Liposome-entrapped plasmid DNA: characterisation studies

Yvonne Perrie, Gregory Gregoriadis

Research output: Contribution to journalArticle

Abstract

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.
Original languageEnglish
Pages (from-to)125-132
Number of pages8
JournalBBA - General Subjects
Volume1475
Issue number2
DOIs
Publication statusPublished - 3 Jul 2000

Fingerprint

Liposomes
Plasmids
DNA
Unilamellar Liposomes
Fluid Therapy
Dehydration
Propane
Complexation
Phosphatidylcholines
Sodium Dodecyl Sulfate
Anions
Association reactions
Phosphatidylglycerols
Zeta potential
Hepatitis B Surface Antigens
Electrophoresis
Transfection
Ovum
Microscopy
Microscopic examination

Keywords

  • electrophoresis
  • fatty acids
  • liposomes
  • microscopy
  • particle size
  • phosphatidylcholines
  • phosphatidylethanolamines
  • plasmids
  • quaternary ammonium compounds
  • agar gel
  • monounsaturated
  • fluorescence

Cite this

Perrie, Yvonne ; Gregoriadis, Gregory. / Liposome-entrapped plasmid DNA : characterisation studies. In: BBA - General Subjects. 2000 ; Vol. 1475, No. 2. pp. 125-132.
@article{fb6ffe79b2894278a4fbbe47cb26f34a,
title = "Liposome-entrapped plasmid DNA: characterisation studies",
abstract = "Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54{\%} of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97{\%}. As expected, the association of DNA with preformed anionic liposomes was low (9{\%}). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.",
keywords = "electrophoresis, fatty acids, liposomes, microscopy, particle size, phosphatidylcholines, phosphatidylethanolamines, plasmids, quaternary ammonium compounds, agar gel, monounsaturated, fluorescence",
author = "Yvonne Perrie and Gregory Gregoriadis",
year = "2000",
month = "7",
day = "3",
doi = "10.1016/S0304-4165(00)00055-6",
language = "English",
volume = "1475",
pages = "125--132",
journal = "BBA - General Subjects",
issn = "0304-4165",
publisher = "Elsevier",
number = "2",

}

Liposome-entrapped plasmid DNA : characterisation studies. / Perrie, Yvonne; Gregoriadis, Gregory.

In: BBA - General Subjects, Vol. 1475, No. 2, 03.07.2000, p. 125-132.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Liposome-entrapped plasmid DNA

T2 - characterisation studies

AU - Perrie, Yvonne

AU - Gregoriadis, Gregory

PY - 2000/7/3

Y1 - 2000/7/3

N2 - Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

AB - Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

KW - electrophoresis

KW - fatty acids

KW - liposomes

KW - microscopy

KW - particle size

KW - phosphatidylcholines

KW - phosphatidylethanolamines

KW - plasmids

KW - quaternary ammonium compounds

KW - agar gel

KW - monounsaturated

KW - fluorescence

UR - http://www.sciencedirect.com/science/article/pii/S0304416500000556

U2 - 10.1016/S0304-4165(00)00055-6

DO - 10.1016/S0304-4165(00)00055-6

M3 - Article

C2 - 10832026

VL - 1475

SP - 125

EP - 132

JO - BBA - General Subjects

JF - BBA - General Subjects

SN - 0304-4165

IS - 2

ER -