Liposome-entrapped plasmid DNA: characterisation studies

Yvonne Perrie, Gregory Gregoriadis

Research output: Contribution to journalArticle

106 Citations (Scopus)

Abstract

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 μg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration–rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmol 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 μg 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.
LanguageEnglish
Pages125-132
Number of pages8
JournalBBA - General Subjects
Volume1475
Issue number2
DOIs
Publication statusPublished - 1 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

  • liposome
  • DNA vaccine
  • plasmid DNA
  • hepatitis B surface antigens
  • microelectrophoresis
  • gel electrophoresis

Cite this

Perrie, Yvonne ; Gregoriadis, Gregory. / Liposome-entrapped plasmid DNA : characterisation studies. In: BBA - General Subjects. 2000 ; Vol. 1475, No. 2. pp. 125-132.
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Liposome-entrapped plasmid DNA : characterisation studies. / Perrie, Yvonne; Gregoriadis, Gregory.

In: BBA - General Subjects, Vol. 1475, No. 2, 01.07.2000, p. 125-132.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Liposome-entrapped plasmid DNA

T2 - BBA - General Subjects

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AU - Gregoriadis, Gregory

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N2 - Plasmid DNA pRc/CMV HBS (5.6 kb) (100 μg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration–rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmol 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 μg 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 μg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration–rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmol 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 μg 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 - liposome

KW - DNA vaccine

KW - plasmid DNA

KW - hepatitis B surface antigens

KW - microelectrophoresis

KW - gel electrophoresis

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

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

M3 - Article

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JO - BBA - General Subjects

JF - BBA - General Subjects

SN - 0304-4165

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ER -