelet adhesion/activation. Nitric oxide releasing (NOrel) materials can be prepared by doping an NO donor species, such as diazeniumdiolated dibutylhexanediamine (DBHD/N2O2), within a polymer coating. The inherent hemocompatibility properties of the base polymer can also influence the efficiency of such NO release coatings. In this study, four biomedical grade polymers were evaluated in a 4 h rabbit model of thrombogenicity for their effects on extracorporeal circuit thrombus formation and circulating platelet count. At the end of 4 h, Elast-Eon E2As was found to preserve 58% of baseline platelets versus 48, 40, and 47% for PVC/DOS, Tecophilic SP-60D-60, and Tecoflex SG80A, respectively. Elast-Eon also had significantly lower clot area of 5.2 cm2 compared to 6.7, 6.1, and 6.9 cm2 for PVC/DOS, SP-60D-60, and SG80A, respectively. Based on the results obtained for the base polymer comparison study, DBHD/N2O2-doped E2As was evaluated in short-term (4 h) rabbit studies to observe the NO effects on prevention of clotting and preservation of platelet function. Platelet preservation for this optimal NO release formulation was 97% of baseline after 4 h, and clot area was 0.9 cm2 compared to 5.2 cm2 for controls, demonstrating that combining E2As with NO release provides a truly advanced hemocompatible polymer coating for extracorporeal circuits and potentially other blood contacting applications.

Reduction in thrombosis and bacterial adhesion with 7 day implantation of S-nitroso-N-acetylpenicillamine (SNAP)-doped Elast-eon E2As catheters in sheep

Thrombosis and infection are two common problems associated with blood-contacting medical devices such as catheters. Nitric oxide (NO) is known to be a potent antimicrobial agent as well as an inhibitor of platelet activation and adhesion. Healthy endothelial cells that line the inner walls of all blood vessels exhibit a NO flux of 0.5~4×10-10 mol cm-2 min-1 that helps prevent thrombosis. Materials with a NO flux that is equivalent to this level are expected to exhibit similar anti-thrombotic properties. In this study, NO-releasing catheters were fabricated by incorporating S-nitroso-N-acetylpenicillamine (SNAP) in the Elast-eon E2As polymer. The SNAP/E2As catheters release physiological levels of NO for up to 20 d, as measured by chemiluminescence. Furthermore, SNAP is stable in the E2As polymer, retaining 89% of the initial SNAP after ethylene oxide (EO) sterilization. The SNAP/E2As and E2As control catheters were implanted in sheep veins for 7 d to examine the effect on thrombosis and bacterial adhesion. The SNAP/E2As catheters reduced the thrombus area when compared to the control (1.56 ± 0.76 and 5.06 ± 1.44 cm2, respectively). A 90% reduction in bacterial adhesion was also observed for the SNAP/E2As catheters as compared to the controls. The results suggest that the SNAP/E2As polymer has the potential to improve the hemocompatibility and bactericidal activity of intravascular catheters, as well as other blood-contacting medical devices (e.g., vascular grafts, extracorporeal circuits).

In vitro and in vivo study of sustained nitric oxide release coating using diazeniumdiolate-doped poly(vinyl chloride) matrix with poly(lactide-co-glycolide) additive

Nitric oxide (NO) is an endogenous vasodilator as well as natural inhibitor of platelet adhesion and activation that can be released from a NO donor species, such as diazeniumdiolated dibutylhexanediamine (DBHD/N2O2) within a polymer coating. In this study, various Food and Drug Administration approved poly(lactic-co-glycolic acid) (PLGA) species were evaluated as additives to promote a prolonged NO release from DBHD/N2O2 within a plasticized poly(vinyl chloride) (PVC) matrix. When using an ester-capped PLGA additive with a slow hydrolysis time, the resulting coatings continuously release between 7-18×10-10 mol cm-2 min-1 NO for 14 d at 37°C in PBS buffer. The corresponding pH changes within the polymer films were visualized using pH sensitive indicators and are shown to correlate with the extended NO release pattern. The optimal combined diazeniumdiolate/PLGA-doped NO release (NOrel) PVC coating was evaluated in vitro and its effect on the hemodynamics was also studied within a 4 h in vivo extracorporeal circulation (ECC) rabbit model of thrombogenicity. Four out of 7 control circuits clotted within 3 h, whereas all the NOrel coated circuits were patent after 4 h. Platelet counts on the NOrel ECC were preserved (79 ± 11% compared to 54 ± 6% controls). The NOrel coatings showed a significant decrease in the thrombus area as compared to the controls. Results suggest that by using ester-capped PLGAs as additives to a conventional plasticized PVC material containing a lipophilic diazeniumdiolates, the NO release can be prolonged for up to 2 weeks by controlling the pH within the organic phase of the coating.

Transfection activity of layer-by-layer plasmid DNA/poly(ethylenimine) films deposited on PLGA microparticles

Layer-by-layer (LbL) assemblies of DNA and polycations on the surface of colloidal templates can be used for gene delivery. Plasmid DNA encoding for secreted alkaline phosphatase (SEAP) was used to deposit LbL films with poly(ethylenimine) (PEI) on the surface of polystyrene and poly(lactide-co-glycolide) microparticles. The formation of LBL films was confirmed by zeta potential analysis and fluorescence and atomic force microscopy techniques. The LbL particles were rapidly internalized in a dose-dependent manner by J774.1 murine macrophages. Transfection activity of the LbL particles was evaluated in J774.1 cells using three different doses (5, 10, 25 particle per cell). The levels of SEAP expression increased with increasing dose but were lower than transfection levels mediated by control PEI/DNA polyplexes at corresponding DNA doses. The LbL particles reported here present a promising platform for delivery of DNA to phagocytic cells.