Sujatha Dokka

Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes.

AbstractPurpose. The objectives of this study are to investigate the toxicityassociated with polycationic liposomes and to elucidate the underlyingmechanism. We tested the hypothesis that the positive charge of liposomesis a key determinant of toxicity by testing differently chargedliposomes in mice. Methods. Differently charged liposomal systems including cationicliposomes, LipofectAMINE and DOTAP, and neutral and negativeliposomes were evaluated for their toxicity after pulmonaryadministration in mice. LDH assay and differential cell counts were performedto measure toxicity and pulmonary inflammation, respectively. Reactiveoxygen intermediates (ROI) were assessed by chemiluminescence. Results. Instillation of cationic liposomes eliciteddose-dependent toxicity and pulmonary inflammation. This effect was more pronouncedwith the multivalent cationic liposome LipofectAMINE as comparedto the monovalent cationic DOTAP. Neutral and negative liposomes didnot exhibit lung toxicity. Toxicity associated with cationic liposomescorrelated with the oxidative burst induced by the liposomes.LipofectAMINE induced a dose-dependent increase in ROI generation. Thiseffect was less pronounced with DOTAP and absent with neutral andnegative liposomes. Conclusions. ROI play a key role in cationic lipid-mediated toxicity.Polyvalent cationic liposomes cause a release of ROI which areresponsible for the pulmonary toxicity.

Novel non-endocytic delivery of antisense oligonucleotides

Antisense oligonucleotides (ONs) have several properties that make them attractive as therapeutic agents. Hybridization of antisense ONs to their complementary nucleic acid sequences by Watson-Crick base pairing is a highly selective and efficient process. Design of therapeutic antisense agents can be made more rationally as compared to most traditional drugs, i.e., they can be designed on the basis of target RNA sequences and their secondary structures. Despite these advantages, the design and use of antisense ONs as therapeutic agents are still faced with several obstacles. One major obstacle is their inefficient cellular uptake and poor accessibility to target sites. In this article, we will discuss key barriers affecting ON delivery and approaches to overcome these barriers. Current methods of ON delivery will be reviewed with an emphasis on novel non-endocytic methods of delivery. ONs are taken up by cells via an endocytic process. The process of ON release from endosomes is a very inefficient process and, hence, ONs end up being degraded in the endosomes. Thus, ONs do not reach their intended site of action in the cytoplasm or nucleus. Delivery systems ensuring a cytoplasmic delivery of ONs have the potential to increase the amount of ON reaching the target. Here, we shall examine various ON delivery methods that bypass the endosomal pathway. The advantages and disadvantages of these methods compared to other existing methods of ON delivery will be discussed.

Induction of neutrophil apoptosis and secondary necrosis during endotoxin-induced pulmonary inflammation in mice.

The present study investigated the relationship between apoptotic and necrotic cell death and their role in pulmonary inflammatory response to endotoxin. Pulmonary administration of lipopolysaccharide (LPS) caused a rapid increase in the levels of pro‐inflammatory cytokine TNF‐α and inflammatory cell influx in the bronchoalveolar lavage (BAL) fluids. Control mice showed only resident alveolar macrophages with no apoptosis, whereas LPS‐treated mice showed clear apoptosis of BAL cells. Microscopic studies confirmed the presence of apoptotic neutrophils and macrophages ingesting apoptotic bodies. The number of apoptotic neutrophils increased concomitantly with the increase in neutrophil influx which peaked 1 day after the treatment. However, necrosis was not detected at this early time, but increased subsequently and peaked at day 3. The levels of necrosis and apoptosis were both elevated and prolonged at high LPS doses. Treatment of mice with phosphatidylserine (PS)‐containing liposome, known to inhibit macrophage phagocytosis of apoptotic cells, increased the level of apoptosis and necrosis caused by LPS, whereas control non‐PS liposome or saline treatment had no effects. We conclude that necrosis occurs secondary to apoptosis in LPS‐treated lung model and that this development is not the result of direct insult by LPS. Instead, our results and previous studies suggest that inefficient clearance of apoptotic cells by macrophages contributes, at least in part, to the levels of apoptosis and necrosis induced by LPS. Because necrosis is associated with cell damage and release of histotoxic contents, this development is likely to play a role in determining the severity and duration of lung toxicity induced by endotoxin.

High-efficiency gene transfection of macrophages by lipoplexes

Macrophage transfection studies are crucial for understanding gene regulation and expression. However, gene transfection in macrophages is difficult. We have shown here that macrophages are more resistant to gene transfection compared with other cell types. To further develop an efficient gene delivery system for macrophages, we evaluated various liposomal and non-liposomal agents including LipofectAMINE(R), Lipofectin(R), DOTAP, DEAE-dextran, and the DNA condensing agent protamine sulfate for their ability to promote gene transfection. CMV-luciferase was used as a reporter plasmid. Macrophage transfection was maximal at the DNA:LipofectAMINE:protamine ratio of 1:12:1 microg/ml. The LipofectAMINE formulation showed a 10-12-fold increase in transfection efficiency over DOTAP and a 4-5-fold increase over Lipofectin. This transfection method showed minimal toxicity at the concentrations tested and was at least 20-25-fold superior to the most frequently used DEAE-dextran method for macrophage transfection.

Cellular Delivery of Oligonucleotides by Synthetic Import Peptide Carrier

AbstractPurpose. Inefficient cellular uptake and endosomal entrapment are among the obstacles impeding the therapeutic use of oligonucleotides (ONs). The objectives of this study are to investigate the feasibility of utilizing a synthetic import peptide as a drug carrier for cytoplasmic delivery of ONs and to study its transport mechanisms. Methods. A molecular conjugate consisting of a signal import peptide (IP) derived from Kaposi fibroblast growth factor (K-FGF) and a polycationic ON linker, polylysine (PL), was synthesized and complexed with 5′ fluorescently-labeled ON. Complex formation was verified by spectral shift assay and cellular uptake of the ON complex was studied fluorometrically. Microscopic studies were performed to visualize the intracellular distribution of the ON. Results. Cells treated with the ON:IP-PL complex exhibited a dose-dependent increase in ON uptake over free ON-treated controls. The uptake of the complex was shown to occur via an energy-independent, non-endocytic, process since metabolic and endocytic inhibitors and low temperature did not prevent the uptake. Microscopic studies revealed a non-punctate fluorescence pattern, consistent with the non-endocytic transport process. Intense nuclear fluorescence was observed in cells treated with the complex but not with free ON, suggesting enhanced cytoplasmic delivery and nuclear accumulation of the ON by the conjugate. Efficient complex uptake was shown to require both the ON-binding moiety PL and the IP moiety. The delivery system was found to be non-toxic at the concentrations used. Conclusions. The peptide carrier was effective in promoting the cellular uptake of ON. The mechanism by which the peptide facilitates ON uptake appears to involve a direct translocation of ON via a non-endocytic process. The peptide carrier has the potential to overcome the problem of ON endosomal entrapment and degradation.

Inhibition of nuclear transcription factor-κB by specific IκB kinase peptide inhibitor

Aberrant activation and expression of genes is the major cause of many human diseases. Most genes are quiescent or have minimal activity in affecting physiologic processes. However, in certain pathologic conditions, these genes are abruptly turned on by a preexisting genetic switch, causing them to overexpress. The nuclear transcription factor-kB (NF-kB) is one such important factor that controls the genetic switch for many important genes that encode cytokines, growth factors, adhesion molecules, and some acute phase proteins (1). Abnormal activation of NF-kB is associated with a number of diseases, including immune and inflammatory diseases (see Ref. 2 for review). Because of its pathophysiologic importance, the NF-kB has been identified as a key target for pharmacologic manipulations. The purpose of this study is to investigate a gene inhibition approach using synthetic peptides that inhibit signal-induced degradation of the NF-kB inhibitory subunit IkB by IkB kinases (IKK), thereby preventing NF-kB activation. NF-kB belongs to a superfamily of protein dimmers frequently composed of two DNA-binding subunits: NF-kB1 (p50) and RelA (p65) (3). It is normally kept in an inactive form in the cytoplasm by attachment of the inhibitory subunit IkB. The activation of NF-kB is accomplished by phosphorylation of the IkB by specific IkB kinases (IKK), which triggers the complete degradation of the inhibitor (4). The activated NF-kB is then translocated into the nucleus where it binds to the promoter region of target genes and activates their transcription. The phosphorylation Of IkB by IKK occurs at two specific serine residues (ser32 and ser36), and mutation of these sites results in an inhibition of signalinduced phosphorylation and IkB degradation (5). Because the interaction between the IKK and IkB is sequence-specific, we hypothesize that specific peptides carrying the same amino acid sequences as those of the IkB recognition sites may be used to selectively inhibit IkB phosphorylation and degradation, and thus NF-kB activation. To test this hypothesis, we used hybrid peptides containing the normal or mutated sequence of the IkB recognition site, covalently linked to a cell-permeable signal peptide to aid their cellular delivery. These peptides were then tested for their NF-kB-inhibiting activity in signal-induced macrophage RAW 264.7 cell model.

Activation of aPKC is required for vanadate-induced phosphorylation of protein kinase B (Akt), but not p70S6k in mouse epidermal JB6 cells.

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms by which vanadate mediates adverse effects are not well understood. The present study investigated the vanadate-induced phosphorylation of Akt and p70S6K, two kinases known to be vital for cell survival, growth, transformation, and transition of the cell cycle in mammals. Exposure of mouse epidermal JB6 cells to vanadium led to phosphorylation of Akt and p70S6K in a time- and dose-dependent manner. Vanadium exposure also caused translocation of atypical isoforms of PKC (λ, ζ) from the cytosol to the membrane, but had no effect on PKCα translocation, suggesting that the atypical PKCs (aPKC) were specifically involved in vanadium-induced cellular response. Importantly, overexpression of a dominant negative mutant PKCλ blocked Akt phosphorylation at Ser473 and Thr308, whereas it did not inhibit p70S6k phosphorylation at Thr389 and Thr421/Ser424, suggesting that aPKC activation is specifically involved in vanadium-induced activation of Akt, but not in activation of p70S6k. Furthermore, vanadium-induced p70S6k phosphorylation at Thr389 and Thr421/Ser424 and Akt phosphorylation at Thr308 occurred through a PI-3K-dependent pathway because a PI-3K dominant negative mutant inhibited induction as compared with vector control cells. These results indicate that there was a differential role of aPKC in vanadate-induced phosphorylation of Akt and p70S6k, suggesting that signal transduction pathways leading to the activation of Akt and p70S6k were different.