Nontoxic Vehicle Research Articles

Efficient nuclear delivery of antisense oligodeoxynucleotides and selective inhibition of CETP expression by apo E peptide in a human CETP-stably transfected CHO cell line.

N,N-Dipalmitylglycyl-apolipoprotein E (129-169) peptide (dpGapoE) is an efficient gene delivery system for both plasmids and antisense oligodeoxynucleotides (ODNs). To develop a new and efficient approach to the regulation of cholesteryl ester transfer protein (CETP) expression, we used dpGapoE to transfect phosphorothioate antisense ODNs against nucleotides 329 to 349 of human CETP cDNA into a human CETP-stably transfected Chinese hamster ovary (CHO) cell line (hCETP-CHO). After transfection, translocation to the nuclei and concentration in nuclear structures were observed in >95% of the cells at 6 and 12 hours by fluorescence microscopy. No membrane disruption was observed after transfection of ODNs by dpGapoE. Although the translocation stability of phosphorothioate ODNs in the nuclei continued for >48 hours, it had weakened after 24 hours. Cellular CETP mRNA levels gradually declined, and the maximum reduction in the mRNA level (>50%) was observed at 36 hours, after which the mRNA level started to recover. CETP activity in the culture medium declined over 72 hours. The maximum reduction in CETP activity was observed at 36 hours (53.8% of control). Neither CETP mRNA nor CETP activities changed throughout the experiment after the transfection of sense phosphorothioate ODNs delivered by dpGapoE complex or naked antisense ODNs. We conclude that (1) the novel synthetic dpGapoE was a highly effective and nontoxic vehicle for the nuclear delivery of antisense ODNs into hCETP-CHO cells and (2) antisense ODNs selectively inhibited both CETP expression and activity in an hCETP-CHO cell line. This approach may enable gene regulation in vivo and could possibly be used as an antiatherosclerotic agent to alter high density lipoprotein metabolism.

Enhancement of liposomal gene delivery in human breast cancer cells by dimethyl sulfoxide.

Non-toxic concentrations ( 1%) of dimethyl sulfoxide (DMSO) enhance the liposomal delivery of DNA to both MCF-7 and MDA-MB-231 human breast tumor cells. Uptake of SV-40-luciferase was enhanced in MCF-7 cells by 14-fold while uptake of CMV-beta-galactosidase was increased 10-fold. In MDA-MB-231 cells, uptake of SV-40-luciferase was increased by approximately 70%. A mixture of ethanol and polyethylene glycol (45:55) at a concentration of 1% produced less pronounced improvements in transgene delivery to MCF-7 cells (a 70% increase in SV-40-luciferase uptake and a 4-fold increase in CMV-beta-galactosidase uptake) but no improvement in SV-40-luciferase gene delivery to MDA-MB-231 cells. These studies suggest that select pharmaceutical adjuvants which dissolve clinically useful drugs may have promise as non-toxic vehicles for improving transgene delivery. However, the relative effectiveness of these adjuvants is likely to vary depending on both the nature of the gene being delivered as well as the tumor cell which is the target for uptake of the exogenous gene.

In vivo effects of β-amyloid implants in rodents: lack of potentiation of damage associated with transient global forebrain ischemia

Recent studies have shown that the principal component of the senile plaque in Alzheimer's disease (AD), β-amyloid protein (βAP) can exert direct and indirect neurotoxicity in vitro. Because of the studies that demonstrated potentiation of excitatory amino acid toxicity by βAP, we decided to test whether βAP was able to potentiate damage in an in vivo model where excitotoxic damage is thought to be important. The present study evaluated the in vivo effects of βAP implants in the brain of rats before and after being subjected to 10 min of transient global forebrain ischemia by 4-vessel occlusion (4-VO). Implants of either synthetic βAP or prolactin (PRL), which was used as a control protein, were made into the striatum and the hippocampus of either the left (βAP) or the right (PRL) cerebral hemisphere. The implants were made in a lipophilic, non-toxic vehicle so as to try and achieve sustained βAP exposure. One group of animals was evaluated for direct in vivo effects within 1 week following implantation; the other group was subjected to 4-VO 3–4 days post-implantation for evaluation of potential indirect effects. This latter group was compared to the histopathology of animals subjected to 4-VO without prior implantation. In the group of animals evaluated for direct effects, no evidence of neurotoxicity was observed. Bielschowsky silver staining and immunostaining for ubiquitin were unremarkable in all lesions. βAP was detected by immunocytochemistry in the parenchymal tissue that received βAP implants. Marked glial activation was observed to be associated with experimental and control implants. Under the experimental conditions employed in this study, significant protection from ischemia rather than potentiation of damage was observed. These results suggest that βAP may not be neurotoxic in rodents in vivo and that the lesions and/or trauma produced by the implantation procedure 3–4 days prior to 4-VO may have induced factors that were protective against ischemia-induced damage.

In vitro and in vivo studies evaluating a liposome system for drug solubilization.

A liposome system was developed which demonstrates suitability as an intravenous drug carrier for a lipophilic drug compound (RS-93522, a dihydropyridine CA2+ channel blocker). An aqueous phospholipid suspension was employed as a nontoxic solubilizing vehicle for this drug. The liposome formulation, composed of a 3% mixture of dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol, produced a physically and chemically stable preparation which solubilized the lipophilic drug compound at a concentration 500 times above its intrinsic aqueous solubility. Characterizing the liposome-drug system by gel filtration chromatography showed that the drug comigrated with the lipid constituents of the liposome. Further in vitro studies established that the liposome-RS-93522 formulation allowed for rapid and complete transfer of the drug from the liposome to bind with albumin when added to human serum. In vivo studies with rats were performed in which the pharmacokinetics of the liposomal-RS-93522 system were compared to those of a cosolvent-solubilized RS-93522 solution. This study showed that the pharmacokinetic profiles of the two solutions were identical. All the evidence indicates that a liposome formulation of this type does not alter the distribution of the drug in serum and is, therefore, not likely to affect the intrinsic pharmacological or toxicological parameters of the drug relative to the conventional solvent/excipient-containing formulation. This liposome system demonstrates utility as a biocompatible, nontoxic drug delivery vehicle.

Tissue Injury Caused by Wound Disinfectants

Wound disinfectants of various kinds were tested on microwounds and on different tissues in animals and in man. The analysis was performed by vital microscopy, electron microscopy, and microangiography. It was found that disinfectants, regardless of type, damaged tissue and interfered with tissue function, thereby increasing the injury already existing in a damaged tissue and delaying wound healing. The significance of these findings for wound healing is discussed. The importance of using the lowest possible concentration of disinfectant, consistent with adequate antiseptic effects, and of a non-toxic vehicle, is emphasized. It should be possible to modify already existing disinfectants according to the requirements of the tissue and to take not only the bacteria but also the injured tissue into consideration when new disinfectants are designed, tested, and selected.