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Incorporation of plasmid DNA into liposomes of varying surface charge and size: Effect on DNA integrity and transfection efficiency
s137
Relating to Invited Lecture I & 7-Drug Delivery (P2.001-P2.113)
Cationic vesicle-DNA complexes have been shown to exhibit a low to modest tmnsfcction in viva (1). Because of potential toxicity of such complexes, exposure of DNA to nucleascs and inability to incorporate other agents which may promote transfection efficiency, their usefulness may not be as promising as when nucleic acids are incorporated within conventional liposomes. To that end, 10-100 pg of pGL2 plasmid DNA (3.99 x 106 Daltons) expressing rhc luciferase reporter gene was incorporated by the dehydration-nhydration method (2) into neutral (44-55%) and negatively charged (45-63% of the amount used) liposomes composed of egg phosphatidylcholinc (PC) and quimolar dioleyl phosphatidylethanolamine (DOPE) supplemented (for negative liposomes) with phosphatidylserinc (PS). Incorporation of pGL2 in similar, positively charged PC/DOPE liposomes supplemented with stearylamine (SA), 1.2-bis (hexadecylcycloxy)-3uimethylaminopropane (BisHOP) or N[l-(2,3-dioleyloxy)propyl]-N,N,Nuiethylammonium (DOTMA) (molar ratio 1:1:0.25) was ever greater (6292%). with most of the DNA located in the interior of the vesicles. Microfluidization (3 cycles) of pGL2-containing liposomes (590-m nm diameter) in the presence of non-entrapped DNA resulted in smaller vesicles (209-383 nm) with n DNA content of 10-2140 (neutral). 41-514s (negative) and 50-83% (positive liposomes) of the original amount used. Much of the DNA content of intact (up to 86%) and microfluidized (up to 79%) liposomes was not degraded on exposure to deoxyribonuclease at 3PC. Plasmid DNA incorporated in intact or micmfluidizcd liposomes retained its sa-uctural integrity and was able to uansfect cells (COS-7) in vitro in relation to the size and surface charge of the vesicles. Such DNAincorporating liposomes are being presently investigated for plasmid DNA expression in viva.
Dipalmitoyl phosphatidylcholine (dppc) liposomes were prepared by standard size
ultrasonication procedures
distribution
diclofenac,
by Coulter
ephedrine,
and checked
counter.
Atropine,
methyl nicotinate,
naproxen
for acceptable benzoic
and nicotine
were incorporated in equimolar proportion to the dppc. were buffered
to suppress
dissociation
acid,
Solutions
of the molecules.
The
liposomes were subjected to differential scanning calorimetty and the enthalpy
change
AHt at about 410C
measured.
AHt was
inversely related to the partition coefficient, Klip of the drug between liposome lipid phase I water, indicating that lipophilic molecules are incorporated
into the bilayer and disrupt its crystalline structure.
Klip can be estimated more conveniently from the linear relationship between log Klip and log btanol.
1. Zhu. N.. Liggit, D., Liu. Y. and Debs. R. (1993) Science 261:209-211 2. Kirby, C. and Gregoriadis. G. (1984) Biofechno[ogy 2:979-984
DuringthelactyarsagreDtintmsthaJbeendcvotedtothepotentialities of liposomes as drug delivery systems sod in this surse enzyme ataapment seems to be really promising. ‘I& activity of enzymes mttappal in liposomcs can be controlled by addition of surfactPnt at sublytic concentrations, avoiding the disruption of the vesicular structure of liposomcs. In this work fl-oplactoaidpre from W&hia C&i was entrappad in soya PC- cxtrudai liposomes (2OOnm) with an encapsulation efficiency of about 10%. The catalytic activity was testal with MU&G (methylumbellifcrylg&ctoside) measuring the formation rate of the fluorescent hydrolisis product (MUB). The activity of atzymc a~trspped in liposomes was latent
(k=6.3x104 min-l/pl of liposotnes against a k=5.7x10s3 midl/pl of liposotnes when the vesicles are disrupted by detergent). The activity was substantially &maaed when s&&ant (T&on X-100) was added in sublytic amcal tmtions: l&tic constants of 1.3x1(r3 min-lljd of liposomes and 2&lfJ3 tnin+jd of liposomes wzm obtained with a surfactant to lipid molar ratio of 0.105 and 0.513, mapectively. At these sutfactaot cxmcentrations turbidity mcasurcments show pracwa tion of the liposome Strucblre.
Theenhanamentofmzymrticactivitymay beduetoanincrraseof membrane parm&ility to small MUB-G molecules.
The affinity of liposomes to human skin and dermal delivery of liposome-encap sulated drugs is thoroughly described (11. It is well known, that the physical and chemical properties of substances, and particularly volubility, influence their penetration through stratified squamous epithelium. In general, lipid-soluble substances move more rapidly across mucosal epithelium than water-soluble and those with a solubility in both penetrate most rapidly. The applicability of liposomal formulations as vehicle for methylene blue was under investigation in this study. Methylene blue (MB) has been used as a vital dye in diagnostic of early squamous cell carcinomas of mucous membranes. MB is a potential photosensitizer for application in photodynamic therapy, because of its high singlet oxygen quantum yield. Liposomes have been used both in vitroand in viva as a drug and dye delivery vehicle [2]. As a first step a ‘remote loading’ of liposomes was intended. As methylene blue is positively charged, liposomes with an expressed negative charge were produced by high pressure homogenisation technique [3]. To increase the negative charge of “classical liposomes” (--2OmV) composed of soy lecithin ascorbic acid palmitate was added and a zeta potential of nearly -4OmV was obtained. These blank liposomes were incubated with an aqueous solution of 10”M methylene blue. Controlled flocculation occured, which was reversible at dilution, though a signif+cant growth in particle size was measured indicating a limited fusion of liposomes. Blank liposomes showed an average PCS-diameter of 75nm. Methylene blue containing samples showed comparable parameters in the range of 210nm. Concentrated preparations for microscopy showed bulb-like shapes like an arrangement of grapes. The in viva model of the chorioallantoic membrane of fertilised chicken embryos (CAM) was introduced by Gottfried et al. for the cultivation of tumorous tissues in the field of photodynamic therapy. 3D-fibrosarcoma tissues’were used and inoculated onto the CAM’s of opened eggs. After incubation for Smin. with MB with and without liposomes. respectively, the tissues were examined by flurescence microscopy (i;,., = 54&m, A, >590nm). In this in vitrostudy distinctly higher fluoresence intensities were detected in the specimen incubated with liposomal MB. In the next step entrapment of MB into positively charged liposomes in statu nascendi will be examined in the same model, because of a potentially higher affinity 01 positive liposomes to negative cell adhesion regions.
Relating to Invited Lecture I & 7-Drug Delivery (P2.001-P2.113)
Cationic vesicle-DNA complexes have been shown to exhibit a low to modest tmnsfcction in viva (1). Because of potential toxicity of such complexes, exposure of DNA to nucleascs and inability to incorporate other agents which may promote transfection efficiency, their usefulness may not be as promising as when nucleic acids are incorporated within conventional liposomes. To that end, 10-100 pg of pGL2 plasmid DNA (3.99 x 106 Daltons) expressing rhc luciferase reporter gene was incorporated by the dehydration-nhydration method (2) into neutral (44-55%) and negatively charged (45-63% of the amount used) liposomes composed of egg phosphatidylcholinc (PC) and quimolar dioleyl phosphatidylethanolamine (DOPE) supplemented (for negative liposomes) with phosphatidylserinc (PS). Incorporation of pGL2 in similar, positively charged PC/DOPE liposomes supplemented with stearylamine (SA), 1.2-bis (hexadecylcycloxy)-3uimethylaminopropane (BisHOP) or N[l-(2,3-dioleyloxy)propyl]-N,N,Nuiethylammonium (DOTMA) (molar ratio 1:1:0.25) was ever greater (6292%). with most of the DNA located in the interior of the vesicles. Microfluidization (3 cycles) of pGL2-containing liposomes (590-m nm diameter) in the presence of non-entrapped DNA resulted in smaller vesicles (209-383 nm) with n DNA content of 10-2140 (neutral). 41-514s (negative) and 50-83% (positive liposomes) of the original amount used. Much of the DNA content of intact (up to 86%) and microfluidized (up to 79%) liposomes was not degraded on exposure to deoxyribonuclease at 3PC. Plasmid DNA incorporated in intact or micmfluidizcd liposomes retained its sa-uctural integrity and was able to uansfect cells (COS-7) in vitro in relation to the size and surface charge of the vesicles. Such DNAincorporating liposomes are being presently investigated for plasmid DNA expression in viva.
Dipalmitoyl phosphatidylcholine (dppc) liposomes were prepared by standard size
ultrasonication procedures
distribution
diclofenac,
by Coulter
ephedrine,
and checked
counter.
Atropine,
methyl nicotinate,
naproxen
for acceptable benzoic
and nicotine
were incorporated in equimolar proportion to the dppc. were buffered
to suppress
dissociation
acid,
Solutions
of the molecules.
The
liposomes were subjected to differential scanning calorimetty and the enthalpy
change
AHt at about 410C
measured.
AHt was
inversely related to the partition coefficient, Klip of the drug between liposome lipid phase I water, indicating that lipophilic molecules are incorporated
into the bilayer and disrupt its crystalline structure.
Klip can be estimated more conveniently from the linear relationship between log Klip and log btanol.
1. Zhu. N.. Liggit, D., Liu. Y. and Debs. R. (1993) Science 261:209-211 2. Kirby, C. and Gregoriadis. G. (1984) Biofechno[ogy 2:979-984
DuringthelactyarsagreDtintmsthaJbeendcvotedtothepotentialities of liposomes as drug delivery systems sod in this surse enzyme ataapment seems to be really promising. ‘I& activity of enzymes mttappal in liposomcs can be controlled by addition of surfactPnt at sublytic concentrations, avoiding the disruption of the vesicular structure of liposomcs. In this work fl-oplactoaidpre from W&hia C&i was entrappad in soya PC- cxtrudai liposomes (2OOnm) with an encapsulation efficiency of about 10%. The catalytic activity was testal with MU&G (methylumbellifcrylg&ctoside) measuring the formation rate of the fluorescent hydrolisis product (MUB). The activity of atzymc a~trspped in liposomes was latent
(k=6.3x104 min-l/pl of liposotnes against a k=5.7x10s3 midl/pl of liposotnes when the vesicles are disrupted by detergent). The activity was substantially &maaed when s&&ant (T&on X-100) was added in sublytic amcal tmtions: l&tic constants of 1.3x1(r3 min-lljd of liposomes and 2&lfJ3 tnin+jd of liposomes wzm obtained with a surfactant to lipid molar ratio of 0.105 and 0.513, mapectively. At these sutfactaot cxmcentrations turbidity mcasurcments show pracwa tion of the liposome Strucblre.
Theenhanamentofmzymrticactivitymay beduetoanincrraseof membrane parm&ility to small MUB-G molecules.
The affinity of liposomes to human skin and dermal delivery of liposome-encap sulated drugs is thoroughly described (11. It is well known, that the physical and chemical properties of substances, and particularly volubility, influence their penetration through stratified squamous epithelium. In general, lipid-soluble substances move more rapidly across mucosal epithelium than water-soluble and those with a solubility in both penetrate most rapidly. The applicability of liposomal formulations as vehicle for methylene blue was under investigation in this study. Methylene blue (MB) has been used as a vital dye in diagnostic of early squamous cell carcinomas of mucous membranes. MB is a potential photosensitizer for application in photodynamic therapy, because of its high singlet oxygen quantum yield. Liposomes have been used both in vitroand in viva as a drug and dye delivery vehicle [2]. As a first step a ‘remote loading’ of liposomes was intended. As methylene blue is positively charged, liposomes with an expressed negative charge were produced by high pressure homogenisation technique [3]. To increase the negative charge of “classical liposomes” (--2OmV) composed of soy lecithin ascorbic acid palmitate was added and a zeta potential of nearly -4OmV was obtained. These blank liposomes were incubated with an aqueous solution of 10”M methylene blue. Controlled flocculation occured, which was reversible at dilution, though a signif+cant growth in particle size was measured indicating a limited fusion of liposomes. Blank liposomes showed an average PCS-diameter of 75nm. Methylene blue containing samples showed comparable parameters in the range of 210nm. Concentrated preparations for microscopy showed bulb-like shapes like an arrangement of grapes. The in viva model of the chorioallantoic membrane of fertilised chicken embryos (CAM) was introduced by Gottfried et al. for the cultivation of tumorous tissues in the field of photodynamic therapy. 3D-fibrosarcoma tissues’were used and inoculated onto the CAM’s of opened eggs. After incubation for Smin. with MB with and without liposomes. respectively, the tissues were examined by flurescence microscopy (i;,., = 54&m, A, >590nm). In this in vitrostudy distinctly higher fluoresence intensities were detected in the specimen incubated with liposomal MB. In the next step entrapment of MB into positively charged liposomes in statu nascendi will be examined in the same model, because of a potentially higher affinity 01 positive liposomes to negative cell adhesion regions.