Study on Liposomal Encapsulation of New Bodipy Sensitizers for Photodynamic Therapy.

We report a series of four efficient photosensitizers (PSs) based on a Bodipy core for photodynamic therapy (PDT). In the absence of hydrophilic functional groups, these PSs have been encapsulated in liposomes and examined for photocytotoxicity against human ovarian carcinoma cell line (SK-OV-3). The IC50 values obtained are as low as 0.350 µM, which compete with the classical photosensitizer chlorine E6 (IC50 = 0.39 µM) under similar experimental conditions.

Systemic delivery of stable siRNA-encapsulating lipid vesicles: optimization, biodistribution, and tumor suppression.

Lipid-based nanoparticles are considered as promising candidates for delivering siRNA into the cytoplasm of targeted cells. However, in vivo efficiency of these nanoparticles is critically dependent on formulation strategies of lipid-siRNA complexes. Adsorption of serum proteins to lipid-siRNA complexes and its charge determine siRNA degradation and serum half-life, thus significantly altering the bioavailability of siRNA. To address these challenges, we developed a formulation comprising dihydroxy cationic lipid, N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium chloride (DHDEAC), cholesterol, and varying concentrations of 1,2-distearoryl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol-2000)] (DSPE-PEG 2000). Using an ethanol dilution method, addition of these lipids to siRNA solution leads to formation of stable and homogeneous population of siRNA-encapsulated vesicles (SEVs). Biodistribution of these SEVs, containing 5 mol % of DSPE-PEG 2000 in xenograft mice, as monitored by live animal imaging and fluorescence microscopy, revealed selective accumulation in the tumor. Remarkably, four intravenous injections of the modified vesicles with equimolar amounts of siRNA targeting ErbB2 and AURKB genes led to significant gene silencing and concomitant tumor suppression in the SK-OV-3 xenograft mouse model. Safety parameters as evaluated by various markers of hepatocellular injury indicated the nontoxic nature of this formulation. These results highlight improved pharmacokinetics and effective in vivo delivery of siRNA by DHDEAC-based vesicles.

Probing protein stability and proteolytic resistance by loop scanning: a comprehensive mutational analysis.

Improvement in protein thermostability was often found to be associated with increase in its proteolytic resistance as revealed by comparative studies of homologous proteins from extremophiles or mutational studies. Structural elements of protein responsible for this association are not firmly established although loops are implicated indirectly due to their structural role in protein stability. To get a better insight, a detailed study of protein wide mutants and their influence on stability and proteolytic resistance would be helpful. To generate such a data set, a model protein, Bacillus subtilis lipase was subjected to loop scanning site-saturation mutagenesis on 86 positions spanning all loops including termini. Upon screening of ~16,000 clones, 17 single mutants with improved thermostability were identified with increment in apparent melting temperature (Tm(app) ) by 1-6°C resulting in an increase in free energy of unfolding (ΔG(unf) ) by 0.04-1.16 kcal/mol. Proteolytic resistance of all single mutants upon incubation with nonspecific protease, Subtilisin A, was determined. Upon comparison, post-proteolysis residual activities as well as kinetics of proteolysis of mutants showed excellent correlation with ΔG(unf) , (r > 0.9), suggesting that proteolysis was strongly correlated with the global stability of this protein. This significant correlation in this set, with least possible sequence changes (single aa substitution), while covering >60% of protein surface strongly argues for the covariance of these two variables. Compared to studies from extremophiles, with large sequence heterogeneity, the observed correlation in such a narrow sequence space (ΔΔG(unf) = 1.57 kcal⁻¹) justifies the robustness of this relation.

Targeted liposomes to deliver DNA to cells expressing 5-HT receptors.

Cell targeted delivery of drugs, including nucleic acids, is known to enhance the therapeutic potential of free drugs. We used serotonin (5-HT) as the targeting ligand to deliver plasmid DNA to cells specifically expressing 5-HT receptor. Our liposomal formulation includes the 5-HT conjugated targeting lipid, a cationic lipid and cholesterol. DNA-binding studies indicate that the targeting 5-HT-lipid binds DNA efficiently. The formulation was tested and found to efficiently deliver DNA into CHO cells stably expressing the human serotonin(1A) receptor (CHO-5-HT(1A)R) compared to control CHO cells. Liposomes without the 5-HT moiety were less efficient in both cell lines. Similar enhancement in transfection efficiency was also observed in human neuroblastoma IMR32 and hepatocellular carcinoma (HepG2) cells. Cell uptake studies using CHO-5-HT(1A)R cells by flow cytometry and confocal microscopy clearly indicated that the targeting liposomes through 5-HT moiety may have a direct role in increasing the cellular uptake of DNA-lipid complexes. To our knowledge this is the first report that demonstrates receptor-targeted nucleic acid delivery into cells expressing 5-HT receptor.

Nanomaterials as in vivo sensors.

The methods of monitoring cellular processes are either snap-shots or based on in vitro measurements. Nanomaterials have specific advantages to be excellent probes to monitor in vivo processes. In this study we have demonstrated that attachment of neurotransmitter, serotonin to iron oxide nanoparticles brings about enhanced contrast in cells that express the receptor for serotonin specifically.

Plasmid DNA delivery into MDA-MB-453 cells mediated by recombinant Her-NLS fusion protein.

A major rate-limiting step in nonviral gene delivery is the entry of nucleic acids across various membrane barriers and eventually into the nucleus where it must be transcribed. Cell-penetrating peptides and proteins are employed to generate formulations that overcome these challenges to facilitate DNA delivery into cells efficiently. However, these are limited by their inability to deliver nucleic acids selectively due to lack of specificity because they deliver to both cancer and normal cells. In this study, through modular design, we generated a recombinant fusion protein designated as Her-nuclear localization sequence (Her-NLS), where heregulin-α (Her), a targeting moiety, was cloned in frame with cationic NLS peptide to obtain a cell-specific targeting biomolecule for nucleic acid delivery. The heregulin-α(1) isoform possesses the epidermal growth factor-like domain and binds to HER2/3 heterodimers which are overexpressed in certain breast cancers. Purified recombinant Her-NLS fusion protein binds plasmid DNA and specifically transfects MDA-MB-453 cells overexpressing the epidermal growth factor receptors HER2/3 in vitro. The approach described would also permit replacement of heregulin ligand with other targeting moieties that would be suited to cell-specific nucleic acid delivery mediated via receptor-ligand interactions.

Cationic lipid-mediated nucleic acid delivery: beyond being cationic.

Realization of the potential of nucleic acids as drugs is intricately linked to their in vivo delivery. Cationic lipids demonstrated tremendous potential as safe, efficient and scalable in vitro carriers of nucleic acids. For in vivo delivery of nucleic acids, the extant two component liposomal preparations consisting of cationic lipids and nucleic acids have been largely found to be insufficient. Being a soft matter, liposomes readily respond to many physiological variables leading to complex component and morphological changes, thus confounding the efforts in a priori identification of a "competent" formulation. In the recent past many chemical moieties that provide advantage in facing the challenges of barriers in vivo, were incorporated into cationic lipids to improve the transfection efficiency. The cationic lipids, essential for DNA condensation and protection, definitely require additional components to be efficient in vivo. In addition, formulations of cationic lipid carriers with non-lipidic components, mainly peptides, have demonstrated success in in vivo transfection. The present review describes some recent successes of in vivo nucleic acid delivery by cationic lipids.

Designed multi-domain protein as a carrier of nucleic acids into cells.

Protein-based nucleic acid carriers offer attractive possibilities to enhance in vitro and in vivo gene delivery to combat diseases. A multi-domain fusion protein, namely TAT-NLS-Mu, designated as TNM, has been designed, cloned, heterologously expressed in E. coli and purified to homogeneity by affinity chromatography. The recombinant chimera TNM harbors three epitopes, a cell-penetrating (TAT) domain, a nuclear localization domain comprising of three nuclear localization sequence (NLS) motifs in tandem and a DNA-binding (Mu) domain. Complexes prepared by combining plasmid DNA with TNM (DP) transfect MCF-7, COS, CHO and HepG2 cells. Ternary complexes prepared with DNA, protein and cationic lipid (DPL) resulted in ~5-7 fold enhancement in reporter gene expression over the DP alone. Treatment of cells with chloroquine during transfection, with DP complexes, resulted in remarkable increases in reporter gene expression suggesting the involvement of endosomal compartments in the uptake process. Interestingly, DPL prepared with Lipofectin or 1, 2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) exhibited enhanced transfection in the presence of serum in MCF-7 and HepG2 cells. Microinjection of DP complexes, with and without NLS sequence, into the cytoplasm and nucleus of smooth muscle cells (SMC) indicated that the presence of NLS sequence in protein carrier significantly enhanced transgene expression. Together the data suggest that modular design of proteins is a promising method to develop gene delivery carriers and also the role of NLS epitopes in mediating nuclear transfer of DNA complexes into various cell types.

Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight.

In vitro evolution methods are now being routinely used to identify protein variants with novel and enhanced properties that are difficult to achieve using rational design. However, one of the limitations is in screening for beneficial mutants through several generations due to the occurrence of neutral/negative mutations occurring in the background of positive ones. While evolving a lipase in vitro from mesophilic Bacillus subtilis to generate thermostable variants, we have designed protocols that combine stringent three-tier testing, sequencing and stability assessments on the protein at the end of each generation. This strategy resulted in a total of six stabilizing mutations in just two generations with three mutations per generation. Each of the six mutants when evaluated individually contributed additively to thermostability. A combination of all of them resulted in the best variant that shows a remarkable 15 degrees C shift in melting temperature and a millionfold decrease in the thermal inactivation rate with only a marginal increase of 3 kcal mol(-1) in free energy of stabilization. Notably, in addition to the dramatic shift in optimum temperature by 20 degrees C, the activity has increased two- to fivefold in the temperature range 25-65 degrees C. High-resolution crystal structures of three of the mutants, each with 5 degrees increments in melting temperature, reveal the structural basis of these mutations in attaining higher thermostability. The structures highlight the importance of water-mediated ionic networks on the protein surface in imparting thermostability. Saturation mutagenesis at each of the six positions did not result in enhanced thermostability in almost all the cases, confirming the crucial role played by each mutation as revealed through the structural study. Overall, our study presents an efficient strategy that can be employed in directed evolution approaches employed for obtaining improved properties of proteins.

Electroporation of adult zebrafish.

We generated transient transgenic zebrafish by applying electrical pulses subsequent to injection of DNA into muscle tissue of 3-6-month old adult zebrafish. Electroporation parameters, such as number of pulses, voltage, and amount of plasmid DNA, were optimized and found that 6 pulses of 40 V/cm at 15 mug/fish increased the luciferase expression by 10-fold compared with those in controls. By measuring the expression of luciferase, in vivo by electroporation in adult zebrafish and in vitro using fish cell line (Xiphophorus xiphidium A2 cells), the strength of three promoters (CMV, human EF-1alpha, and Xenopus EF-1alpha) was compared. Subsequent to electroporation after injecting DNA in the mid region of zebrafish, expression of green fluorescent protein was found far away from the site of injection in the head and the tail sections. Thus, electroporation in adult zebrafish provides a rapid way of testing the behavior of gene sequences in the whole organism.

EPR and susceptibility studies on (ZnS)1-x(MnTe)x powders.

(ZnS)1-x(MnTe)x luminescent powder samples with x=0.02, 0.05, 0.10, 0.15, 0.20 and 0.25 were prepared by solid-state reaction method. EPR spectra were recorded at room temperature (300K) in the frequency range 8.8-9.6GHz for samples of all compositions. The line width (DeltaH) and the number of spins increased with MnTe concentration. Room temperature dc magnetic susceptibility measurements were carried out using vibrating sample magnetometer. Susceptibility of the samples increased with MnTe content.

Cell biological and biophysical aspects of lipid-mediated gene delivery.

Cationic lipids are conceptually and methodologically simple tools to deliver nucleic acids into the cells. Strategies based on cationic lipids are viable alternatives to viral vectors and are becoming increasingly popular owing to their minimal toxicity. The first-generation cationic lipids were built around the quaternary nitrogen primarily for binding and condensing DNA. A large number of lipids with variations in the hydrophobic and hydrophilic region were generated with excellent transfection efficiencies in vitro. These cationic lipids had reduced efficiencies when tested for gene delivery in vivo. Efforts in the last decade delineated the cell biological basis of the cationic lipid gene delivery to a significant detail. The application of techniques such as small angle X-ray spectroscopy (SAXS) and fluorescence microscopy, helped in linking the physical properties of lipid:DNA complex (lipoplex) with its intracellular fate. This biological knowledge has been incorporated in the design of the second-generation cationic lipids. Lipid-peptide conjugates (peptoids) are effective strategies to overcome the various cellular barriers along with the lipoplex formulations methodologies. In this context, cationic lipid-mediated gene delivery is considerably benefited by the methodologies of liposome-mediated drug delivery. Lipid mediated gene delivery has an intrinsic advantage of being a biomimetic platform on which considerable variations could be built to develop efficient in vivo gene delivery protocols.

Efficient expression of transgenes in adult zebrafish by electroporation.

BACKGROUND: Expression of transgenes in muscle by injection of naked DNA is widely practiced. Application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. Zebrafish is a precious model to investigate developmental biology in vertebrates. In this study we investigated the effect of electroporation on expression of transgenes in 3-6 month old adult zebrafish. RESULTS: Electroporation parameters such as number of pulses, voltage and amount of plasmid DNA were optimized and it was found that 6 pulses of 40 V.cm(-1) at 15 microg of plasmid DNA per fish increased the luciferase expression 10-fold compared to controls. Similar enhancement in transgene expression was also observed in Indian carp (Labeo rohita). To establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in A2 cells and adult zebrafish after electroporation. The relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. GFP fluorescence in tissues after electroporation was also studied by fluorescence microscopy. CONCLUSION: Electroporation after DNA injection enhances gene expression 10-fold in adult zebrafish. Electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. Enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish.

Cationic lipids and cationic ligands induce DNA helix denaturation: detection of single stranded regions by KMnO4 probing.

Cationic lipids and cationic polymers are widely used in gene delivery. Using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid, we have investigated the stability of the DNA in DOTAP:DNA complexes by probing with potassium permanganate (KMnO4). Interestingly, thymidines followed by a purine showed higher susceptibility to cationic ligand-mediated melting. Similar studies performed with other water-soluble cationic ligands such as polylysine, protamine sulfate and polyethyleneimine also demonstrated melting of the DNA but with variations. Small cations such as spermine and spermidine and a cationic detergent, cetyl trimethylammonium bromide, also rendered the DNA susceptible to modification by KMnO4. The data presented here provide direct proof for melting of DNA upon interaction with cationic lipids. Structural changes subsequent to binding of cationic lipids/ligands to DNA may lead to instability and formation of DNA bubbles in double-stranded DNA.

Stability studies on a lipase from Bacillus subtilis in guanidinium chloride.

Lipase from Bacillus subtilis is a "lidless" lipase that does not show interfacial activation. Due to exposure of the active site to solvent, the lipase tends to aggregate. We have investigated the solution properties and unfolding of the lipase in guanidinium chloride (GdmCl) to understand its aggregation behavior and stability. Dynamic light scattering (DLS), near- and far-UV circular dichroism, activity and intrinsic fluorescence of lipase suggest that the protein undergoes unfolding between 1 M and 2 M GdmCl. The polarity sensitive dye, 1,1',-bis-(4anilino)naphthalene-5,5"-disulfonic acid (bis-ANS), a probe for hydrophobic pockets, binds cooperatively to the native lipase. An intermediate populated in 1.75 M GdmCl that strongly binds bis-ANS was identified. Tendency of the native protein to aggregate in solution and specific binding to bis-ANS confirms that the lipase has exposed hydrophobic pockets and this surface hydrophobicity strongly influences the unfolding pathway of the lipase in GdmCl.

Cell membrane hybrid bilayers containing the G-protein-coupled receptor CCR5.

A hybrid bilayer membrane is a planar model membrane that is formed at an alkanethiol monolayer-coated gold surface by the spontaneous reorganization of phospholipid vesicles. Membrane vesicles from monkey kidney COS-1 cells also reorganize at an alkanethiol/lipid monolayer-coated surface resulting in the formation of a cell membrane hybrid bilayer. Atomic force microscopy and spectroscopic ellipsometry indicate that the cell membrane layer is equivalent to the thickness of one leaflet of the membrane and is continuous over large areas. Cell membrane hybrid bilayers were formed from membrane vesicles from COS-1 cells that were transiently transfected with a synthetic human CCR5 chemokine receptor gene. Preparations that contained "inside out" and "right side out" membrane vesicles were used. Binding of monoclonal antibodies to either the amino- or carboxyl-terminus of CCR5 was observed by surface plasmon resonance and confirmed the presence and the random orientation of these integral membrane receptors. Specific and concentration-dependent binding of the beta-chemokine RANTES to the cell membrane hybrid confirmed that CCR5 retained ligand-binding activity. The ability to form cell membrane hybrid bilayers that contain functional G-protein-coupled or other multispanning receptors without requiring protein isolation, purification, and reconstitution offers a promising method for the rapid screening of potential ligands.