WO1998023260A2 - Vesicules de tensioactifs non ioniques en tant qu'agent de transport d'acide nucleique - Google Patents

Vesicules de tensioactifs non ioniques en tant qu'agent de transport d'acide nucleique Download PDF

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Publication number
WO1998023260A2
WO1998023260A2 PCT/GB1997/003235 GB9703235W WO9823260A2 WO 1998023260 A2 WO1998023260 A2 WO 1998023260A2 GB 9703235 W GB9703235 W GB 9703235W WO 9823260 A2 WO9823260 A2 WO 9823260A2
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Prior art keywords
formulation according
vesicles
polynucleotide
ionic surfactant
cells
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PCT/GB1997/003235
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English (en)
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WO1998023260A3 (fr
Inventor
Katharine Christine Carter
Alexander Balfour Mullen
Alan James Baillie
John Douglas Ansell
William Gerard Murphy
Kay Samuel
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University Of Strathclyde
University Of Edinburgh
Common Services Agency
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Priority to JP52442098A priority Critical patent/JP2001510459A/ja
Priority to EP97945945A priority patent/EP0946148A2/fr
Publication of WO1998023260A2 publication Critical patent/WO1998023260A2/fr
Publication of WO1998023260A3 publication Critical patent/WO1998023260A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • Non-Ionic Surfactant Vesicles as a Delivery Agent for
  • the present invention relates to an aqueous formulation (such as an injectable formulation) comprising a polynucleotide and a carrier component suspended in an aqueous delivery vehicle.
  • a aqueous formulation such as an injectable formulation
  • the formulation particularly though not exclusively comprises DNA or RNA in combination with vesicles comprised of non-ionic surfactants.
  • Factor IX deficiency Many diseases, such as Factor IX deficiency, are genetic disorders in which there is an inability functionally to express the gene or genes responsible for the production of corresponding peptide (s) or protein(s) .
  • Factor IX deficiency Haemophilia B
  • Factor IX deficiency gives rise to a life long bleeding disorder, requiring therapeutic transfusion with the missing plasma-derived clotting factors.
  • the clinical and economic cost of the disease to both patient and community is substantial.
  • Factor IX is normally produced in the liver, although it has been shown that non-hepatic cells are capable of expression and post-translational modification of Factor IX.
  • haematopoietic stem precursors which are more accessible than liver cells
  • transfection kits could be used as an in vitro , ex vivo or in vivo research tool.
  • USP 4217344 describes a process for the production of spheres which are capable of encapsulating water-soluble pharmaceuticals and the like, although, the entrapment of polynucleotides is neither described nor suggested.
  • USP 4235871 describes a method of encapsulating polynucleotides in synthetic, oligolamellar lipid vesicles or liposomes, however, the said vesicles or liposomes do not comprise non-ionic surfactants and there is no description of transfection efficiency or any indication of the stability of the system.
  • non-ionic surfactants can be used to form vesicles with potential therapeutic applications such as drug delivery (Ozer et al., 1991) and immunological adjuvants (Brewer and Alexander, 1993) .
  • drug delivery Opt et al., 1991
  • immunological adjuvants Bosset and Alexander, 1993
  • the present invention resides in the finding that improved transfection efficacy and/or stability of expression can be achieved by providing the polynucleotide carried by, non-ionic surfactant vesicles such as entrapped within and/or associated with the non- ionic surfactant vesicle walls.
  • the polynucleotide may also be further located in the aqueous carrier vehicle.
  • the present invention provides a formulation which comprises :- an aqueous vehicle; vesicles comprising a non-ionic surfactant suspended in the aqueous vehicle, and polynucleotide fragments carried by said vesicles.
  • DNA carried by vesicles comprising non-ionic surfactants demonstrate better transfection efficiencies and/or greater stability of expression/integration than vesicles hitherto described in the art.
  • formulations of the present invention attain stable transfection efficiencies in vitro of between 7.5% to 96%, preferably between 20% to 90%.
  • Transfection efficiency may be taken to mean a percentage of cells in a given population which are transformed by a polynucleotide of interest.
  • formulation of the present invention may be used to transfect cells in vitro or in vivo.
  • formulations of the present invention for use in the preparation of medicaments for use in therapy.
  • the polynucleotide fragments carried by said vesicles may be entrapped within the vesicle aqueous phase and/or associated with the surfactant bi-layer(s) of the vesicle walls.
  • the polynucleotide fragment may be located within the vesicle bi-layer(s) , in the inter-lamellar space (s), and/or it may be located on the surface of the vesicle bi-layers.
  • the polynucleotide fragments may also be found in the aqueous vehicle.
  • the concentration of polynucleotide in the aqueous vehicle may be the same, greater or lower than the concentration thereof carried by the vesicles.
  • the aqueous vehicle containing the polynucleotide fragments will generally be that which is used to load the polynucleotide fragments into and/or onto the vesicles, where such a method is used to introduce the polynucleotide fragments into and/or onto the vesicles.
  • the formulation containing the vesicles may be formed in any manner known in the art and appropriate to the polynucleotide fragments to be delivered.
  • vesicle formulations can be formed using either a "homogenisation” method or a “freeze-dried” method, both methods being known in the art.
  • a required quantity of vesicle components in a desired molar ratio can be processed in one of the following ways: Dry powdered vesicle components are hydrated with a solution of the polynucleotide for entrapment at a temperature which does not result in the denaturation of the polynucleotide fragments, for example, in the range from 0 up to 70°C and homogenised at the required speed and for the required length of time to produce the desired vesicle characteristics.
  • lipid material can be melted by the application of heat (e.g. temperature range 40-150°C) prior to hydration with the required solution at the necessary temperature.
  • the suspension can then be homogenised at the required speed to produce vesicles having the desired characteristics.
  • Polynucleotide-containing vesicle suspensions can be produced using the homogenisation method outlined above by heating the vesicle constituents, for example, at 135°C. The molten lipid can then be cooled to, for example 70°C prior to hydration with preheated polynucleotide solution. Vesicle size reduction is achieved by mechanical homogenisation of the sample at a specific temperature. For example homogenisation at 8000 rpm for 15 minutes on a Silverson mixer result in vesicles suspensions with a mean diameter of 1030 ⁇ 24.8 nm when hydrated with a solution containing 40 ⁇ g/ml of a hygro ycin plasmid construct.
  • a freeze dried preparation can be made in one of the following ways:
  • the required quantity of vesicle constituents in a desired molar ratio can be dissolved in an organic solvent (e.g. t-butyl alcohol) .
  • This solution can then be frozen and freeze-dried for the time required for complete removal of organic solvent.
  • the resultant lyophilised product can then be hydrated with a solution of the polynucleotide and agitated/homogenised at the required temperature to produce a vesicle suspension.
  • vesicle suspensions are produced by the homogenisation process described above, and then lyophilised to remove the aqueous solvent.
  • the resultant lyophilised product can then be hydrated with the required polynucleotide solution and agitated at the required temperature to produce a vesicle suspension.
  • a vesicle preparation of the present invention may be obtained from a so-called "whole preparation" which comprises vesicles which remain in suspension, together with vesicles which have become associated with the surface of the receptacle; vesicles which have become associated with the surface of the receptacle may be removed by washing the surface of the receptacle and are thus termed the "flask wash" preparation; or vesicles which only remain in suspension.
  • the vesicles are preferably formed of a sterol such as cholesterol or ergosterol, together with a non-ionic surfactant. It is generally necessary to include a charged amphophile such as a fatty acid within the vesicle formulation in order to prevent vesicle aggregation. Suitable charged amphophiles include dicetylphosphate, stearic acid and palmitic acid.
  • a non-ionic surfactant may be a mono, di-, tri-, or poly (up to 10) glycerol mono- or di-fatty acid ester (e.g. a C 10 - C 20 fatty acid ester) such as triglycerol monostearate; or a polyoxyethylene ether preferably comprising from 1 to 10 oxyethylene moieties with a C 10 - C 20 normal or branched alkyl chain.
  • Surfactant V triglycerol monostearate
  • Surfactant VI hexaglycerol distearate
  • Surfactant VII diethylene glycol mono n-hexadecylether
  • Surfactant VIII tetraethylene glycol mono n-hexadecylether
  • a formulation comprising; i) an aqueous vehicle ii) vesicles comprising non-ionic surfactants suspended in the aqueous vehicle, wherein the non-ionic surfactants are selected from the group consisting of mono-, di-, tri-, or poly (up to 10) glycerol mono- or di-fatty acid ester, or a polyoxyethylene ether comprising from 1 to 10 oxyethylene moieties with a C I0 -C 20 normal or branched alkyl chain; and iii) polynucleotide fragment (s) carried by said vesicles.
  • the non-ionic surfactant is a fatty acid ester as defined above
  • the fatty acid ester conforms to general formula (I) :
  • R is independently selected from H or C 10 -C 20 alkyl carbonyl and n is 1 to 10.
  • the non-ionic surfactant is a polyoxyethylene ether as defined above
  • the polyoxyethylene ether to general formula (II) is a polyoxyethylene ether as defined above
  • Vesicle formulations of the invention may comprise non-ionic surfactant, cholesterol and dicetyl phosphate, or a fatty acid (for example, stearic or palmitic acid) , and these are advantageously preferably present in molar ratios of 1-5:1-5:0-3 respectively.
  • the mean vesicle diameter determined as described herein has now been found to be in the range of from 100 to 7000 nm and may therefore be considerably larger than 1000 nm.
  • the vesicle mean diameter when applied in vivo lies in the range of from 200 to 1000 nm and more preferably from 300 to 600 nm.
  • the polynucleotide fragments may in principle be any polynucleotide fragments which may be effectively delivered in a vesicle suspension.
  • examples, of polynucleotide fragments include any naturally occurring or synthetic DNA molecules, cDNA molecules, and RNA molecules as well as mixtures thereof.
  • the polynucleotide fragments can be selected from so-called sense, anti-sense and/or ambisense polynucleotide fragments or mixtures thereof.
  • the polynucleotide fragments may be of any length from a few nucleotides up to about 10's of kilobases or base pair (b or bp) lengths.
  • the length of the polynucleotide fragments will be from about 12 nucleotides in length up to about 30000 b or bp.
  • the length of the polynucleotide fragments will be in the range of from 400 b or bp up to about 30000 b or bp, more preferably still from about 400 b or bp up to about 6000 b or bp.
  • Hydrophilic active agents will generally be soluble in the aqueous solution and entrapped (with) in intra- bilayer spaces, whereas those of a lipophilic nature will generally be present in the vesicular bilayer.
  • the concentration of polynucleotide fragments in the vesicle phase is generally from 0.01 to 10% wt/wt.
  • the formulation is generally prepared by forming a mixture of the vesicle components- usually by melting these together and allowing to cool.
  • an aqueous liquid containing the polynucleotide fragments may be added to the melted vesicle formulation (e.g. at a temperature upto 70°C) followed by vigorous agitation.
  • the formulation may be used as produced, or the concentration of polynucleotide fragments in the aqueous phase may be varied as required.
  • Unentrapped/unassociated polynucleotides may be removed from the vesicle formulation by column filtration techniques known in the art. However, this may lead to dilution of the vesicles and a possibility of contamination of the vesicle preparation.
  • the vesicle formulation may be centrifuged in order to pellet the vesicles whilst not substantially pelleting unentrapped/unassociated polynucleotide fragments, or non- ionic surfactant material which has not been suitably incorporated into the vesicles. The pellet comprising the vesicles and associated polynucleotide fragments may then be resuspended and used for transfection.
  • the formulation may further be prepared by including a freeze/thaw procedure prior to centrifugation of the vesicle formulation.
  • the vesicle formulation may be frozen at -20°C to -90°C for 18-24 hr and then allowed to thaw slowly at room temperature for 18-24 hr.
  • a rapid freezing process such as in liquid nitrogen, may be employed. The inclusion of such a freeze/thaw procedure has been found to increase the frequency of successful transfections.
  • formulations of the present invention when used in vivo may include those adapted for oral, rectal, nasal, vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • Formulations can also be used for ex vivo cellular transfection, for example haemopoietic stem cells, lymphocytes, neoplastic cells, fibroblasts, keratinocytes, for reinfusion and/or reinplantation following transfection.
  • Surfactant V triglycerol monostearate
  • Surfactant VI hexaglycerol distearate
  • Surfactant VII diethylene glycol mono n- hexadecylether
  • Surfactant VIII tetraethylene glycol mono n-hexadecylether
  • the cell line KGla (ECACC no: 88113006, human leukaemia cell line) was transfected with non-ionic surfactant vesicles containing the luciferase construct pG12-basic vector (about 5.4 kb) (Promega) to test for transient gene expression or a hygromycin construct (4.45 kb) (linear and circular plasmids, prepared inhouse by The Institute of Cell, Animal and Population Biology, Edinburgh University, using the pSP72 vector [Promega] with a pPGK-Hyg insert, to test for transient and stable integration.
  • Cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal calf serum (Sigma) , or medium containing serum and 500 ⁇ g/ml hygromycin B (Boeringher-Mannheim) for selection. Cells were also treated with non-ionic surfactant vesicles containing fluoroscein diacetate (FDA, Sigma) , a fluorescent stain, to confirm that non-ionic surfactant vesicles delivered a compound intracellularly. In some cases cells were grown in agar (base layer 0.6%, top layer containing cells 0.3%), prepared using cell culture medium containing hygromycin.
  • FDA fluoroscein diacetate
  • NAV Non-Ionic Surfactant Vesicle
  • a vesicular melt was prepared by mixing surfactant, cholesterol and dicetyl phosphate (750 ⁇ moles) in a 5:4:1 molar ratio, and heating the mixture at 130°C for 5 minutes.
  • the melt was allowed to cool to 70°C prior to hydration with 5 ml of the appropriate solution (PBS for empty non-ionic surfactant vesicles or the appropriate concentration, ⁇ g/ml, of a gene construct or calf thy us DNA in PBS) at the same temperature.
  • the hydrated lipid mixture was subjected to mechanical agitation using a Silverson mixer set at 8000 rpm for 15 minutes using a temperature of 70°C.
  • Unentrapped/unassociated DNA was removed from vesicle suspensions by gel filtration using an 18 x 2.6cm Sephadex G50 column with PBS as the eluant.
  • the cholesterol content of filtered non-ionic surfactant vesicle preparations was determined using a cholesterol assay kit (Sigma) to determine the amount the initial suspensions were diluted by filtration. In some cases dicetyl phosphate was replaced with palmitic or stearic acid. Cells for transfection were either treated with non- ionic surfactant vesicle suspensions as prepared or with non-ionic surfactant vesicle suspensions which had been gel filtered.
  • Example 2 Determination of Non-Ionic Surfactant Vesicle DNA Content
  • 0.1ml of a columned vesicle suspension was diluted 1:1 with propanol to release the vesicular contents.
  • the amount of DNA present in the resulting solution was determined using the method of Daxhelet et al . (1989, Analytical Biochemistry 179: 401-403) and the Hoechst 33258 DNA- specific fluorescent dye. Alternatively, 14 C labelled DNA was used to determine DNA entrapment efficiency.
  • Cells at the required concentration (2 x 10 5 - 2 x 10 6 /ml) , were incubated in 24 well tissue culture plates or 25 cm 2 culture flasks, with the appropriate suspensions, with or without gel filtration, of "empty" PBS loaded non- ionic surfactant vesicle or DNA loaded non-ionic surfactant vesicle, or a solution containing the equivalent amount of the relevant DNA construct. Cells were incubated at 37 °C in a humidified incubator (5% C0 2 ) for various time periods (3 hours - 48 hours) , harvested and washed twice with PBS and then incubated for 48 hours in fresh cell medium.
  • Cells were then harvested and resuspended at the required concentration for cloning or for colony growth under selection in the presence of hygromycin if cells had been transfected with the hygromycin gene construct. Hygromycin selection was continued for up to 5 days then the cells were harvested, washed and resuspended in fresh medium. In some cases, cells were exposed to a second round of hygromycin selection. If cells had been transfected with the luciferase plasmid then the luciferase activity of the cell population was assessed at different time points post- transfection using a standard assay kit (luciferase assay system, Promega) . Cell number and cell viability was monitored throughout experiments.
  • luciferase assay system Promega
  • lysis buffer lOmM Tris pH 7.4, lOmM EDTA, 150mM NaCl and 0.4% SDS
  • proteinase K lmg/ml
  • non-ionic surfactant vesicle treatment was assessed by flow cytometry (FACscan, Becton Dickinson) using the vital stain propidium iodide (Sigma) , or the characteristics of the cell population were compared with untreated controls.
  • Non-ionic surfactant vesicle suspensions contained vesicles with mean diameters in the range 400-900 nm.
  • Non-ionic surfactant vesicles (5:4:1 molar ratio surfactant: cholesterol: dicetyl phosphate 150 ⁇ moles/ml) , hydrated with 40 ⁇ g calf thymus DNA/ml gave 40% entrapment and hydrated with lO ⁇ g DNA/ml gave 20% entrapment.
  • Non-ionic vesicles (5:4:1 molar ratio of Surfactant VII: cholesterol: dicetyl phosphate, 150 ⁇ moles/ml) , hydrated with 40 ⁇ g spleen cell 14 C labelled DNA/ml, had an entrapment efficiency of 4.7%.
  • the detection limits of the assay method meant that it was impossible to determine the entrapment efficiency for a similar suspension hydrated with 4 ⁇ g spleen cell 14 C labelled DNA/ml. 3.
  • Flow cytometry results showed that one hour incubation with gel filtered, FDA loaded non-ionic surfactant vesicles gave incorporation of the dye into 100% of KGla cells in the test suspension.
  • Example 2 Effect of Treatment of KGla Cells with Non- Ionic Surfactant Vesicles loaded with Circular Hygromycin Plasmid on Transfection Efficiency
  • the cells were washed as before, resuspended in culture medium (1 x 10 5 cells/ml) supplemented with 0.3% agar and 500 ⁇ g/ml hygromycin and plated over medium containing 0.6% agar in Petri dishes. Untransfected control cultures were also set up with/without hygromycin. After 5 days the number of colonies present was determined. Results are shown in Table 2.
  • Example 3 Effect of Treatment of KGla Cells with Non- Ionic Surfactant Vesicles Loaded with Linear Hygromycin Plasmid on Transfection Efficiency.
  • 2 x 10 6 KGla cells were incubated with 2 ml gel filtered NIV (5:4:1 molar ratio of Surfactant VII: cholesterol: dicetyl phosphate, 150 ⁇ moles/ml) , containing a linear hygromycin plasmid, in 25 cm 2 tissue culture flasks containing 8 ml medium. After 24 hours the cells were harvested, washed twice in PBS, resuspended in the 10 ml fresh culture medium and cultured for a further 48 hours. The cells were then washed as before, and plated out at 1 cell/well in 96 well round-bottomed micro- titre plates containing 200 ⁇ l culture medium supplemented with 500 ⁇ g/ml hygromycin for selection.
  • Rl and R2 are defined on the basis of the forward and side scatter distribution characteristics of the cell population. The distribution of cells between Rl and R2 was essentially the same for treated and untreated cells. Green fluorescence is obtained in untreated cells due to the intrinsic enzyme activity and was deducted from the results of treated cells. Empty NIV formulations (i.e. PBS loaded) gave results similar to those of untreated controls i.e. treatment did not cause an increase in the % positive cells or the peak fluorescence obtained (data not shown) .
  • Example 5 Modified Production of Non-ionic Surfactant Vesicles (see flow chart 1, Figure 1)
  • the mixture of components in the stoppered vessel was heated at 135°C - 140°C for 20 -30 minutes in a hot air melt oven.
  • the hydration solution usually 5ml PBS alone or containing an appropriate concentration of DNA of interest was heated to 70°C in a waterbath for 15 - 20 minutes.
  • the hydrated material was then loaded into a bench top shaker (Stuart Scientific) , such that the vessel was immersed in a waterbath at 70°C.
  • the shaker was run at 800rpm for 2 hours, after which time the shaker and waterbath were switched off and the flasks left immersed in the waterbath to cool for 18 -24 hours, after which the contents of the flask was recovered.
  • each preparation consisted of two separate recoverable fractions - the whole preparation (WP) and the flask wash (FW) .
  • the whole preparation this was recovered by pipetting as much material from the flask as possible.
  • the flask wash this was material closely associated with the inner surface of the flask following removal of the whole preparation.
  • Plasmid DNA (pEGFP) was labelled with 32 P-dCTP (Amersham) using a High Prime labelling kit (Boehringer) according to the manufacturers instructions.Unincorporated 32 P-dCTP was removed by centrifugation through a Sephadex G10 column (prepared in house) .
  • Experiment 1 DNA labelled and vesicles prepared according to Example 1 were subsequently centrifuged and assayed, lml of preparation was diluted with 5ml PBS and centrifuged as described above ie 10, OOOg x 30 minutes, pellet and supernatant were collected and samples taken for analysis. See Table 5 for results.
  • Experiment 2 DNA labelled and vesicles prepared according to Example 1, including freeze/thaw step and centrifugation. See Table 6 and 7 for results.
  • Example 7 FACS analysis of 4 NISV preparations for size distribution
  • freeze/thaw appears to increase the number of vesicles in Rl and R2 (smaller) for both whole preparation and flask wash as compared to that in untreated samples, when cooled quickly, but causes little change in distribution when cooled slowly. Effect of free/thaw on supernatant
  • Freeze/thaw and slow cooling therefore do not appear to alter the size distribution of NISV, but do result in the production of a pellet containing a higher proportion of large vesicles on centrifugation, whilst leaving distribution in the supernatant little changed.
  • Example 8 Comparison of the effects of vesicle fractions on viability of K562 cells in vitro
  • K562 cells were cultured in 24 well tissue culture plates as described previously, with the exception that cells were not washed after 3 hours.
  • table 9 separation of preparations into pellet and supernatant following freeze/thaw reduces toxicity of the whole preparation considerably - whilst flask wash preparations are generally non-toxic even before the freeze/thaw and centrifugation steps.
  • Example 9 In vivo delivery of pEGFP to cells of haematopoietic origin using DNA loaded NISV
  • mice Groups of 3-5 mice were injected either ip or iv with 0.5ml washed pEGFP loaded vesicles or empty vesicles. After 18 hours mice were killed and haematopoietic tissues removed. After preparation of single cell suspensions and washing, samples were taken for the assessment of fluoresence using a FACScan (Becton Dickinson) .
  • Cells were transfected as described previously, using pEGFP loaded NISV which were not harvested as whole preparation or flask wash fractions nor freeze/thawed, but were separated by centrifugation to give a pellet and supernatant.
  • Figure 3 demonstrates the level of transfection achieved following incubation of cell lines with pEGFP loaded vesicles which were separated by centrifugation following a freeze/thaw step, the expression of GFP is increased possibly due to the reduced toxicity observed following inclusion of a freeze/thaw step.

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Abstract

L'invention concerne une formulation aqueuse (particulièrement sous forme injectable) contenant un polynucléotide et un constituant-entraîneur suspendus dans un véhicule aqueux. Cette formulation, bien que non exclusivement, contient de l'ADN ou de l'ARN combiné à des vésicules composées de tensioactifs non ioniques.
PCT/GB1997/003235 1996-11-26 1997-11-26 Vesicules de tensioactifs non ioniques en tant qu'agent de transport d'acide nucleique WO1998023260A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP52442098A JP2001510459A (ja) 1996-11-26 1997-11-26 核酸の放出剤としての非イオン性界面活性剤小胞
EP97945945A EP0946148A2 (fr) 1996-11-26 1997-11-26 Vesicules de tensioactifs non ioniques en tant qu'agent de transport d'acide nucleique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9624536.0 1996-11-26
GBGB9624536.0A GB9624536D0 (en) 1996-11-26 1996-11-26 Non-ionic surfactant vesicles as a delivery agent for nucleic acid

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WO1998023260A2 true WO1998023260A2 (fr) 1998-06-04
WO1998023260A3 WO1998023260A3 (fr) 1998-08-27

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2802422A1 (fr) * 1999-12-21 2001-06-22 Capsulis Structures mixtes resultant de l'incorporation d'une macromolecule biologique, en particulier d'adn, dans une phase structuree d'amphiphiles et vesicules obtenues a partir de ces structures
US20090324743A1 (en) * 2008-06-27 2009-12-31 University Of Strathclyde Pulmonary drug delivery

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995016437A1 (fr) * 1993-12-17 1995-06-22 Micro-Pak, Inc. Procede de transmission, a une cellule, d'une substance a activite biologique
WO1996004890A1 (fr) * 1994-08-10 1996-02-22 University Of Strathclyde Formulation renfermant des vesicules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995016437A1 (fr) * 1993-12-17 1995-06-22 Micro-Pak, Inc. Procede de transmission, a une cellule, d'une substance a activite biologique
WO1996004890A1 (fr) * 1994-08-10 1996-02-22 University Of Strathclyde Formulation renfermant des vesicules

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2802422A1 (fr) * 1999-12-21 2001-06-22 Capsulis Structures mixtes resultant de l'incorporation d'une macromolecule biologique, en particulier d'adn, dans une phase structuree d'amphiphiles et vesicules obtenues a partir de ces structures
WO2001045672A2 (fr) * 1999-12-21 2001-06-28 Capsulis Structures mixtes resultant de l'incorporation d'une macromolecule biologique dans une phase cristal-liquide d'amphiphiles
WO2001045672A3 (fr) * 1999-12-21 2001-12-27 Capsulis Structures mixtes resultant de l'incorporation d'une macromolecule biologique dans une phase cristal-liquide d'amphiphiles
US7208173B2 (en) 1999-12-21 2007-04-24 Capsulis Mixed structures resulting from the incorporation of a biological macromolecule, especially of DNA, in a liquid crystal phase of amphiphiles, and vesicles obtained using these structures
US20090324743A1 (en) * 2008-06-27 2009-12-31 University Of Strathclyde Pulmonary drug delivery

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GB9624536D0 (en) 1997-01-15
WO1998023260A3 (fr) 1998-08-27
EP0946148A2 (fr) 1999-10-06
JP2001510459A (ja) 2001-07-31

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