Guangzhao Mao

Gene delivery in vitro and in vivo from bioreducible multilayered polyelectrolyte films of plasmid DNA

Layer-by-layer (LbL) films were assembled on flexible stainless steel substrate using plasmid DNA and reducible hyperbranched poly(amido amine) (RHB) polycation. The films were characterized by XPS and their disassembly in reducing conditions confirmed by ellipsometry. Fibroblast and smooth muscle cell attachment and proliferation on DNA/RHB films were indistinguishable from those on control DNA/poly(ethylenimine) (PEI) films. In vitro transfection activity was evaluated using reporter plasmids encoding for secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP). DNA/RHB films showed higher and longer lasting transfection activity than control DNA/PEI films using SEAP plasmid. It was revealed through the use of GFP plasmid that DNA/RHB films transfected almost the entire cell population growing on the films. In vivo transfection activity was evaluated by subcutaneously implanting a stainless steel substrate coated with the DNA/RHB films containing SEAP plasmid DNA and measuring the levels of SEAP secreted into the blood circulation of rats. It was found that the plasma levels of SEAP peaked at approximately 160 ng SEAP/mL five days post-implantation.

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.

Hyaluronic acid-conjugated polyamidoamine dendrimers for targeted delivery of 3,4-difluorobenzylidene curcumin to CD44 overexpressing pancreatic cancer cells.

The current study was aimed to develop a targeted dendrimer formulation of 3, 4-difluorobenzylidene curcumin (CDF) and evaluate its potential in CD44 targeted therapy for pancreatic cancer. Using amine terminated fourth generation poly(amidoamine) (PAMAM) dendrimer nanocarrier and hyaluronic acid (HA) as a targeting ligand, we engineered a CD44-targeted PAMAM dendrimer (HA-PAMAM) formulation of CDF. The resulting dendrimer nanosystem (HA-PAMAM-CDF) had a particle size and surface charge of 9.3 ± 1.5 nm and -7.02 ± 9.53 mV, respectively. When CD44 receptor overexpressing MiaPaCa-2 and AsPC-1 human pancreatic cancer cells were treated with HA-PAMAM-CDF, a dose-dependent cytotoxicity was observed. Furthermore, blocking the CD44 receptors present on the MiaPaCa-2 cells using free excess soluble HA prior to treatment with HA-PAMAM-CDF nano-formulation resulted in 1.71 fold increase in the IC50 value compared to non-targeted formulation (PAMAM-CDF), confirming target specificity of HA-PAMAM-CDF. Additionally, HA-PAMAM-CDF formulation when compared to PAMAM-CDF, displayed higher cellular uptake in MiaPaCa-2 cancer cell lines as shown by fluorescence studies. In summary, the novel CD44 targeted dendrimer based nanocarriers appear to be proficient in mediating site-specific delivery of CDF via CD44 receptors, with an improved therapeutic margin and safety.

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.

Stepwise adsorption of a long trichlorosilane and a short aminosilane

Abstract A mixed film of microscopic heterogeneity was formed by stepwise deposition of a long-chain organosilane n -octadecyltrichlorosilane (OTS) and a short-chain organosilane aminobutyldimethylmethoxysilane (ABS). The amount of silane deposited on an oxidized silicon substrate was controlled kinetically by varying the dip time in each silane solution. The binary film was characterized by the atomic force microscopy (AFM), infrared attenuated total internal reflection (IR-ATR), and contact angle goniometry. OTS forms a monolayer structure whose fractional surface coverage and surface energy can be precisely controlled. ABS agglomerates slowly to form island structure and is ineffective in surface energy or composition variation. The contact angles on sub-monolayers of OTS follows the Cassie equation, while the contact angle of the binary OTS and ABS film deviates positively from the equation.

DNA release dynamics from reducible polyplexes by atomic force microscopy.

Controlled intracellular disassembly of polyelectrolyte complexes of polycations and DNA (polyplexes) is a crucial step for the success of nonviral gene delivery. Motivated by our previous observation of different gene delivery performances among multiblock reducible copolypeptide vectors ( Manickam, D. S. ; Oupicky, D. Bioconjugate Chem. 2006, 17, 1395- 1403 ), atomic force microscopy is used to visualize plasmid DNA in various decondensed states from reducible polypeptide polyplexes under simulated physiological reducing conditions. DNA decondensation is triggered by reductive degradation of disulfide-containing cationic polypeptides. Striking differences in DNA release dynamics between polyplexes based on polypeptides of histidine-rich peptide (HRP, CKHHHKHHHKC) and nuclear localization signal (NLS, CGAGPKKKRKVC) peptide are presented. The HRP and NLS polyplexes are similar to each other in their initial morphology with a majority of them containing only one DNA plasmid. Upon reductive degradation by dithiothreitol, DNA is released from NLS abruptly regardless of the initial polyplex morphology, while DNA release from HRP polyplexes displays a gradual decondensation that is dependent on the size of polyplexes. The release rate is higher for larger HRP polyplexes. The smaller HRP polyplexes become unstable when they are in contact with expanding chains nearby. The results reveal potentially rich DNA release dynamics that can be controlled by subtle variation in multivalent counterion binding to DNA as well as the cellular matrix.

DNA Release Dynamics from Bioreducible Poly(amido amine) Polyplexes

The DNA release dynamics of bioreducible poly(amido amine) polyplexes were studied in real time by atomic force microscopy (AFM). DNA release is triggered by a depolymerization of high-molecular-weight polycations into low-molecular-weight oligocations that occurs by means of the thiol and disulfide exchange reaction mechanism. AFM images were captured in a simulated physiological reducing environment that used dithiothreitol. Distinctive stages of disassembly are common among various polyplexes that have different disulfide content, molecular weight, and polymer architecture, while the DNA release rate depends upon the disulfide content. In the first stage, polyplexes evolve from metastable structures into the more stable toroid structure upon the depolymerization. In the second stage, toroids either aggregate or fuse into larger toroids. In the last stage, DNA wormlike chains and loops are held by a central compact core. The results confirm the prospect of bioreducible poly(amido amine)s as controlled DNA delivery vectors. The study offers new physical insights into the DNA release pathway including intermediate structures that have a high degree of structural heterogeneity and disassembly induced particle growth. The study identifies disassembly induced colloidal and morphological instability as an important issue to be addressed.

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.

Immobilization and molecular interactions between bacteriophage and lipopolysaccharide bilayers

The paper describes immobilization methods of bacteriophage P22 and tailspike gp9 proteins isolated from P22 on atomic force microscope (AFM) probes. The paper also reports single molecule force spectroscopy (SMFS) using AFM of the immobilized P22 (or gp9) interactions with substrate-supported O-antigenic lipopolysaccharides (LPS) bilayers. LPS covers the outer membrane of gram-negative bacteria, such as Salmonella typhimurium. Evidence from AFM imaging and SMFS shows that immobilized P22 (or gp9) are capable of strong and multivalent binding to supported LPS. The most common rupture forces between P22 and LPS were identified to be 72, 130, 206, and 279 pN at force loading rate of 12,000 pN/s. The quantized unbinding force was found to decrease with decreasing force loading rate as predicted by the Bell model. By fitting the force data with the Bell model, an energy barrier of 55 kJ/mol was obtained. Evidence is also provided that demonstrates the resilience of phage to pH and temperature fluctuation as well as dehydration/rehydration cycles. The biospecific interactions between P22 and the LPS are relevant to cell infection, inflammation, cancer progression and metastasis, food safety, pharmaceuticals, and biosensor development.