ARBUTUS - EXHIBIT 2009
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation
`IPR2019-00554
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`U.S. Patent
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`Feb. 17, 2009
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`Sheet 1 of 23
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`Sheet 2 of 23
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`US 7,491,409 B1
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`1
`ENCAPSULATION OF BIOACTIVE
`COMPLEXES IN LIPOSOMES
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`FIELD OF THE INVENTION
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`This invention is directed to a methodfor the encapsulation
`of complexes, such as polycation-condensed nucleic acids, in
`liposomes, using an emulsion stabilized by amphipathic lip-
`ids as an intermediary within which the complex forms. This
`inventionis also directed to the liposome encapsulated com-
`plexes so formed. The methodof this invention is applicable
`for providing liposomes loaded with a variety of compounds
`which heretofore have been difficult to load into liposomesat
`high compoundto lipid ratios.
`
`BACKGROUND OF THE INVENTION
`
`In order to be useful as pharmaceutical preparations, bio-
`active agents must be able to reach the therapeutic site in an
`adequate therapeutic effective amount. While many bioactive
`agents and drugsarestable in vivo, others are often degraded.
`When such degradation occurs prior to the drug or bioactive
`agent reaching its target site, a non-therapeutic amount of
`drug will reach thetarget site. Other drugsor bioactive agents
`are taken up by non-target systems, once again resulting in the
`lack of a therapeutic amount of a drug or bioactive agent
`reaching the targetsite at therapeutically effective amounts.
`Certain polar drugs can not enter cells at all because of their
`inability to cross the target cell membrane. The only waythat
`these polar drugs may enter a cell is by uptake by the process
`of endocytosis, exposing them to degradative lysosomal
`enzymes in the cell. Yet another problem in the therapeutic
`delivery of drugsor bioactive agents is the inability to admin-
`ister a high enough concentration of the drug or bioactive
`agent to be therapeutic, while avoiding toxicities often asso-
`ciated with some drugsor bioactive agents. These problems
`have been approached by a number of different methods.
`When a drug or bioactive agent has no toxicity associated
`with it,
`it may be administered in high enough doses to
`account for degradation, removal by non-target organs and
`lack of targeting to the site where the therapeutic drug or
`bioactive agent is required. However, many drugsor bioactive
`agents are either too expensive to allow such waste or have
`toxicities that prevent administration of such high dosages.
`Numerous methods have been used to overcome some of the
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`problems encountered in administering therapeutic amounts
`of drugs or bioactive agents.
`One such methodis the encapsulation of drugsor bioactive
`agents in liposomes. While somedrugs or bioactive agents
`can be encapsulated in liposomesat therapeutically effective
`doses by passive loading or by gradient loading, these meth-
`ods are limited either to drugs or bioactive agents with spe-
`cific chemical properties or to drugs or bioactive agents that
`can be administered in relatively low concentrations. Some
`bioactive compounds such as weak bases or weak acids can
`be loaded remotely into preformed liposomes to form highly
`concentrated complexes. This type of loading, referred to as
`remote or gradient loading, requires that the drug or bioactive
`agent be temporarily able to pass through the lipid bilayer of
`the liposome. However, this is not the case for all bioactive
`molecules, many of which cannotpass through the liposomal
`bilayer.
`Onearea in which attempts to administer therapeutic levels
`of drugs or bioactive agents have been only partially success-
`ful is the area of gene therapy. Gene therapy involves the
`introduction of an exogenous gene into an appropriate cell
`type, followed by enablementofthe gene’s expression within
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`the cell at therapeutically relevant levels. Such therapy has
`progressed, in a relatively short period of time, from basic
`research to the introduction into cells of a variety of genes,
`including those useful for treating cancers (Duqueetal.,
`Histol Histopathol, 13: 231-242 (1998); Runnebaumetal.,
`Anticancer Res., 17: 2887-2890 (1997)). While naked DNA,
`in some cases has been taken up into cells (Wolff et al.,
`Science, 247: 1465-1468 (1990)), it generally cannot be, due
`to its large size and high degree ofnegative charge; moreover,
`naked DNA cannotbe designedso asto be targeted to specific
`cells. Accordingly, successful gene therapy generally is reli-
`ant upon the availability of “vectors” for introducing DNA
`and other nucleic acids into cells.
`
`Presently, there are two major groups of DNA delivery
`systems, viral and non-viral. Viral vectors, including replica-
`tion-deficient viruses, such as retroviruses, adenoviruses, and
`adeno-associated viruses, have thus far been the most widely
`described gene delivery vehicles (Robbins et al., Trends in
`Biotech, 16: 3540 (1998)). However, their use has been ham-
`pered by the immunogenicity of their viral components,
`potential risk of reversion to a replication-competentstate,
`potential introduction of tumorgenic mutations, lack of tar-
`geting mechanisms, limitations in DNA capacity, difficulty in
`large scale production and other factors (see, e.g., Lee and
`Huang, J Biol Chem, 271: 8481-8487 (1996)).
`Two majortypes of nonviral vehicles have been developed
`as alternatives to viral vectors. Cationic liposome-DNA com-
`plexes (or “lipoplexes,” Feigner et al., Proc Natl Acad Sci
`USA, 84: 7413-7417 (1987)), consisting of cationic lipids and
`DNAhavethusfar been the most widely described alternative
`to viral vectors for gene delivery. However, such lipoplexes
`suffer from several major drawbacks when used in gene
`therapy, including low stability, high cytotoxicity, non-biode-
`gradability, poor condensation and protection of DNA, serum
`sensitivity, large size and lack oftissue specificity. Moreover,
`as the lipoplexesare positively charged, they generally inter-
`act nonspecifically with the negatively charged surfaces of
`mostcells; accordingly, it is generally not possible to target
`such lipoplexes to specific sites in vivo.
`Anothervariation of lipoplexes and DNA involves polyl-
`ysine-condensed DNA boundto anionic liposomes (Lee and
`Huang, J Biol Chem, 271: 8481-8487 (1996)). These require
`certain anionic lipids to form the active structure. The
`lipoplexes formed either do not completely encapsulate the
`DNA or must form two or more bilayers around the con-
`densed DNA. In the latter case delivery to the cytoplasm
`would require the DNA to cross at least three membranes.
`This would be expected to inhibit transfection efficiency. In
`the formercase, stability may be compromised by exposure
`of the DNAin physiological salt solutions.
`Liposomesare an additional type of nonviral vectoralter-
`native, and offer several advantages for such use in compari-
`son to the lipoplexes. For example, liposomal bilayers form
`around encapsulated nucleic acids, thereby protecting the
`nucleic acids from degradation by environmental nucleases;
`lipoplexes, by contrast, do not encapsulate nucleic acids, and
`hence, cannot completely sequester them away from environ-
`mental nucleases. Moreover, liposomes can encapsulate, in
`their aqueous compartments, other bioactive agents in addi-
`tion to nucleic acids; lipoplexes, by contrast, cannot because
`they do not encapsulate aqueous volume. Furthermore, lipo-
`somes can be made to be neutrally charged or anionic, as
`opposedto the restricted ionic nature of the aforementioned
`lipoplexes. Thus, liposomes can be designed so as to avoid
`cytotoxicities induced by the delivery vehicle itself and to
`enhancetheir accumulation at specific sites of interest.
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`US 7,491,409 B1
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`3
`While the concept of encapsulating bioactive agents in
`liposomes is not new, many agents have been difficult to
`encapsulate in liposomesat any level and others have proven
`difficult to encapsulate in liposomesat levels that would be
`therapeutically effective. Many small molecules can be
`encapsulated in liposomes but leak out. Thus,it has also been
`difficult to encapsulate some bioactive agents and have them
`retained within the liposomesat a therapeutically effective
`dose for a therapeutically effective time. For instance, it has
`been difficult to encapsulate particularly large molecules into
`a complex within a liposome. It has also been difficult to use
`many water soluble molecules as therapeutic agents because
`they are unable to penetrate the cell membrane. When encap-
`sulated stably into liposomesthat can fuse to cell membranes,
`it is possible to deliver these drugs at therapeutically effective
`doses into the target cells. The method ofthe present inven-
`tion enables formation of liposomescontaining such drugs or
`bioactive agents in a therapeutically useful form.
`Several attempts have been made to encapsulate nucleic
`acids in liposomes, these including use of the reverse-phase
`evaporation (Fraley et al., J Biol Chem, 255: 10431-10435
`(1980)), dehydration-rehydration (Alizo et al., JMicroencap,
`7: 497-503 (1990)) and freeze-thaw (Monnard et al., Biochem
`Biophys Acta, 1329: 39-50 (1997)) methodsof liposomefor-
`mation. However, each of these methods has several limita-
`tions, including requirements for low starting concentrations
`of nucleic acid, resulting in significant percentages of empty
`vesicles in the product liposomes, inability to reproducibly
`encapsulate sufficient quantity of DNA in liposomes to be
`therapeutically effective at the desiredtarget site and difficul-
`ties in optimizing the vehicles for protection of their encap-
`sulated nucleic acids from nuclease-mediated degradation.
`Attempts have also been made to complex DNA with com-
`plexing agents and subsequently encapsulate the complexed
`DNA in liposomes. Complexing agents are agents that react
`with other molecules causing the precipitation or condensa-
`tion of the molecules. Complexing agents useful in the prac-
`tice of the present invention are selected from the group
`consisting of charged molecules that have a charge opposite
`to the charge on the bioactive agent. The complexing agent
`may beselected from the group of charged molecules con-
`sisting of spermine, spermidine, hexammine cobalt, calctum
`ions, magnesium ions, polylysines, polyhistidines, prota-
`mines, polyanions such as heparin and dextran sulfate, citrate
`ions, or sulfate ions. For instance, polycations of charge +3 or
`higher, e.g., polyamines, polylysine and hexammine cobalt
`(IIT) are known(see Chattoraj et al., JMo/ Biol, 121: 327-337
`(1978); Gosule L C and Schellman J A. Nature 259: 333-335
`(1976); Vitello et al., Gene Therapy, 3: 396-404 (1996);
`Widom et al. J. Mol. Biol., 144: 431-453 (1980); Arscott et al.,
`Biopolymers, 30: 619-630 (1990); Wilsonet al., Biochem, 18:
`2192-2196 (1979)) to be able to condense DNA molecules,
`through interaction with multiple negative charges on the
`DNA.Polyamines, e.g., spermidine (3+) and spermine(4+),
`have, unlike other types of polycations, been found to occur
`naturally in all living cells (see, e.g., Ames and Dubin, J Biol
`Chem, 253: 769-775 (1960); Tabor and Tabor, Annu Rev
`Biochem, 53: 749-790 (1984)). High polyamine levels are
`knownto exist in actively proliferating animal cells, and are
`believed to be essential therein for maintaining normalcell
`growth (Amesand Dubin, JBiol Chem, 253: 769-775 (1960);
`Tabor and Tabor, Annu Rev Biochem, 53: 749-790 (1984);
`Hafneret al., J Biol Chem, 254: 12419-12426 (1979); Pegg,
`Biochem J, 234: 249-262 (1986)).
`Liposome encapsulation of spermine-condensed linear
`DNAin liposomeshas been attempted by Tikchonenkoetal.,
`Gene, 63: 321-330 (1988). However, the starting DNA con-
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`centration therein was low, with the consequence that the
`resulting liposomesalso had a low ratio of encapsulated DNA
`to liposomallipid (0.02-0.2 micrograms DNAper micromole
`lipid). Moreover, such condensation oflinear DNA molecules
`in the absence of intermolecular DNA aggregation required
`control over spermine concentrations to an impracticable
`degree of precision. Additionally, Baeza et al., Ori Life Evol
`Biosphere, 21: 225-252 (1992) and Ibanez et al., Biochem
`Cell Biol, 74: 633-643 (1996) both report encapsulation of
`1-4 microgramsper micromole of spermine-condensed SV40
`plasmid DNAin liposomes. However,neitherof their prepa-
`rations were dialyzed against high salt buffers subsequentto
`liposome formation, the reported amounts of encapsulated
`DNAactually may include a significant percentage of unen-
`capsulated DNA.Since these liposomal formulations were
`not exposed to DNAase degradation to determine the percent-
`age of DNA actually sequestered in the liposomes, the high
`reported amounts probably do notreflect actually encapsu-
`lated DNA.
`
`Efficient preparation and use of liposomal encapsulated
`nucleic acids requires the use of high-concentration suspen-
`sions of nucleic acids, in order to minimize the percentage of
`empty liposomesresulting from the process and to maximize
`the DNA:liposomallipid ratios. However, condensation of
`DNAat high concentrations during known methodsoflipo-
`some formation generally results in intermolecular aggrega-
`tion, leading to the formation ofnucleic acid-based structures
`unsuitable for gene delivery. Large aggregates formed by
`condensation of DNA directly with a complexing agent can-
`not be easily encapsulated in liposomeand such large aggre-
`gate structures (on the order of the size of cells) can not
`efficiently deliver materials to target cells. For instance, if the
`aggregates are larger than 500 nm,they are rapidly cleared
`from the circulation because of their size after intravenous
`administration. On the other hand, larger aggregates may be
`administered to cells in vitro. However, sometimesthe aggre-
`gates as too large too be taken up bycells.
`Thus, in order to deliver a variety of drugs in therapeuti-
`cally effective amounts into target cells, it was necessary to
`provide a method of making liposomesthat contain bioactive
`agents complexed so as to decrease their permeability
`through the lipid bilayer, while providing a methodthat also
`limits the size of the complex to be encapsulated in thelipo-
`some so that the resultant therapeutic product is in a thera-
`peutic size range.
`
`SUMMARY OF THE INVENTION
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`The present invention provides a method of encapsulating
`a bioactive complex in a liposome which comprises the steps
`of:
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`(a) dissolving at least one amphipathiclipid in one or more
`organic solvents
`(b) combiningat least one aqueous suspension comprising
`a solution containing a first molecule selected from the
`group consisting of a bioactive agent and a complexing
`agent with the lipid-containing organic solution of step
`(a) so as to form an emulsion in the form of a reverse
`micelle comprising the first molecule andthe lipid;
`(c) adding a second aqueoussuspension comprising a sec-
`ond molecule selected from the group consisting of a
`bioactive agent and a complexing agent wherein if the
`first molecule is a bioactive agent, the second molecule
`is a complexing agent andvice versa, to the emulsion of
`step (b),
`(d) incubating the emulsion of step (c) to allow the com-
`plexing agent to contact the bioactive agent thereby
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`US 7,491,409 B1
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`5
`forming a complex ofthe bioactive agent with the com-
`plexing agent within lipid stabilized water droplets;
`wherein said complex is no greater in diameter than the
`diameter of the droplet and,
`(e) removing the organic solvent from the suspension of
`step (d), so as to form liposomes comprising the com-
`plexed bioactive agent andthe lipid.
`The methodofthe present invention 1s usefulfor the prepa-
`ration of therapeutically useful liposomes containing a wide
`range of bioactive molecules complexed with complexing
`agent within the liposome. Preferably, the liposomesare fuso-
`genic liposomes which by the methodofthe present invention
`can encapsulate a variety of molecules. These fusogeniclipo-
`somes are able to fuse with cell membranes and enable the
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`delivery of bioactive agents in therapeutically effective
`amounts to cells and organs. In addition, the method of the
`present invention also allows morethan one bioactive agent to
`be encapsulated in a liposome. One or more bioactive agents
`may be encapsulated in the same liposomesat the same time
`by the method of the present invention. If more than one
`bioactive agent is encapsulated in a liposome by the method
`of the present invention, it is not necessary for each of the
`bioactive agents to be in the form of complexes.
`Somebioactive agents easily pass throughthelipid bilayer
`and therefore, are not stably sequestered in liposomes. By
`forming complexesofthe bioactive agents with a complexing
`agent, the bioactive agent remains in the liposomes. A major
`hurdle has been the problem of encapsulating complexed
`bioactive agents into liposomes. When the bioactive agent
`and complexing agent are mixed in solution prior to encap-
`sulation in liposomes, many complexes that are uncontrolla-
`bly large are formed at the concentrations necessary foreffi-
`cient loading of liposomes. The term bioactive complex is any
`bioactive agent bound to a complexing agent such that the
`complex thus formed results in a change in the physical
`properties such as decreasing the size of the bioactive mol-
`ecule, decreasing the solubility of the bioactive agent, pre-
`cipitating the bioactive agents, condensing the bioactive
`agent, or increasing the size of the complex. Liposomesthat
`fuse with cell membranesare able to deliver a vast category of
`molecules to the inside of cells. One advantage of the inven-
`tion is that, by forming the complexofthe bioactive agent in
`the reverse micelles, the formation of unsuitable large com-
`plexes incapable of being encapsulated in therapeutically
`useful liposomesis prevented.
`The formation of complexes comprising a bioactive com-
`pound within liposomes has the advantage that such com-
`plexes are less likely to leak out of the liposome before
`delivery to the desired target cell. Furthermore, the formation
`of a complex can concentrate a large amountof the bioactive
`agent within the liposome such that the ratio of bioactive
`agent-to-lipid is high and delivery is efficacious. The dis-
`closed method provides for complexation of bioactive mate-
`rials with complexing agents within an emulsion followed by
`encapsulation within a liposome in a manner that prevents the
`formation of extremely large, detrimental aggregates, greater
`than several microns, of the bioactive agent and complexing
`agent.
`In one embodiment, the method of the present invention
`has provided a method to encapsulate nucleic acid com-
`plexes. For instance, nucleic acids, such as DNA, are com-
`plexed with a condensing agent within reverse (inverted)
`micelles, followed by formation of liposomes from the
`micelles. While, as described above, previous attempts have
`been made to encapsulate DNA in liposomes, none of said
`methods were successfulat efficiently preparing therapeuti-
`cally useful liposomal DNA.
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`This invention provides a method to prepare a liposome
`comprising a condensed nucleic acid, in amountsofat least
`about 0.5 microgramsnucleic acid per micromole ofliposo-
`mallipid.
`The liposomes’ lipid component preferably comprises a
`derivatized phospholipid and an additionallipid, generally in
`proportions of about 20-80 mole % derivatized phospholipid
`to about 80-20 mole % additional lipid. Preferred derivatized
`phospholipids include: phosphatidylethanolamine (PE)-bi-
`otin conjugates; N-acylated phosphatidylethanolamines
`(NAPEs), such as N-C12 DOPE; and, peptide-phosphatidyle-
`thanolamine conjugates, such as Ala-Ala-Pro-Val DOPE. The
`additional lipid can be anyofthe variety of lipids commonly
`incorporated into liposomes; however, where the derivatized
`phospholipid is a NAPE,the additional lipid is preferably a
`phosphatidylcholine (e.g., DOPC). Preferably, the nucleic
`acid is DNA.
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`Also provided herein is a method to prepare a pharmaceu-
`tical composition comprising the liposome and a pharmaceu-
`tically acceptable carrier; said composition can be used to
`deliver the nucleic acid to the cells of an animal.
`
`Other and further objects, features and advantages will be
`apparent from the following description of the preferred
`embodiments of the invention given for the purpose ofdis-
`closure when taken in conjunction with the following draw-
`ings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1. Micrographs of spermine-mediated plasmid DNA
`aggregation (200 micrograms plasmid DNA in 125 microli-
`ters LSB was mixed gently with 7 mM spermine in 125
`microliters LSB). (A) Light microscope observation after 15
`minutes incubation at room temperature (bar represents 10
`microns). (B) Cryo TEM observation (bar represents 100
`nm).
`FIG. 2. Schematic representation of method of DNA
`encapsulation. Condensation of DNA occurs(I) within phos-
`pholipid-stabilized water droplets that have formed around
`the DNA in a bulk organic solvent. Separate spermine-con-
`taining droplets transfer (II) spermine into the DNA contain-
`ing droplets by transient (III) contact and exchange. After
`condensation within the emulsion (IV), vesicles are formed
`by solvent evaporation and further extruded to smaller sizes
`(V).
`FIG. 3. Effect of liposomal N-C12 DOPE on spermine-
`mediated aggregation of plasmid DNA.Equilibrium dialysis
`was performed in a three-chamber dialysis device (see
`Example 4). The curve on theleft is from dialyses without
`liposomes, while the curve shifted to the right is from dialyses
`that included a chamber with liposomes. X-axis: spermine
`concentration (mM); y-axis: turbidity (O.D. 400 nm).
`FIG.4. Agarose gel analysis of plasmid DNAprotection in
`N-C12 DOPE/DOPC(70:30) formulations—analiquot from
`each preparation after is extrusion and dialysis was divided,
`and one part digested with DNase I (see Example 9). Lane 1.
`Preparation without spermine. Lane 2. Sameas lane 1 but
`digested with DNase I. Lane 3. Preparation with spermine.
`Lane 4. Sameaslane 3, but digested with DNaseI.
`FIG. 5. Light micrographsofthe particles in N-C12 DOPE/
`DOPC (70:30) sample prepared as described in Example 3
`with pZeoLacZ plasmid and spermine (A) versus polystyrene
`beads with an average diameter of 269+7 nm (B) (bars rep-
`resent 10 nm).
`FIG.6. Freeze-fracture TEM micrographs (see Example 7)
`of N-C12 DOPE/DOPC(70:30) samples prepared with plas-
`
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`

`US 7,491,409 B1
`
`7
`midand spermine,as described in Example 3. Arrow points to
`the particle with apparently encapsulated material (bar rep-
`resents 400 nm).
`FIG. 7. Cryo TEM micrographs (see Example 8) of lipo-
`somes with N-C12 DOPE/DOPCand the pZeoLacZ plasmid
`without spermine (a), or with spermine(b), said liposomes
`being prepared as described in Example 3. In (a) fiber-like
`structures are seen outside (star) and apparently inside (ar-
`row) liposomes. In (b), an arrow points to a toroid that
`resembles polycation condensed plasmid DNA (bars in (a)
`and (b) represent 100 nm). Photomicrograph(c) represents an
`EPC sample made with spermine. Toroid (arrow) and bent rod
`(star) structures are compared with multilamellar liposomes
`(poundsign) [bar represents 50 nm].
`FIG. 8. Fluorescence photomicrographs of confluent
`OVCAR3cells after transfection (see Example 11) with the
`N-C12 DOPE/DOPC (70:30) preparations. Liposomal
`samples were prepared (see Example 3) with pEGFP-C1
`plasmid DNA (a) with spermineor (b) without spermine; a
`sample (c) of empty N-C12 DOPE/DOPC(70:30) liposomes
`without spermineplus free pEGFP-C1 plasmid DNA added
`outside the preformed liposomeswasalso tested. The amount
`of plasmid DNA addedto the empty liposomes in sample c
`was equal to the total amount in each of the other prepara-
`tions. Equal liposome concentrations were usedin the experi-
`ments.
`
`FIG. 9. Quantitation of EGFP expression in OVCAR 3
`cells transfected with pEGFP-C1, as measured by the EGFP
`fluorescence level. Transfection experiments(a, b and c, see
`Example 11) were the sameas in the previousfigure legend.
`In addition, formulations tested were: d) egg PC liposomes
`prepared with spermine and pEGFP-Cl plasmid (see
`Example 3); and, e) no additions. The cells were washed and
`labeled with CBAM,andthen dissolved in detergent to mea-
`sure the fluorescence of EGFPandcalcein blue (see Example
`10; error bars are +s.d).
`FIG. 10. Association of transfection activity with the lipid
`pellet of N-C12-DOPE/DOPC (70:30) prepared with sper-
`mine and pEGFP-C1 plasmid DNA (see Example 3); the
`initial plasmid DNA and spermine solutions contained 200
`mMsucrose. After extrusion anddialysis, half of the sample
`wasusedfor transfection withoutfurther handling(a), and the
`lipid particles from the rest of the sample were pelleted by
`centrifugation and washed once with HBSS before being
`used for transfection (b). An N-C12-DOPE/DOPC(70:30)
`sample with only the 200 mM sucrose wasalso prepared, and
`plasmid DNA and spermine were both added externally just
`before dialysis at an amount equal to that used in the other
`samples. The pellet of this empty sample (c) was prepared the
`same way, then, an equal lipid amount of each of the samples
`was used for transfection under the conditions described in
`the previous figure legends. After overnight incubation, the
`cells were labeled with CBAM andthefluorescence of EGFP
`and calcein blue were measured(error bars are +s.d).
`FIG. 11. Transfection via N-C12 DOPE/DOPC (70:30)
`liposomes in mouseascites fluid compared to buffer. Ascites
`wasobtained from the lavage of a tumor-bearing SCID mouse
`as described in Example 13. Cells were incubated with plas-
`mid DNA-containing liposomes(nota pellet) at a final con-
`centration 10 mMtotal lipid in HBSS or HBSS with ascites
`fluid, at a final protein concentration of approximately 3.5
`mg/ml (see Example 11). After 3 hr. of incubation,the trans-
`fection solution was replaced with serum- and butyrate-con-
`taining medium for approximately 20 hr. Expression of EGFP
`was measuredvia its fluorescence(error bars are +s.d).
`FIG. 12. Fluorescent photomicrographs of OVCAR-3cells
`transfected (see Example 11) with to N-C12 DOPE/DOPC
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`8
`(70:30) liposomes in buffer or mouse ascites fluid. Cells
`treated as described in the legend to FIG. 12 were photo-
`graphed. Photograph A represents transfection without peri-
`toneal ascites fluid and photograph B with peritoneal ascites
`fluid; cells are confluent in these views.
`FIG. 13. Fluorescent probe determination of liposome
`lamellarity.
`FIG. 14. Fluorescent photomicrographs of OVCAR-3
`tumortransfected in vivo with N-C12 DOPE/DOPC(70:30)
`liposomes containing pEGFP-C1. Panel A depicts the expres-
`sion of EGFP. Panel B depicts the red fluorescence from
`rhodamine—labeled liposomes.
`FIG. 15. Fluorescent photomicrographs of OVCAR-3
`tumortaken from a differentsite than FIG. 14 transfected in
`vivo with N-C12 DOPE/DOPC(70:30) liposomes containing
`pEGFP-C1. Panel A depicts the expression of EGFP. Panel B
`depicts the red fluorescence from rhodamine—labeled lipo-
`somes.
`
`FIG. 16. Fluorescent photomicrographs of control tumor
`tissue. Panel A depicts diffuse green fluorescence. Panel B
`depicts the lack ofred fluorescence from rhodamine—labeled
`liposomes
`FIG. 17. Graph depicting expression of (f-galactosidase
`activity in muscletissue after transfection in vivo.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Following are abbreviations, and the corresponding terms,
`used throughoutthis application: PE, phosphatidylethanola-
`mine; PC, phosphatidyicholine; EPC, egg phosphatidylcho-
`line; DO-, dioleoyl-; DOPC, dioleoyl phosphatidylcholine;
`DOPE, dioleoyl phosphatidylethanolamine; NAPE, N-acy-
`lated phosphatidylethanolamine; N-C12 DOPE, N-dode-
`canoyl dioleoyl phosphatidylethanolamine; AAPV-DOPE,
`Ala-Ala-Pro-Val-dioleoy]
`phosphatidylethanolamine;
`CBAM,calcein blue acetoxy methyl ester, PBS, phosphate
`buffered saline; LSB, low salt buffer; HBSS, Hank’s balanced
`salt solution; EGFP, enhanced green fluorescence protein;
`SPLYV, stable plurilamellar liposomes; MLVs, multilamellar
`liposomes; ULVs, unilamellar liposomes; LUVs, large unila-
`mellar liposomes; SUVs, small unilamellar liposomes; ds
`DNA, double stranded DNA; TEM,transmission electron
`microscopy.
`The present invention provides a method of encapsulating
`a bioactive complex in a liposome which comprises the steps
`of:
`
`(a) dissolving at least one amphipathiclipid in one or more
`organic solvents
`(b) combiningat least one aqueous suspension comprising
`a solution containing a first molecule selected from the
`group consisting of a bioactive agent and a complexing
`agent with the lipid-containing organic solution of step
`(a) so as to form an emulsion in the form of a reverse
`micelle comprising the first molecule andthe lipid;
`(c) adding a second aqueoussuspension comprising a sec-
`ond molecule selected from the group consisting of a
`bioactive agent and a complexing agent wherein if the
`first molecule is a bioactive agent, the second molecule
`is a complexing agent or vice versa, to the emulsion of
`step (b),
`(d) incubating the emulsion of step (c) to allow the com-
`plexing agent to contact the bioactive agent thereby
`forming a complex ofthe bioactive agent with the com-
`plexing agent within lipid stabilized water droplets;
`wherein said complex is no greater in diameter than the
`diameter of the droplet and,
`
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`US 7,491,409 B1
`
`9
`(e) removing the organic solvent from the suspension of
`step (d), so as to form liposomes comprising the com-
`plexed bioactive agent andthe lipid.
`The methodofthe present invention 1s usefulfor the prepa-
`ration of therapeutically useful liposomes containing a wide
`range of bioactive molecules complexed with complexing
`agent within the liposome. Preferably, the liposomesare fuso-
`genic liposomes which by the methodofthe present invention
`can encapsulate a variety of molecules. These fusogeniclipo-
`somes are able to fuse with cell membranes and enable the
`
`delivery of bioactive agents in therapeutically effective
`amounts to cells and organs. In addition, the method of the
`present invention also allows morethan one bioactive agent to
`be encapsulated in a liposome. One or more bioactive agents
`may be encapsulated in the same liposomesat the same time
`by the method of the present invention. If more than one
`bioactive agent is encapsu