MXPA01008804A - Encapsulation of bioactive complexes in liposomes - Google Patents
Encapsulation of bioactive complexes in liposomesInfo
- Publication number
- MXPA01008804A MXPA01008804A MXPA/A/2001/008804A MXPA01008804A MXPA01008804A MX PA01008804 A MXPA01008804 A MX PA01008804A MX PA01008804 A MXPA01008804 A MX PA01008804A MX PA01008804 A MXPA01008804 A MX PA01008804A
- Authority
- MX
- Mexico
- Prior art keywords
- liposomes
- dna
- lipid
- bioactive
- spermine
- Prior art date
Links
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Abstract
This invention provides a method to prepare liposome-encapsulated bioactive agents such as nucleic acids, comprising complexation of the bioactive agents in reverse micelles prior to forming liposomes, as well as methods of using the liposomes so formed and formulations to deliver nucleic acids to cells.
Description
ENCAPSULATION OF BIOACTIVE COMPOUNDS IN LIPOSOMES
FIELD OF THE INVENTION This invention is directed to a method for the encapsulation of complexes, such as nucleic acids condensed with polycation, in liposomes, using an emulsion stabilized by antipathetic lipids as intermediates within which the complex is formed. This invention is also directed to the complexes encapsulated in liposomes thus formed. The method of this invention is applicable to provide liposomes loaded with a variety of compounds that have hitherto been difficult to load into liposomes in high proportions of the compound to lipid.
BACKGROUND OF THE INVENTION In order to be useful as pharmaceutical preparations, bioactive agents must be able to reach the therapeutic site in an effective, adequate therapeutic amount. Although many of the bioactive agents and drugs are stable in vivo, others are degraded. When such degradation occurs before the drug or bioactive agent reaches its chosen site, a non-therapeutic amount of the drug will arrive at the chosen site. Other medications or bioactive agents are
captured by the non-chosen systems, once again giving rise to the lack of a therapeutic amount of a drug or bioactive agent that reaches the chosen site in effective therapeutic quantities. Some polar drugs can not enter cells due to their inability to cross the chosen cell membrane. The only way in which these polar drugs can enter a cell is by uptake through the process of endocytosis, exposing them to degrading lysosomal enzymes in the cell. Yet another problem in the therapeutic delivery of drugs or bioactive agents is the inability to administer a rather high concentration of the drug or the bioactive agent to be therapeutic, thereby avoiding toxicities often associated with some drugs or bioactive agents. These problems have been treated by different methods. When a drug or bioactive agent has no toxicity associated with it, it can be administered in very high doses taking into account degradation, elimination by non-elected organs and lack of direction to the site where the therapeutic drug or bioactive agent is required. However, many drugs or bioactive agents are too expensive to allow such waste or may have toxicities that prevent
administration at such high doses. Some methods have been used to solve some of the problems found in the administration of therapeutic quantities of drugs or bioactive agents. One of these methods is the encapsulation of drugs or bioactive agents in liposomes. Although some drugs or bioactive agents can be encapsulated in liposomes at effective therapeutic doses by passive loading or gradient loading, these methods are limited to drugs or bioactive agents with specific chemical properties or to drugs or bioactive agents that can be administered at relatively low concentrations . Some bioactive compounds such as weak bases or weak acids can be remotely loaded [sic] in preformed liposomes to form highly concentrated complexes. This type of charge, known as remote or gradient loading, requires that the drug or bioactive agent temporarily pass through the lipid bilayer of the liposome. However, this is not the case for all bioactive molecules, many of which can not cross the liposomal bilayer. An area in which attempts to administer therapeutic concentrations of drugs or bioactive agents have been partially successful is in the area of
the gene therapy. Gene therapy includes the introduction of an exogenous gene into an appropriate cell type, followed by the habilitation of gene expression within the cell at therapeutically relevant concentrations. Such treatment has progressed, in a relatively short time, from basic research to introduction into cells to a variety of genes, including those that are useful for the treatment of cancers (Duque et al., Histol Histopa thol, 13: 231- 242 (1998); Runnebaum et al., Anticancer Res. 17: 2887-2890 (1997)). Although naked DNA, in some cases, has been taken up in cells (Wolff et al., Science, 247: 1465-1468 (1990)), this generally can not be, due to its large size and high degree of negative charge; In addition, naked DNA can not be designed to be targeted to specific cells. Therefore, successful gene therapy generally depends on the availability of "vectors" to introduce DNA and other nucleic acids into cells. At present, there are two main groups of DNA supply systems, viral and non-rival. Viral vectors, including replication-deficient viruses such as retroviruses, adenoviruses and adeno-associated viruses, have thus far been the vehicles for gene delivery more widely
described (Robbins et al., Trends in Biotech, 16: 35-40 (1998)). However, its use has been impeded by the immunogenicity of its viral components, the potential risk of reversion to a competent state of replication, the potential introduction of tumorigenic mutations, the lack of steering mechanisms, the limitations in DNA capacity, the difficulty in large-scale production and other factors (see, for example, Lee and Huang, J Biol Chem, 271: 8481-8497 (1996)). As alternatives for viral vectors, two main types of non-viral vehicles have been developed. The cationic liposome-DNA complexes (or "lipoplejos", Felgner et al., Proc Na ti, Acad Sci USA, 84: 7413-7417 (1987)), consisting of cationic lipids and DNA in this way have been the most widely available alternative. described for viral vectors for gene delivery. However, such lipoplejos present some important disadvantages when used in gene therapy, including low stability, high cytotoxicity, non-biodegradability, poor condensation and DNA protection, sensitivity to serum, large size and lack of tissue specificity. In addition, since lipoplejos have a positive charge, they generally interact in a non-specific manner with the
negatively charged surfaces of most cells; therefore, in general it is not possible to direct such lipoplejos to specific sites in vivo. Another variation of lipoplexes and DNA includes DNA condensed with polylysine bound to anionic liposomes (Lee and Huang, J Biol Chem, 271: 8481-8497 (1996)). These require certain anionic lipids to form the active structure. The lipoplejos formed do not completely encapsulate the DNA or they must form two or more bilayers around the condensed DNA. In the latter case, the cytoplasm supply would require the DNA to cross at least three membranes. This would be expected to inhibit the efficiency of transfection. In the first case, stability may be compromised by exposing the DNA to physiological saline solutions. Liposomes are an additional type of alternative non-viral vector, and offer some advantages for such use compared to lipoplexes. For example, liposomal bilayers are formed around the encapsulated nucleic acids, thus protecting the nucleic acids from degradation by the surrounding nucleases; lipoplejos, on the contrary, do not encapsulate the nucleic acids and, therefore, can not sequester them completely away from the nucleases of the environment. In addition, liposomes can encapsulate, in their compartments
aqueous, other bioactive agents in addition to nucleic acids; On the contrary, lipoplejos can not because they do not encapsulate aqueous volumes. In addition, liposomes can be prepared to be neutral charge or anionic, contrary to the limited ionic nature of the aforementioned lipoplexes. Thus, liposomes can be designed to prevent cytotoxicities induced by the delivery vehicle itself and improve its accumulation at specific sites of interest. Although the concept of encapsulating bioactive agents in liposomes is not new, many agents have been difficult to encapsulate in liposomes at any concentration and others have proven difficult to encapsulate in liposomes at concentrations that would be effective from a therapeutic point of view. Many small molecules can be encapsulated in liposomes but escape. Thus, it is also difficult to encapsulate some bioactive agents and retain them within the liposomes at an effective therapeutic dose for a therapeutically effective time. For example, it has been difficult to encapsulate particularly large molecules in a complex within a liposome. It has also been difficult to use many water-soluble molecules as therapeutic agents because they can not penetrate the cell membrane. When they are encapsulated in a stable way
in liposomes that can fuse to the membranes, it is possible to deliver these drugs in effective therapeutic doses to the chosen cells. The method of the present invention allows the formation of liposomes containing such drugs or bioactive agents in a form useful for therapeutic use. Some attempts have been made to encapsulate nucleic acids in liposomes, these include the use of reverse phase evaporation (Fraley et al., J Biol Chem, 255: 10431-10435 (1980)), dehydration-rehydration (Alizo et al., J My croencap, 7: 497-503 (1990)) and freeze-thawing (Monnard et al., Biochem Biophys Acta, 1329: 39-50 (1997)) methods for liposome formation. However, each of these methods has some limitations, which includes the requirement of low initial concentrations of the nucleic acid, giving rise to significant percentages of empty vesicles in the product liposomes, the inability to reproducibly encapsulate sufficient quantities of the DNA in the liposomes to have therapeutic efficacy in the chosen site, desired and difficulties in the optimization of the vehicles for protection of their encapsulated nucleic acids against the degradation mediated by the nucleases. Attempts have also been made to complex DNA
with complexing agents and then encapsulating the DNA complexed in liposomes. The complexing agents are agents that react with other molecules causing the precipitation or condensation of the molecules. The complexing agents useful in the practice of the present invention are selected from the group consisting of charged molecules having a charge opposite to the charge of a bioactive agent. The complexing agent may be selected from the group of charged molecules consisting of spermine, spermidine, hexaminecobalt, calcium ions, magnesium ions, polylysines, polyhistidines, protamines, polyanions such as heparin and dextran sulfate, citrate ions or sulfate ions. For example, it is known that charge polycations +3 or greater, for example polyamines, polylysine and cobalt (III) examinations (see Chattoraj et al., J Mol Biol, 121: 327-337 (1978); Gosule LC and Schellman JA Naure 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); Wilson et al., Biochem, 18: 2192-2196 (1979)) can condense DNA molecules by interacting with multiple negative charges on the DNA. Polyamines, for example, spermidine (3+) and spermine (4+), unlike other types of polycations, as we have seen, occur in nature in all living cells (see, for example, Ames and Dubin). , J Biol Chem, 253: 769-775 (1960); Tabor and Tabor, Annu Rev Biochem, 53: 749-790 (1984)). It is known that high concentrations of polyamine exist in actively proliferating animal cells, and are considered to be essential in these to maintain normal cell growth (Ames and Dubin, J Biol Chem, 253: 769-775 (1960); Tabor, Annu Rev Biochem, 53: 749-790 (1984), Hafner et al., J Biol Chem, 254: 12419-12426 (1979), Pegg, Biochem J, 234: 249-262 (1986)). Encapsulation in linear DNA liposomes condensed with spermine has already been attempted by Tikchonenko et al., Gene 63: 321-330 (1988). However, the initial concentration of DNA in these was low, with the consequence that the resulting liposomes also had a low ratio of the DNA encapsulated to the liposomal lipid (0.02-0.2 micrograms of DNA per micromole of the lipid). In addition, such condensation of linear DNA molecules in the absence of intermolecular DNA aggregation necessitated control over spermine concentrations to an impracticable degree of precision. In addition, Baeza et al., Ori Lige Evol Biosphere, 21: 225-252 (1992) and Ibanez et al., Biochem Cell Biol, 74: 633-643 (1996) reported the encapsulation of 1-4 micrograms per micromol of DNA plasmid of SV40 condensed with spermine in liposomes. However, none of their preparations were dialyzed against buffer solutions with high salt concentration after liposome formation, the reported amounts of encapsulated DNA can actually include a significant percentage of the unencapsulated DNA. In view of the fact that these liposomal formulations were not exposed to DNAase degradation to determine the percentage of DNA actually sequestered in liposomes, the high reported amounts probably do not reflect the truly encapsulated DNA. The preparation and efficient use of nucleic acids encapsulated in liposomes requires the use of suspensions with a high concentration of nucleic acids to minimize the percentage of empty liposomes resulting from the process and to maximize the DNA: liposomal lipid proportions. However, the condensation of DNA at high concentrations during the known methods of liposome formation generally leads to intermolecular aggregation, resulting in the formation of nucleic acid-based structures unsuitable for gene delivery. Large aggregates that are formed by the condensation of DNA directly with a complexing agent can not be easily encapsulated in liposomes and such large aggregated structures (in order of cell size) can not be effective delivery materials for the chosen cells. For example, if the aggregates are larger than 500 nm, these are quickly cleared of circulation due to their size after intravenous administration. On the other hand, larger aggregates can be administered to cells in vitro. However, sometimes the aggregates are too large to be captured by the cells. Thus, in order to deliver a variety of drugs in effective therapeutic quantities to the chosen cells, it was necessary to provide a method for manufacturing liposomes containing the bioactive agents complexed in order to decrease their permeability through the lipid bilayer while providing a This method also limits the size of the complex to be encapsulated in the liposomes so that the resulting therapeutic product is within a range of therapeutic size.
SUMMARY OF THE INVENTION The present invention proposes a method for encapsulating a bioactive complex in a liposome, which comprises the steps of: (a) dissolving at least one unfriendly lipid in one or more organic solvents
(b) combining at 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 organic solution containing the lipid of step (a) to form an emulsion in the form of a reverse micelle containing the first molecule and the lipid
(c) adding a second aqueous suspension comprising a second 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 and vice versa; step emulsion (b)
(d) incubating the emulsion of step (c) to allow the complexing agent to make contact with the bioactive agent thereby forming a complex of the bioactive agent with the complexing agent within the water drops stabilized with lipid
where the complex has a diameter no greater than the diameter of the drop and
(e) removing the organic solvent from the suspension of step (d), to form the liposomes containing the bioactive agent complexed and the lipid.
The method of the present invention is useful for the preparation of therapeutic use liposomes containing a wide variety of bioactive molecules complexed with complexing agent within the liposome. Preferably, the liposomes are fusogenic liposomes which by the method of the present invention can encapsulate a variety of molecules. These fusogenic liposomes can be fused with cell membranes and allow the delivery of bioactive agents in effective therapeutic amounts to cells and organs. In addition, the method of the present invention also allows more than one bioactive agent to be encapsulated in a liposome. One or more bioactive agents may be encapsulated in the same liposomes at 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 that each of the bioactive agents be in the form of a complex.
Some bioactive agents easily cross the lipid bilayer and, therefore, do not sequester stably in the liposomes. By forming complexes of the bioactive agents with a complexing agent, the bioactive agent remains in the liposomes. One major obstacle has been the problem of encapsulating the complexed bioactive agents in liposomes. When the bioactive agent and the complexing agent are mixed in solution prior to encapsulation in the liposomes, multiple complexes are formed which are uncontrollably large at the concentrations necessary for the efficient loading of the liposomes. The term bioactive complex is any bioactive agent bound to the complexing agent so that the complex thus formed of origin to a change in the physical properties such as the decrease in the size of the bioactive molecule, decrease in the solubility of the bioactive agent, precipitation of the agents bioactive, condensation of the bioactive agent or increase in the size of the complex. Liposomes that fuse with cell membranes can supply a vast categories of molecules to the interior of cells. An advantage of this invention is that, by forming the complex of the bioactive agent in the reverse micelles, the formation of inadequately large complexes unable to be encapsulated in useful therapeutic liposomes is avoided. The formation of complexes comprising a bioactive compound within the liposomes has the advantage that such complexes are less likely to leak out of the liposome prior to delivery to the desired chosen cell. further, the formation of a complex can concentrate a large amount of the bioactive agent within the liposome so that the ratio of bioactive agent to lipid is very high and the delivery is effective. The method described proposes the complexing of bioactive materials with complexing agents within an emulsion followed by encapsulation within a liposome in a form that prevents the formation of extremely large, harmful aggregates, greater than some microns, of the bioactive agent and the agent complex In one embodiment, the method of the present invention has provided a method for encapsulating nucleic acid complexes. For example, nucleic acids such as DNA are complexed with a condensing agent inside reverse micelles (inverted), followed by the formation of the liposomes of the micelles. Although, as already described, previous attempts have been made to encapsulate DNA in liposomes, none of these methods were useful in efficiently preparing liposomal DNA for therapeutic use. This invention provides a method for preparing a liposome comprising a condensed nucleic acid, in amounts of at least about 0.5 micrograms of nucleic acid per micromole of liposomal lipid. The lipid component of the liposomes preferably consists of a phospholipid derivative and an additional lipid, generally in proportions of about 20-80 mol% of the phospholipid derivative up to about 80-20 mol% of the additional lipid. Preferred derivatized phospholipids include: phosphatidylethanolamine (PE) -biotin conjugates; N-acylated phosphatidyl ethanolamines (NAPE), such as N-C12 DOPE; and peptide-phosphatidyl ethanolamine conjugates, such as Ala-Ala-Pro-Val DOPE. The additional lipid can be any of the variety of lipids commonly incorporated in liposomes; however, where the phospholipid derivative is a NAPE, the additional lipid is preferably phosphatidyl choline (e.g., DOPC). Preferably, the nucleic acid is DNA. Also proposed herein is a method for preparing a pharmaceutical composition comprising the liposome and a carrier acceptable for pharmaceutical use; the composition can be used to deliver the nucleic acid to the cells of an animal. Other and more objects, features and advantages will be apparent from the following description of the preferred embodiments of the invention which are provided for description purposes when taken in conjunction with the following drawings.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Micrographs of the aggregation of spermine-mediated plasmid DNA (200 micrograms of plasmid DNA in 125 microliters of LSB were mixed lightly with 7 mM spermine in 125 microliters of LSB). (A) Observation to the light microscope after 15 minutes of incubation at room temperature (the bar represents 10 microns). (B) Cryo TEM observation (the bar represents 100 nm).
Figure 2. Schematic representation of the method of DNA encapsulation. DNA condensation occurs (I) within water droplets stabilized with the phospholipid that have formed around the DNA in a bulk organic solvent. Drops containing spermine, separated, transfer (II) spermine to the drops containing DNA by transient contact (III) and exchange. After condensation within the emulsion (IV), the vesicles are formed by evaporation of the solvent and furthermore they are extruded to smaller sizes (V).
Figure 3. Effect of liposomal N-C12 DOPE on DNA aggregation mediated by spermine. The equilibrium dialysis was performed in a three chamber dialysis device (see Example 4). The curve to the left is for dialysis without liposomes, while the curve displaced to the right is for dialysis that included a camera with liposomes. X axis: spermine concentration (mM); Y axis: turbidity (D. O. 400 nm).
Figure 4. Agaroza gel analysis of plasmid DNA protection in formulations of N-C12 DOPE / DOPC
(70:30) -An aliquot of each preparation after extrusion and dialysis was divided, and a part was digested with Dnasa I. (see Example 9). Strip 1. Preparation without spermine. Strip 2. The same as strip 1 but digested with Dnasa I. Strip 3. Preparation with spermine. Strip 4. Same as Strip 3 but digested with Dnasa I.
Figure 5. Luminous micrographs of the particles in N-C12 DOPE / DOPC (70:30) sample prepared as described in Example 3 with the plasmid pZeoLacZ and spermine (A) against polystyrene beads with an average diameter of 269 ± 7 nm (B) (the bars represent 10 nm).
Figure 6. Micrographs of the freeze-fractured TEM (see Example 7) of samples N-C12 DOPE / DOPC (70:30) prepared with plasmid and spermine, as described in Example 3. The arrow indicates the particle with material apparently encapsulated (the bar represents 400 nm).
Figure 7. Cryo TEM micrographs (see Example 8) of liposomes with N-C12 DOPE / DOPC and the plasmid pZeoLacZ without spermine (a), or with spermine (b), the liposomes being prepared as described in Example 3. In (a) the fiber-like structures are observed outside (star) and apparently inside (arrow) of the liposomes. In (b), an arrow indicates a toroid that resembles plasmid DNA condensed with polycation (bars in (a) and (b) represent 100 nm). The photomicrograph (c) represents an EPC sample made with spermine. The structures of toroid (arrow) and flexed rod (star) are compared with multilamellar liposomes (pound symbol) [the bar represents 50 nm].
Figure 8. Fluorescence photomicrographs of confluent 0VCAR3 cells after transfection (see Example 11) with N-C12 DOPE / DOPC preparations (70:30). Liposomal samples were prepared (see Example 3) with plasmid DNA pEGFP-Cl (a) with spermine or (b) without spermine; a sample (c) of the empty liposomes N-C12 DOPE / DOPC (70:30) without spermine plus the plasmid DNA pEGFP-Cl free adding outside the preformed liposomes was also tested. The amount of plasmid DNA added to the empty liposomes in sample C was equal to the total amount in each of the other preparations. In the experiments, equal concentrations of liposomes were used.
Figure 9. Quantification of EGFP expression in OVCAR3 cells transfected with pEGFP-Cl, as measured by the fluorescence level of EGFP. The transfection experiments (a, b and c, see Example 11) were the same as in the legend of the previous figure. In addition, the formulations tested were: (d) egg PC liposomes prepared with spermine and plasmid pEGFP-Cl see Example 3); and (e) without additions. The cells were washed and labeled with CBAM, and then dissolved in detergent to measure the fluorescence of EGFP and calcein blue (see Example 10, the error bars are ± d, e.).
Figure 10. Asstion of transfection activity with lipid pellets of N-C12 DOPE / DOPC (70:30) prepared with spermine and plasmid DNA pEGFP-Cl (see Example 3); the initial plasmid DNA and the spermine solutions contained 200 mM sucrose. After extrusion and dialysis, half of the sample was used for transfection without further manipulation (a), and the lipid particles of the rest of the sample were packed by centrifugation and washed once with HBSS before being used for transfection (b) ) a sample N-C12 DOPE / DOPC (70:30) only with 200 mM sucrose was also 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 package of this empty sample (c) was prepared in the same way, then, an equal amount of lipid of each of the samples was used for the transfection under the conditions described in the legends of the previous figures. After incubation overnight, the cells were labeled with CBAM and the fluorescence of EGFP and calcein blue was measured (the error bars are ± d. E.).
Figure 11. Transfection through the N-C12 DOPE / DOPC liposomes (70:30) in fluid of mouse ascites compared with buffer solution. The ascites was obtained from washing a tumor-bearing SCID mouse as described in Example 13. The cells were incubated with liposomes containing plasmid DNA (not a pack) at a final concentration of 10 mM total lipids in HBSS or HBSS with fluid of ascites, at a final protein concentration of approximately 3.5 mg / ml (see Example 11). After three hours of incubation, the transfection solution was replaced with medium containing serum and butyrate for about 20 hours. The expression of EGFP was measured by its fluorescence (error bars are ± d, e.).
Figure 12. Fluorescent photomicrographs of transfected OVCAR-3 cells (see Example 11) with N-C12 DOPE / DOPC liposomes (70:30) in buffer solution or mouse ascites fluid. Cells treated as described in the legend for Figure 12 were photographed. Photograph A represents transfection without peritoneal ascites fluid and photograph B with peritoneal ascites fluid; the cells with fluents in these views. Figure 13. Determination of the lamellarity of the liposome with a fluorescent probe.
Figure 14. Fluorescent photomicrographs of OVCAR-3 tumor transfected in vivo with N-C12 DOPE / DOPC liposomes (70:30) containing pEGFP-Cl. Figure A represents EGFP expression. Board B represents the red fluorescence of the rhodamine-labeled liposomes.
Figure 15. Fluorescent photomicrographs of OVCAR-3 tumor taken from a site different from that of Figure 14 transfected in vivo with N-C12 DOPE / DOPC liposomes (70:30) containing pEGFP-Cl. Figure A represents EGFP expression. Figure B represents the red fluorescence of the rhodamine-labeled liposomes.
Figure 16. Fluorescent photomicrographs of control tumor tissue. Figure A represents diffuse green fluorescence. Figure B represents the lack of red fluorescence of the rhodamine-labeled liposomes.
Figure 17. Graph showing the expression of β-galactosidase activity in mouse tissue after transfection in vivo.
DETAILED DESCRIPTION OF THE INVENTION The following are the abbreviations and corresponding terms that were used throughout this application: PE, phosphatidylethanolamine; PC, phosphatidylcholine; EPC egg phosphatidylcholine; DO-, dioleoyl; DOPC, dioleoyl phosphatidylcholine; DOPE, dioleoyl phosphatidylethanolamine; NAPE, N-acylated phosphatidylethanolamine; N-C12 DOPE, N-dodecanoyl dioleoyl phosphatidylethanolamine; AAPV-DOPE, Ala-Ala-Pro-Val-dioleoyl phosphatidylethanolamine; CBAM, calcein blue acetoxymethyl ester; PBS, salt buffered with phosphates; LSB, low-salt buffer solution; HBSS, Hank's balanced salt solution :; EGFP, improved green fluorescence protein; SPLV, stable plurilamellar liposomes, MLV multilamellar liposomes, ULV, unilamellar liposomes; LUV, large unilamellar liposomes; SUV small unilamellar liposomes; ds DNA, double-stranded DNA; TEM electronic transmission microscopy.
The present invention provides a method for encapsulating a bioactive complex in a liposome, which comprises the steps of: (a) dissolving at least one antipathic lipid in one or more organic solvents (b) combining at 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 organic solution containing lipid from step (a) to form an emulsion in the form of a reverse micelle containing the first molecule and the lipid
(c) adding a second aqueous suspension comprising a second 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 from step (b) (d) incubating the emulsion of step (c) to allow the complexing agent to make contact with the bioactive agent thereby forming a complex of the bioactive agent with the complexing agent within the water drops stabilized with the lipid wherein the complex is no larger in diameter than the diameter of the drop, and (e) removing the organic solvent from the suspension of step (d) to form liposomes comprising the bioactive agent complexed and the lipid.
The method of the present invention is useful for the preparation of liposomes for therapeutic use containing a wide range of bioactive molecules complexed with complexing agent within the liposome. Preferably, the liposomes are fusogenic liposomes which, by the method of the present invention, can encapsulate a variety of molecules. These fusogenic liposomes can be fused with cell membranes and allow the delivery of bioactive agents in effective amounts for therapeutic use to cells and organs. In addition, the method of the present invention also allows more than one bioactive agent to be encapsulated in a liposome. One or more bioactive agents can be encapsulated in the same liposomes at 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 that each of the bioactive agents be in the form of complexes. The term "bioactive agents" means any compound or composition of matter that can be administered to animals, preferably humans, for therapeutic or diagnostic purposes. The method of the present invention is useful for encapsulating bioactive agents including, but not limited to, water-soluble membrane waterproofing agents such as nucleic acids, nucleotides or nucleoside analogues such as cytosine β-D-arabinofuranoside 5'-triphosphate (araCTP), proteins such as cytochrome c, anticancer polar agents such as cisplatin, N-phosphono-acetyl-L-aspartic acid or 5'-fluoro-orotonic acid, polar derivatives or loaded with anticancer agents, polar peptides, histone inhibitors, Acetylase as butyrate, etc. Bioactive agents also include, but are not limited to, agents selected from the group consisting of nucleic acids such as DNA and RNA, antiviral agents such as acyclovir, zidovudine and the interferons; antibacterial agents such as amino glycosides, cephalosporins and tetracyclines, antifungal agents such as polyene antibiotics, imidazoles and triazoles; antimetabolic agents such as folic acid, and purine and pyrimidine analogues; antineoplastic agents such as anthracycline antibiotics and plant alkaloids; carbohydrates, for example sugars and starches; amino acids, peptides, proteins such as cellular receptor proteins, immunoglobulins, enzymes, hormones, neurotransmitters and glycoproteins; colorants; radioactive labels such as radioisotopes and compounds labeled with radioisotopes; radio opaque compounds; fluorescent compounds; mydriatic compounds; bronchodilators; local anesthetics and the like.
The term bioactive complex is any bioactive agent bound to a complexing agent so that the complex thus formed leads to a change in the physical properties such as the decrease in the size of the bioactive molecule, the decrease in the solubility of the bioactive agent, the precipitation of the bioactive agents, the condensation of the bioactive agent or the increase in the size of the complex. Water-in-oil emulsions containing reverse micelles have previously been used to study enzyme kinetics (eg, Bru et al., Biochem. J, 310: 721-739 (1995)) and to form liposomes (eg, Szoka and col., Proc. Na t Acad Sci USA, 75: 4194-4198 (1978), Gruner et al., Biochem, 24: 2833-2842 (1984)), but the use of these emulsions to modulate the complexation of two compounds for purposes of loading liposomes has not previously been reported. It is possible to form the emulsions by different methodologies, which are within the abilities of the person skilled in the art. sonication, vortex formation, mechanical agitation, static mixing, homogenization, injection, microfluidization, colloid mills, pressure emulsifiers and / or Kady mills can be used to prepare emulsions of different types including various orders of material addition. The emulsions of the present invention are formed in two steps so that at least one component, the bioactive agent or the complexing agent is previously sequestered within the water droplets of the emulsion stabilized with the lipid prior to the addition of the aqueous dispersion of the other agents. With the elimination of the solvent of the emulsion stabilized with the lipid, the "liposomes" are formed. The solvent can be removed by various methods including, but not limited to, rotary evaporation and nitrogen flow. The "liposomes" are self-assembled structures comprising one or more lipid bilayers, each of which comprises two monolayers containing antipathetic lipid molecules oriented in opposite directions. The antipathetic lipids comprise a polar (hydrophilic) major group region covalently linked to one or two non-polar (hydrophobic) acyl chains. The energetically unfavorable contacts between the hydrophobic acyl chains and the surrounding aqueous medium induces the antipathetic lipid molecules to arrange themselves so that their polar major groups are oriented towards the surface of the bilayer, while the acyl chains are reoriented towards the interior of the bilayer. In this way an energetically stable structure is formed in which the acyl chains are effectively protected from contact with the aqueous environment. Liposomes (see, for example, Cullis et al., Biochim Biophys Acta, 559: 399-420 (1987); New, 1995) may have a single lipid bilayer (unilamellar liposomes, "ULV"), or multiple lipid bilayers. (multilamellar liposomes, "MLV" or "SPLV"). Each bilayer surrounds or encapsulates an aqueous compartment. Given this encapsulation of the aqueous volume within a protective barrier of lipid molecules, the liposomes can sequester encapsulated molecules, for example nucleic acids, away from the degradative effects of factors such as nuclease enzymes, present in the external environment. Such protection of the encapsulated content, in the case of nucleic acid molecules, is demonstrated for example by the type of agarose gel analysis set forth in Example 9, the results of which are presented in the Figure. The liposomes can have different sizes, for example, an average diameter as low as 25 nm or as high as 10,000 nm or more. Size is affected by some factors, such as lipid composition and method of preparation, factors such that are within the skills of the expert who will determine and take into account, and is determined by different techniques such as quasi-elastic light scattering, also within the skills of the expert. To prepare liposomes of a smaller size, from larger liposomes it is possible to use some methodologies, also within the skills of the expert, such as sonication, homogenization, French press application and grinding. Extrusion (see, for example, U.S. Patent No. 5,008,050) can be used to reduce the size of liposomes, i.e., to produce liposomes having a predetermined average diameter by forcing liposomes, under pressure, through filter pores. of a defined size, selected. Tangential flow filtration (WO 89/008846) can also be used to regularize the size of the liposomes, that is, to produce a population of liposomes that have less heterogeneity in size, and a more homogeneous, defined size distribution. The contents of these documents are incorporated herein by reference. The liposomes of this invention may be unilamellar, or oligolamellar, and may have a size equal to that of the liposomes produced by any of the methods set forth in the foregoing. However, in the preferred embodiments of this invention, liposomes are unilamellar liposomes with average numerical sizes of about 50-300 nm. Liposomes are composed of a variety of lipid, both antipathic and non-antipathetic, obtained from some sources, natural and synthetic. Suitable liposomal lipids include, without limitation, phospholipids such as phosphatidylcholines ("PC"), phosphatidyl ethanolamines ("PE"), phosphatidylserines ("PS"), phosphatidylglycerols ("PG"), phosphatidylinositol ("Pl"), and phosphatidic acids ( "PA"). These phospholipids generally have two acyl chains, both saturated, both unsaturated or one saturated and one unsaturated; These chains include, without limitation: myristate, palmitate, stearate, oleate, linoleate, linolenate, arachididate, arachidonate, behenate and lignocerate chains. Phospholipids can also be obtained by binding to these a convenient reactive group. Such a group is generally an amino group and, therefore, the phospholipids derived or obtained are usually phosphatidylethanolamines. The different portions suitable for PE binding include, without limitation: acyl chains (WO 98/16199), useful for improving the fusion ability of liposomes to biological membranes; peptides (WO 98/16240), useful for destabilizing the liposomes in the vicinity of the chosen cells; biotin and maleimide portions (U.S. Patent Nos. 5,059,421 and 5,399,331, respectively) useful for ligating selected portions as the antibodies to the liposomes; and, various molecules such as gangliosides, polyalkyl ethers, polyethylene glycols and organic dicarboxylic acids (see, for example, U.S. Patent Nos. 5,013,555, 4,920,016 and 4,837,028). The contents of the aforementioned documents are incorporated herein by reference. Accordingly, in the most preferred embodiments of this invention, the liposomes prepared by the method of the present invention comprise a phospholipid derivative, adapted to improve the delivery of its contents. Liposomes may also, but are not required to, comprise additional lipids as well, with additional lipids being incorporated into liposomes for various reasons apparent to those skilled in the art in the field of liposomology. Such reasons include, without limitation, stabilizing or directing the liposomes, as well as others that acquire the pharmacokinetic behavior of the liposomes. Additional suitable lipids include any of the lipids commonly known to be suitable for incorporation into liposomes, including, without limitation, phospholipids, glycolipids, and sterols. Preferably, the liposomes of this invention have a lipid component comprising a phospholipid derivative and an additional lipid. The derivatized phospholipid has the formula:
CH2-R1
CH-R2 I CH2-0-P (0) 2-0-CH2CH2NH-Z,
wherein: Z is selected from the group consisting of 3 biotin, a maleimide portion, a group designated R and a group having the formula XY; X is a linker selected from the group consisting of a single link and
4 the group R; and Y is a peptide that can be dissociated with an enzyme comprising an amino acid sequence that is the substance of a peptidase secreted by the cell.
Each of R 1, R 2, R 3 and R 4 is a group having the formula OC (0) (CH 2) ni (CH = CH) n 2 (CH 2) n 3 (CH = CH) n 4 (CH 2) n 5 (CH = CH) ) n6 (CH2) n7 (CH = CH) n8 (CH2) n9CH3, where: ni is zero or an integer from 1 to 22; n3 is zero or an integer from 1 to 19; n5 is zero or an integer from 1 to 16; n7 is zero or an integer from 1 to 13; n9 is zero or an integer from 1 to 10; and each of n2, n4, n6 and n8 is zero or 1. Each of ni, n2, n3, n4, n5, n6, n7, n8 and n9 is the same or different in each occurrence. For R1 and R2, the sum of ni + 2n2 + n3 + 2n4 + n5 + 2n6 + n7 + 2n8 + n9 is independently an integer from 12 to 22, while for R and R, the sum of ni + 2n2 + n3 + 2n4 + n5 + 2n6 + n7 + 2n8 + n9 is independently an integer from 2 to 22. The phospholipid derivative preferably comprises about 20 to 80 mol% of the liposomal lipid. Where R3 is - C (O) (CH2) ni (CH = CH) n2 (CH2) n3 (CH = CH) n4 (CH2) n5 (CH = CH) n6 (CH2) n7 (CH = CH) n8 (CH2 ) n9CH3, the phospholipid derivative is an N-acylated phosphotidylethanolamine ("NAPE", see WO 98/16199). Preferably, R is then -OC (O) (CH2) n? CH3, more preferably -OC (O) (CH2)? 0CH3. Preferably, the phospholipid derivative is an N-acylated PE. Such NAPEs are useful for preparing fusogenic liposomes and are preferred for preparing liposomes containing the medicament or complexes of the bioactive agent of the present invention. The destabilization of the bilayer induced by NAPE induces the bilayers to fuse to the biological membranes in the vicinity and, therefore, improves the fusogenicity of the bilayers (Shangguan et al., Biochim Biophys Acta, 1368: 171-183 (1998)). ). The improved fusogenicity, in turn, can be used to deliver encapsulated bioactive agents, such as nucleic acids or other agents that can not traverse the cell membrane, to the cells, combining the cells with the liposomes under conditions, for example, the presence of suitable concentrations such as Ca + and Mg +. The liposome-cell contact gives rise to the release of the bioactive agents encapsulated in the liposome into the cells, and / or directly into the cytoplasm of the cells as a result of the fusion between the liposome and the cell membranes. This supply is in vivo or in vi tro. Where R is the acyl chain or the peptide and, therefore, where the phospholipid derivative is a NAPE or peptide-lipid conjugate, at least one of R 1 or R 2 is preferably an unsaturated acyl chain, i.e. at least one of n2, n4, n6 or n8 in this is equal to 1. The unsaturated acyl chains include, without limitation, palmitoleate, oleate, linoleate, linolenate, and arachidonate chains. Preferably, the unsaturated acyl chain is an oleate chain ("-OC (O) (CH2) 7CH = CH (CH2) 7CH3"). More preferably, R 1 and R 2 are oleate chains, that is, the phospholipid derivative is then:
CH2-OC (0) (CH2) 7 (CH = CH) (CH2) 7CH3 I
CH-OC (O) (CH2) 7 (CH = CH) (CH2) 7CH3
CH2-0-P (O) 2-0-CH2CH2NH-Z,
3 where Z is R or X-Y. Most preferably, the phospholipid derivative is then:
CH2-0C (0) (CH2) 7 (CH = CH) (CH2) 7CH3 I
CH-OC (O) (CH2) 7 (CH = CH) (CH2) 7CH3 I
CH2-0-P (O) 2-0-CH2CH2NH-OC (O) (CH2)? 0CH3
that is, "N-C12 DOPE". Where the phospholipid derivative is N-C12 DOPE, the liposomal lipid preferably also comprises a phosphatidyl choline, preferably a PC having at least one unsaturated acyl chain and, most preferably, dioleoyl phosphatidylcholine. Preferably, the liposomal lipid comprises approximately 70 mol% of N-C12 DOPE and approximately 30 mol% DOPC, ie, it is a "70:30" formulation of N-C12 DOPE and DOPC, wherein the concentrations of the liposomal lipid are mentioned herein by relation, and wherein such ratios are an indication of the relative percentages in the liposomal lipid of the particular lipids mentioned).
The liposomal lipid may also comprise a "lipid modified in the main group", ie, a lipid having a polar group derived by binding thereto a portion capable of inhibiting the binding of the serum proteins to a liposome incorporating the lipid The incorporation of the modified lipids in the main group in the liposomes in this way alters their pharmacokinetic behavior, so that the liposomes remain in the circulation of an animal for a longer time with a normal lipid (see, for example, Blume et al. col., Biochim, Biophys, Acta., 1149: 180
(1993); Gabizon et al., Pharm. Res. , 10 (5): 703 81993);
Park et al., Biochim. Biophys Acta 257: 1108 (1992);
Woodle et al., U.S. Patent No. 5, 013, 556; Alien et al., U.S. Patent Nos. 4,837,028 and 4,920,016; the contents of these documents are incorporated herein by reference). The lipids modified in the main group are usually phosphatidylethanolamines (PE "), for example, dipalmitoylphosphatidylethanolamine (" DPPE "), palmitoleoyl phosphatidylethanolamine (" POPE "), and dioleoyl phosphatidylethanolamine (" DOPE "), among others. major groups generally derived with a polyethylene glycol, or with an organic dicarboxylic acid such as succinic or glutaric acid ("GA"), or their corresponding anhydrides The amount of lipid modified with the main group incorporated in the lipid carrier generally depends on certain factors known to those skilled in the art or that is within their abilities to determine without due experimentation.These include, but are not limited to: the type of lipid and the type of modification of the main group, the type and size of the carrier; The therapeutic use of the proposed formulation, usually from about 5 mol% to about 20 mol% of the lipid in u A lipid carrier containing modified lipid in the main group is the modified lipid in the main group. Complexing agents generally include, but are not limited to, a group with opposite charge to the bioactive agent including spermine, spermidine, cobalt hexamine, calcium ions, magnesium ions, polylysines, polyhistidines, protamines, polyanions such as heparin and dextran sulfate, citrate ions and sulfate ions. A person skilled in the art will also know other complexing agents useful in the method of the present invention. The condensed nucleic acids encapsulated in the liposome are DNA, which includes genomic DNA, plasmid DNA and cDNA, or RNA; preferably the encapsulated nucleic acid is DNA, more preferably, closed (circular) plasmid DNA. The condensed nucleic acids are encapsulated in the liposomes at a concentration of at least about 0.5 micrograms per micromole of the liposome lipid, or at least about 0.75, 1.0, 1.25, 1.5, 1.75 or 2 micrograms per micromole. More preferably, the liposomes contain about 2 micrograms of nucleic acid per micromole of lipid to about 20 micrograms per micromole. "Condensed" as used herein in connection with nucleic acids refers to nucleic acids that have been combined with one or more polycations so that the nucleic acid strands are more tightly packed than would be the case in the absence of the nucleic acids. polycations. Such packaging allows the nucleic acids to be encapsulated in liposomes, but leaves the nucleic acids in a transfectable conformation, ready for transcription. Accordingly, in the preferred embodiments of this invention, the method prepares liposomes comprising a condensed DNA and liposomal lipid comprising approximately 70 mol% of N-C12 DOPE and approximately 30 mol% of DOPC. Such liposomes contain approximately 0.5 micrograms of condensed DNA per micromole of the lipid. The liposomes provided by the method of the present invention may contain one or more bioactive agents in addition to the complexed bioactive agent. Bioactive agents that may be associated with liposomes include, but are not limited to: antiviral agents such as acyclovir, zidovudine and interferons; antibacterial agents such as aminoglycosides, cephalosporins and tetracyclines; antifungal agents such as polyene, imidazole and triazole antibiotics; antimetabolic agents such as folic acid, and purine and pyrimidine analogues; antineoplastic agents such as anthracycline antibiotics and plant alkaloids; sterols as cholesterol; carbohydrates such as sugars and starches; amino acids, peptides, proteins such as cellular receptor proteins, immunoglobulins, enzymes, hormones, neurotransmitters and glycoproteins; colorants; radioactive labels such as radioisotopes and compounds labeled with radioisotopes; radio opaque compounds; fluorescent compounds; mydriatic compounds; bronchodilators; local anesthetics; and similar. Formulations of the bioactive liposomal agent can improve the therapeutic index of the bioactive agent, for example, by buffering the toxicity of the agent. Liposomes can also reduce the rate at which the bioactive agent is removed from the circulation of animals. Accordingly, the liposomal formulation of the bioactive agents may mean that less of the agent needs to be administered to obtain the desired effect. The liposome of this invention can be dehydrated, stored, and then reconstituted so as to retain a substantial portion of its internal contents. Liposomal dehydration generally requires the use of a hydrophilic drying protector such as disaccharide sugar on the inner and outer surfaces of the bilayers of liposomes (see U.S. Patent No. 4,880,635, the contents of which are incorporated herein by reference). This hydrophilic compound is generally considered to prevent rearrangement of the lipids in the liposomes, so that their size and content is maintained during the drying process, and through subsequent rehydration. The suitable qualities of such secant protectants are that they must be strong hydrogen bond acceptors, and possess stereochemical characteristics that preserve the intermolecular separation of the components of the liposomal bilayer. Otherwise, it is possible to omit the secant protector if the liposomal preparation does not freeze before dehydration, and enough water remains in the preparation after dehydration. Also provided herein is a pharmaceutical composition comprising an acceptable carrier for pharmaceutical use and the liposome of this invention. This composition is useful, for example, in the delivery of nucleic acids to the cells of an animal. "Carrier acceptable for pharmaceutical use" when used in the present invention are those generally acceptable for use in connection with the administration of lipids and liposomes, including liposomal formulations of bioactive agents, to animals, including humans. Acceptable carriers for pharmaceutical use are generally formulated according to some factors that are within the expert's abilities to determine and take into account, which includes without limitation: the particular liposomal bioactive agent that is used, its stability concentration and the proposed bioavailability; the disease, disorder or condition that is to be treated with the liposomal composition; the individual, their age, size and general condition; and the proposed route of administration of the composition, for example nasal, oral, ophthalmic, topical, transdermal, vaginal, subcutaneous, intramammary, intraperitoneal, intravenous or intramuscular (see, for example, Nairn (1985), the contents of which incorporated herein by reference). Acceptable carriers for common pharmaceutical use which are used in the parenteral administration of the bioactive agent include, for example, D5W, an aqueous solution containing 5% by volume weight of dextrose and physiological saline. Acceptable carriers for pharmaceutical use may contain additional ingredients, for example those that improve the stability of the active ingredients included, such as preservatives and antioxidants. Further provided herein is a method for encapsulating a nucleic acid, e.g., DNA, in a liposome, comprising the steps of: (a) combining an aqueous suspension of the nucleic acid with an organic solution comprising a lipid, e.g. , a phospholipid derivative and an additional lipid, to form a suspension of inverted micelles (inverted) comprising the nucleic acid and the lipid; (b) adding a polycation to the micellar suspension to condense the nucleic acid within the reverse micelles; and (c) removing the organic solvent from the suspension of step (b), to form liposomes containing the nucleic acid and the lipid of the reverse micelles. The ratio of the nucleic acid to the liposomal lipid obtained by the encapsulation method is at least about 0.5 micrograms of nucleic acid per micromole of lipid. Lipids useful in the practice of this invention are, as described above, those lipids recognized as suitable for incorporation into liposomes, by themselves or in connection with additional lipids; These include, phospholipids, glycolipids, sterols or their derivatives. The organic solvents that are used in this method are any of a variety of solvents useful in dissolving the lipids during the course of liposome preparation; these include, without limitation, methanol, ethanol, dimethisulfoxide, chloroform and mixtures thereof. Preferably, the organic solvent is chloroform. The polycations useful in the method of the present invention for condensing nucleic acids are any of the chemical compounds having three or more ionizable groups that can be used to condense nucleic acids, other bioactive agents or drugs; these include, without limitation, polylysine, polyamines (e.g., spermine and spermidine), cobalt (III) hexamine, polyhistidine, polyethylene imine, and the like. Preferably, the polycation is spermine. Nucleic acids useful in the practice of this invention include DNA, for example, genomic DNA, cDNA and plasmid DNA, linear or closed, as well as RNA. The nucleic acids are suspended in aqueous medium by the methods commonly understood and easily practiced, for example by vortexing the macromolecules in suspension. Suitable aqueous media are aqueous solutions of some additives as buffering agents, and are virtually free of certain ingredients such as salts and nuclease enzymes; such means include, without limitation, low salt buffer ("LSB", see Example 3 below). Water-in-oil emulsions stabilized by phospholipids contain reverse micelles. Reverse micelles (see Bru et al., Biochem J 310: 721-739 (1995)) are structures based on antipathetic lipids in which the hydrophilic domains of the lipids are sequestered within the surfaces of the micelles, whereas the Hydrophobic domains of lipids are arranged outside. Emulsions with reverse micelles are formed, as already described and in Figure 2 below, as protected bioactive agents that include nucleic acids sequestered therein from the intermolecular contacts which would otherwise give rise to their aggregation in the presence of agent complexing and inadequately by incorporation into liposomes. Such a process is carried out to maximize the percentage of the resulting liposomes containing the desired complexes. Within the emulsion, the complexes are formed by the exchange of the added complexing agents, such as polycations, or the bioactive agents between the aqueous compartment of the inverse micelles in the emulsion (see, for example, Bru et al., FEBS , 282, 170-174 (1991), Fletcher et al., J Chem Soc Faraday Trans I, 83: 985-1006 (1987)). In the case of encapsulation of DNA complexes, convenient polycations are any of the polycations useful for condensing nucleic acids. For example, spermine and spermidine have been used (see, for example, Chattoraj et al., J Mol Biol, 121: 327-337 (1978) and Gosule et al., Nature, 259: 333-335 (1976), the contents of which are incorporated herein by reference), in vi tro to condense individual plasmids, but only at low concentrations of DNA, to avoid the aggregation of the condensed plasmids. Such concentrations were minimal that, having attempted the liposomal encapsulation of the condensed nucleic acids, there would be a significant number of empty liposomes, ie, without containing DNA. Polylysine and cobalt (III) hexamine are also available for condensation of nucleic acids. The concentrations of suitable polycations for condensing nucleic acids are those concentrations that lead to the neutralization of a sufficient number of negative charges of the nucleic acid, for example, about 90% or more of the negative charges in the case of DNA (Wilson et al. ., Biochem, 18: 2192-2196 (1979)). Those skilled in the art will be able to determine convenient or optimal concentrations of the polycations given the nucleic acid to be condensed, the polycation used, the nucleic acid concentrations and the polycation valence. In addition, additional factors that are within the skills of the expert to determine and take into account may affect the concentrations of polycations useful for the condensation of bioactive agents such as nucleic acids. For example, NAPEs such as N-C12 DOPE carry a net negative charge, through the additional acyl chain; therefore, such lipids can interact with positively charged molecules, thus decreasing the combination of available polycations for the condensation of nucleic acids. Accordingly, in such cases, it may be necessary to add an amount of the above polycation that would otherwise require the condensation of the nucleic acid. Such sufficient additional amounts of the polycations can be determined by different means including, for example, the type of partition experiments set forth in Example 4. Such experiments provide data (see Figure 3) showing the concentrations of the additional polycation required for the nucleic acid condensation. For example, with the nucleic acid and lipid concentrations used in Example 3, 0.6 mM spermine was sufficient for the condensation of plasmid DNA, but this amount increased to 0.85 mM in the presence of NAPE N-C12 DOPE, in the concentration established However, polycation concentrations greater than these, ie, higher than minimally necessary, can be used, for example, again by observing the conditions of Example 3 as an example, a final spermine concentration of 8-20 mM in the emulsion it was found to be optimal for the neutralization of the charge of the nucleic acid and the lipid. Those skilled in the art will be able to determine suitable concentrations for lipids and nucleic acids for the practice of this invention. For example (see Example 3, below) to encapsulate condensed plasmid DNA within 200 nm spherical liposomes, 200 micrograms of pZeoLacZ plasmid DNA in 125 microliters of LSB were combined with 30 micromoles of a combination of N-C12 DOPE and DOPC in a molar ratio 70:30. Accordingly, the preferred embodiments of this invention are practiced with a condensed nucleic acid which is plasmid DNA, a lipid comprising a lipid derivative, for example N-C12 DOPE, chloroform and spermine, for example, in a concentration of about 1 mM or older. Still further, a method of transfecting the cells of an animal with a bioactive agent such as a nucleic acid is provided herein, the method comprising the step of contacting the cells with a liposome of this invention containing the nucleic acid. complexed Such contact is in vi tro, in which case a composition comprising the liposome is added to the culture medium surrounding the cells, or in vivo, in which case the liposome is administered in a pharmaceutical composition which also contains an acceptable carrier for use. pharmaceutical, and the animal is administered by any of the normal means of administering such compositions to the animals. In vivo contact, especially where specificity or choice is desired, is added by incorporating into the liposome a means for directing the liposome to a specific site, for example, conjugating an antibody to the liposomes via streptavidin, causing the contents of the liposome are preferentially released at a certain site, for example, by incorporating the NAPE or peptide / lipid conjugates into the liposomes, and / or causing the liposomes to accumulate at the sites, e.g., tumors, by incorporating a modified lipid therein in the main group. The efficiency of the transfection, that is, the efficiency of the actual introduction of a nucleic acid molecule encapsulated in liposome to a cell is assisted, in vi tro or in vivo, by incorporating in the liposome a means for inducing the bilayers of the liposome to merge to the membranes of the cells. Such means include, without limitation, the incorporation of NAPEs, peptide-lipid conjugates and ionizable lipids (see WO 87/07530 and WO 95/27478, the contents of which are incorporated herein by reference). Transfection, in vi tro or in vivo, is aimed at introducing the nucleic acids in the cells so that they are transcribed and translated into them. Such expression of proteins by the introduction of exogenous nucleic acid can be used to cope with various defects in the cell caused by lack of expression, or by over-expression, of a gene in this, or otherwise to modify cellular proteins and their expression. Transfection of the cells with nucleic acids encapsulated in liposomes provided herein is useful for treating animals affected with diseases or disorders characterized by abnormal expression; or there is no either abnormally low, abnormally high or inadequate, of a protein. Such diseases and disorders include, for example and without limitation, some cancers and disorders due to gene defects. Therapeutically relevant transfection can also give rise to the expression of a protein previously not expressed in the chosen cells. The successful result of the transfection, and the expression of the transfected nucleic acid in a cell, can be detected in different ways, these generally depending on the detection of the physical presence of the nucleic acid in the cell, for example, by the incorporation of radionucleotides in the cell. the nucleic acid, or by detecting the expression of the protein encoded by the nucleic acid. This can be done in different ways; which includes, without limitation, where the protein is a detectable marker, for example fluorescent, or where the protein is a selected marker, for example resistance to a cytotoxic agent. For example, plasmid pEGFP-1 contains a DNA sequence encoding the improved green fluorescence protein, the presence of which is detected by fluorescence microscopy. Accordingly, successful transfection of the cells with this plasmid (see Examples 10-12) is easily determined by assessing the amount of fluorescence exhibited by the cells. The results of these experiments (see Figures 8-12), both the successful transfection of OVCAR-3 cells with the plasmid pEGFP-1, as well as the high level of expression of the transfected plasmid in a significant percentage of the transfected cells. Such a satisfactory expression was observed only where the transfected DNA had been condensed with polycation; the samples not processed with spermine showed none, or almost none, fluorescence (see Figure 8). The quantification of the fluorescent protein expression (Figure 9) showed that transfection with DNA fused with polycation gave rise to significant expression levels, while transfection of the samples processed without spermine did not originate quantifiable fluorescence. In addition, transfection with free, ie, non-encapsulated, DNA also led to no observable or quantifiable fluorescence (Figures 8c and 9c). This invention will be better understood from the following examples, which are only illustrative of the invention as defined in the clauses that are presented below.
Examples
Example 1 - Materials
N- (rhodamine sulfamyl-lysamine) -phosphatidylethanolamine (transesterified from egg PC), DOPC, EPC and N-C12 DOPE were purchased from Avanti Polar Lipids (Alabaster, AL). The OVCAR3 ovarian carcinoma cells were purchased from the NCI-Frederick Cancer Research Laboratory (Frederick, MD). Plasmid pEGFP-Cl and DH5a competent cells of E. coli were purchased from Clontech Laboratories (Palo Alto, CA). Plasmid pZeoSVLacZ, competent cells and S.O.C from Hanahan were purchased from Invitrogen (San Diego, CA). Hank's balanced salt solution (HBSS), RPMI 1640 and heat-inactivated fetal bovine serum and lipofectin were purchased from Gibco / BRL (Grand Island, NY). The DNase-free RNase and the RNase-free DNase I were purchased from Boehringer Mannheim (GMBH, Germany). Agarose was obtained from FMC Bioproducts (Rockland, ME). Bacto agar, Tryptone bacto and yeast extract were purchased from DIFCO Laboratories (Detroit, MI). The blue calcein acetoxy methyl ester (CBAM) dyes, PicoGreen and SybrGreen I were from Molecular Probes (Eugene, OR).
Example 2 - Purification of the plasmid
In this study two plasmids were used: the plasmid pZeoSVLacZ which is 6.5 kb, and expresses the lacZ gene for β-galactosidase in mammalian cells from the SV40 enhancer-promoter early, allowing selection in mammalian cells and E. coli using the antibiotic zeocin; and, the plasmid pEGFP-Cl which is 4.7 kb and expresses the enhanced green fluorescent protein (EGFP) of a human cytomegalovirus immediate early promoter, allowing selection in E. coli using kanamycin, and in mammalian cells using G418. The plasmids were purified from E. coli (Baumann and Bloomfield, Biotechniques, 19: 884-890 (1995)) -the final ratio of D.O. at 260 nm to D.O. at 280 nm was greater than 1.9 for all preparations; agarose gel electrophoresis indicated DNA in the expected size range.
Example 3 - Liposomal DNA Formulations
Samples were prepared by diluting 200 μg of DNA in 125 μl of LSB, and then combining the resulting suspension with 1 ml of CHCl3 containing 30 μmol of the molar ratio 70:30 of N-C12 DOPE and DOPC, in a Pyrex tube of 13 x 100 with vortex shaking. The sample was immediately sonicated for 12 seconds in a sonicator bath (Laboratory Supplies Co. Hicksville, NY) with the maximum energy, to form an emulsion with the first plasmid DNA. Subsequently, an aliquot of 125 μl of LSB containing various concentrations of spermine (16 to 40 millimolar) was added to this emulsion with vortexing and sonication. The samples without spermine were prepared in the same manner, except that the spermine was omitted from the second 125 μl aliquot. The preparation of the samples with EPC was also identical, except that 7M spermine was used.
The resulting emulsions were placed, for a few minutes, in a flask on a Rotovap (Büchi Laboratoriums-Technik AG, Sweden). The organic solvent was removed by rotating the flask at its maximum velocity, while the vacuum was modulated with a pin valve. Initially a vacuum of approximately 600-650 mm was established, subsequently increasing it as fast as possible without excessive bubbling, until a maximum vacuum was reached (approximately 730 mm); the flask was then evacuated for another 25 minutes. The film remaining in the flask was resuspended in 1 ml of sucrose in 300 mM LSB, and the sample was extruded 5 times through 0.4 micron polycarbonate membrane filters (Poretics, Livemore, CA). The sample was then dialyzed against Hank's balanced salt buffer (HBSS) without Ca + / Mg +, overnight at 4 ° C. Other lipid compositions were used to encapsulate the condensed DNA according to the present invention. The plasmids were condensed and encapsulated in liposomes as described in this previous example and pelleted as in Example 12. The lipid composition of the liposomes was cholesterol hemisuccinate: cholesterol: l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine: 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine: dioleoyl dimethylammonium propane: oleoyl acetate in a ratio of 12.5: 2.5: 50: 12: 10.5: 10.5. After pelleting and washing, the DNA / lipid ratio for these liposomes was determined as in Example 10. The DNA / lipid common ratios were 1.4-2.1 μg of DNA per micromole of lipid.
Example 4 - Partitioning spermine
The N-C12 DOPE carries a net negative charge that could potentially interact with positively charged spermine and affect the condensation process. Therefore, it was necessary to test the partition of the spermine between DNA and the liposomes of this composition in a dialysis experiment with buffer solution with low salt concentration. Experiments designed to measure spermine partition between negatively charged phospholipids and DNA were performed with a three-chamber dialysis device (Sialomed, MD) - each chamber contained 250 μl of liquid. The desired amount of spermine was diluted in LSB and placed in the central chamber, which is flanked by two dialysis membranes cut 100,000 p.m. The chamber on one side of the spermine chamber contained 400 μg of pZeoLacZ plasmid DNA in a total volume of 200 μl of LSB. The chamber on the other side contained 250 μl of LSB alone, or empty N-C12 DOPE / DOPC (70:30) liposomes, prepared as described in Example 3 at a total lipid concentration of 30 mM, in 250 μl of LSB - in this arrangement, only the espermina had access to the three cameras. Since it is known that the neutralization of plasmid DNA by spermine gives rise to aggregation (Figure 1), the turbidity of the solution in the chamber containing DNA was used as a means to monitor the partition of spermine. If the liposomes completely sequestered the spermine away from the DNA, the DNA would not be added. The amount of negatively charged lipid available was approximately twice the amount of the negative charge on the DNA in these experiments. Each dialysis device had a rotation on a 12-inch motorized wheel during the night
(approximately 20 hours). The chamber containing DNA was then evacuated with repeated pipetting to mix the mixture, and placed in a 250 μl volume bucket. The absorbance at 400 nm was used to monitor the turbidity against the bottom of the buffer solution.
The spermine titration curves of DNA turbidity were constructed for dialysis with and without liposomes present (Figure 3). The approximate shift in the curve due to the presence of liposomes was used to calculate the relative binding constants for lipids and DNA, assuming that each spermine molecule binds to four phosphate groups of the nucleotide or four phospholipids, in simple equilibrium with the association constants KDNA and KiiPid0, respectively. It is known that at a low salt concentration, the DNA spermine dissociation constant is in the micromolar range (Wilson et al., Biochem, 18: 2192-2196 (1079); Gosule et al., J Mol Biol, 121: 327-337 (1978)). Therefore, the free concentration of spermine was taken as negligible at the inimolar concentrations of spermine required for DNA aggregation in these experiments. The fractional neutralization of the phosphate groups of the DNA by the spermine required for the DNA aggregation,?, Was taken as 0.9, based on the data obtained in the absence of the liposomes. This is the same value reported as necessary for DNA condensation, coinciding with the previous observation that aggregation accompanies condensation at high DNA concentrations (Wilson et al., Biochem, 18: 2192-2196 (1979); Gosule et al. ., J Mol Biol, 121: 327-337 (1978)). Assuming [DNA-spm] =? [DNA] T0TAL at the point of aggregation and [lipid-spm] = the displacement on the curve, it is possible to use the equation:
KCNA / K? IPido = [? / (1 ~ 7)]? [(lipidototal displacement) / displacement]]
When the lipidototai is taken as the total concentration of the negatively charged lipid exposed on the outside of the liposomes divided by 4, the ratio of the apparent equilibrium constants is 178, that is, the binding of the spermine to the DNA is very much avid that the binding to lipids. The relation of the union constants and the first factor in to the right with constants.
Therefore, the last factor on the right can be used to calculate the displacement in the spermine titration curve for DNA condensation for any concentration of total lipids, including the highest effective concentration used in the emulsions. The data presented in Figure 3 demonstrate that the presence of the liposomes only displaces slightly for the curve for DNA aggregation. Thus, approximately 0.6 mM spermine in the initial emulsion of 250 μl was sufficient to condense the plasmid DNA, while a total of 0.85 mM would be sufficient to condense the DNA in the presence of the amount of N-C12 DOPE used. Therefore, it would be expected that the plasmid DNA could actually condense in these preparations without the complication of neutralizing the negatively charged lipids which would destabilize the liposomes.
Example 5 - Luminous microscopy of the liposome samples
The pre-condensation of the DNA was tested for potential encapsulation in the liposomes. Mass aggregation occurred as expected by a large change in the turbidity of the solution. This situation was not unexpected given that similar problems had been reported. Light microscopy of the plasmid aggregates (Figure 1) was performed using 200 μg of the pZeoLacZ plasmid in 125 μl of LSB, mixed lightly with 7 mM spermine in 125 μl of LSB and incubated for 15 minutes at room temperature. Observation under the microscope (Figure IA) showed that the aggregates in general were much larger than 1 μ and often as large as 5-10 μm. The large size of these aggregates was also confirmed by cryo-electron microscopy (Figure IB). Of particular interest to this amplification are the regular arrangements of the fibers, perhaps as a result of the condensation induced by the spermine to a partially ordered structure. There were also some canes or curved rods suggesting the start of the toroidal structures, but not complete toroids. The aggregates formed in this way were too large to be useful for a supply system. To calculate the size of the N-C12 DOPE / DOPC liposomes (70:30) containing the DNA (Figure 5), the polystyrene beads with an average diameter of 269 ± 7 nm (Duke Scientific Corp. Palo Alto CA) were diluted with H20 at a concentration suitable for microscopy, and samples of N-C12 DOPE / DOPC (70:30) liposomes containing DNA were used after extrusion and dialysis without further dilution (approximately 20 mM lipid). Samples were examined under an Olympus BH-2 fluorescence microscope (Olympus, Lake Success, NY) at lOOOx. The results are presented in Figure 5. The liposome particles containing the DNA appeared relatively uniform in size and shape to this amplification, and the approximate size of the sample particles appeared very similar to those obtained from the dispersion studies of dynamic light. Comparison of this sample of liposome particles containing DNA with the spermine-DNA added in Figure IA demonstrates the benefit of condensing the DNA in the reverse micelles according to the present invention before forming the liposomes. There was no evidence of the very large aggregates observed when the spermine interacted directly with DNA in aqueous solution, indicating that the emulsion condensation method can greatly inhibit such aggregate formation.
Example 6 - Analysis of the particles by light scattering
The preparations of N-C12 DOPE / DOPC were characterized by quasi-elastic light scattering. The particle size analysis was performed using a particle size analyzer Nicomp 370 (Particle Sizing Systems, Santa Barbara CA). The samples were diluted approximately 10 times for the analysis. A Gaussian analysis was performed in the vesicle mode, and weighted numerical averages were reported. The data for pZeoLacZ condensed spermine-DNA plasmid prepared as in Example 3 can be adjusted by a Gaussian size distribution with a numerical average particle size of 222.6 nm.
Example 7 - TEM frozen-fracture
Preparations of N-C12 DOPE / Fusogenic DOPC with encapsulated DNA were further characterized by freezing TEM fracture. Approximately 2 μl of sample were deposited between two Balzers copper containers in double replication and frozen in liquid propane. The sample was fractured at -100 ° C, 10 -10"bar and shaded with platinum (<45 ° C) and carbon in a Balzers BAF 400 freezing-fracture device. The replicas were digested with 5% bleach during the overnight, they were washed with distilled water and mounted on 300 mesh grids. Images were obtained as a Phillips 300 TEM. The results are presented in Figure 6. Most of the particles were small (less than 400 nm) , consistent with NICOMP results Due to the fracture plane prevalent through the lipid bilayers, observation of internal contents is rare with this technique, however, a small number of the particles seemed to have some encapsulated structures that would represent DNA condensed.
Example 8 - Cryo-transmission electron microscopy
Cryo-EM was used to confirm the liposomal nature of the preparations and to possibly visualize any of the encapsulated materials. For the aggregate EPC and spermine-DNA sample, the copper grids covered with a carbon support with holes were used without treatment. For liposome samples containing N-C12 DOPE / DOPC DNA (70:30), EM slits with a carbon film with holes or screen were positively charged by placing a drop of 0.1 mM polylysine solution on the surface of the grate, and leaving it at rest for one minute. The polylysine was dried and the grid rinsed with a few drops of distilled water, followed by a few drops of the sample's buffer solution. Then a 5 μl sample aliquot was placed on the grid, dried with a thin film filter and immediately immersed in liquid ethane. The grids were stored in liquid nitrogen until use. These were observed in a Philips CM 12 electronic transmission microscope (Mahwah, NJ), operating at an accelerating voltage of 120 kV. A cryo-container 626 (Werrendale, PA) was used to maintain the temperature of the sample between -177 ° C to -175 ° C during the observation of the image. Electron micrographs were recorded from the areas suspended above the holes under low electronic dose conditions. Amplifications of 35,000x or 60,000x and subfocus values of 1.8-2.5 microns were used. The results are presented in Figure 7. When spermine was omitted from the procedure for the N-C12 DOPE / DOPC liposomes, relatively unilamellar, relatively small but structurally heterogeneous liposomes were observed (Figure 7a), consistent with the Nicomp analysis. Some liposomes appeared tubular, probably as a result of the osmotic gradient generated during the preparation procedure. Some liposomes showed fiber-like interior structures possibly representative of non-condensed DNA (left arrow). Non-encapsulated free fibers (right arrow) could also be observed.
The samples of N-C12 DOPE / DOPC liposomes containing DNA prepared with spermine (Figure 7b, c) were also heterogeneous in size, shape and lamellarity. Some particles were normal-looking liposomes without visible encapsulated material. However, others contained electronically dense, well-defined toroidal structures (Figure 7b, arrows) that were not observed in samples without spermine. Such structures were not related to the particular lipid that was used, such as the toroidal structures (Figure 7c, right arrow) and the flexed rod structures (Figure 7c, left arrow) were also observed in egg PC preparations, which tended to be more stable under the conditions of cryo-EM sample preparation. The separation between the fine lines within the rods and the toroids was uniform and significantly smaller than the separation between two membranes in the multilamellar (star) liposomes. These toroidal structures have greater similarity to the toroids and rods observed when the free DNA is condensed by spermine (Chattoraj et al., J Mol Biol, 121: 327-337 (1978)) or other condensing agents (Arscott et al., Biopoly erß, 30: 619-630 (1990); Gosule and Schellman, J Mol Biol, 121: 327-337 (1978)) in diluted solutions. The parallel and concentric fine lines visible within the rods and toroids also resemble the lines observed within the plasmid aggregates (Figure lb). The membranes could be clearly observed around one of the toroidal structures (for example, Figure 7b). It is very likely that all the observed toroids are encapsulated within an ion-impermeable barrier, since condensed DNA toroids can not exist in the high-salt buffer solution in which the liposomes were finally suspended. Therefore, it would appear that an important portion of these preparations consists of plasmid DNA encapsulated in the liposomes.
Example 9 - Analysis of agarose gel
The protection of plasmid DNA with DNase digestion was evaluated by agarose gel electrophoresis for plasmid DNA encapsulated in liposomes prepared with spermine and a control sample prepared without spermine. An aliquot of 50 μl of the desired preparation was diluted in 145 μl of HBSS without Ca 2+ / Mg 2+, and 1 μl of 0.2 M MgCl 2 plus 2 μl of DNase I (20 units) were added with mixing. After incubation for 6 hours at room temperature, 2 μl 0.5 M EDTA was added to interrupt the reaction. For undigested controls, an aliquot of 50 μl of each sample was mixed with 150 μl of HBSS (without Ca2 + / Mg2 +). The samples were then extracted with phenol / CHC13 / isoamyl alcohol and precipitated with ethanol as described (Sambrook et al., Molecular cloning: A labora tory manual, 2nd edition Cold Spring Harbor Laboratory: Cold Spring Harbor NY, pp B4-B5 (1984)). The pack was dissolved in 20 μl of TE (pH 8.0), 5 μl of which was loaded onto a 0.8% agarose gel. The gels were stained with a 1: 10,000 dilution of SYBR Green I nucleic acid gel staining (Molecular Probes) for 30 minutes, and visualized on a FotoSpectrum® ultraviolet transilluminator (luminous box). The photographs were taken in the light box with a Polaroid MP 4+ camera system. These photographs were then scanned on a ScanJet IIC® (Hewlett Packard, Palo Alto CA) and digitized with Aldus Photostyler® (U-Lead Systems, Torrance, CA). The results are presented in Figure 4 and demonstrate that both preparations allowed significant DNA protection or apparent encapsulation.
Example 10 - Quantification analysis
To quantify DNA protection, DNA was extracted from each aliquot and measured by a fluorescent assay. Fluorescent PicoGreen assays (Haugland, Handbook of fluorescent probes and research chemicals, 6th edition of Molecular Probes, Inc., pp. 161-162 (1996)) were used to quantify the DNA that had been extracted by the phenol / chloroform procedure established in the Example 9. A working solution was prepared by adding 100 μl of PicoGreen standard solution (Molecular Probes) to 20 ml of TE (pH 7.5). The extracted sample was first diluted 100 μg with TE (pH 7.5). Then an aliquot of 14 μl of the diluted sample was mixed with 986 μl of TE (pH 7.5) and 1 ml of the working solution PicoGreen. The mixture was incubated in the dark at room temperature for 4 minutes. The fluorescence of PicoGreen was recorded at room temperature in a PTl Alphascan fluorometer (South Brunswick, NJ), with an excitation wavelength of 480 nm, and an emission wavelength of 520 nm, with a high-pass filter > 500 nm (Schott Glass Technologies, Duryea, PA). The fluorescence of a mixture of 1 ml of TE (pH 7.5) and 1 ml of the working solution PicoGreen was used as target. The percentage of DNA protected from digestion with DNase I was calculated by subtracting the blank and taking the undigested sample as 100%. In our experimental conditions, the fluorescent signal of the digested DNA was negligible. The sample with spermine showed 10.1 + 5.6 percent protection of the plasmid, while 19.0 + 4.5 was protected in the sample without spermine.
Example 11 - Transfection assays
The transfection activity of the liposomal preparations encapsulating the plasmid DNA pEGFP-Cl was then tested. OVCAR3 cells were plated at 1 x
5 cells per ml in 24 well plates, or 2 x 105
cells per ml in 96-well plates in one ml or 0.1 ml per well, respectively, of RPMI 1640 with 10% heat-inactivated fetal bovine serum. The cells were allowed to grow for 2 days (approximately 40-48 hours) before carrying out the transfections; at this point, the cells were in confluence. Transfection solutions were prepared by diluting suitable samples of liposome or DNA in serum-free medium. The plates were aspirated to remove the medium and washed once with saline buffered with Dulbecco's phosphates followed by aspiration. The solutions for transfection (0.5 ml per well for 24-well plates), 0.1 ml per well for 96 well plates) were prepared by diluting the dialyzed samples containing the pEGFP-Cl plasmid 10 times in serum free medium (approximately 2 mM total lipid unless otherwise indicated), and then they were added to the wells and incubated at 37 ° C for 3 hours. The wells were aspirated, and the medium containing 10% heat inactivated fetal bovine serum was added to each well. Due to the silencing of the transgenes previously demonstrated under the CMV promoter (Tang et al., Human Gene Therapy, 8: 2117-2124 (1997)); Dion et al., Virology, 231: 201-209 (1997)) 5 μM of the histone deacetylase inhibitor, trichostatin A, was added to each well to improve expression. In the experiments presented in the last two figures, another inhibitor of acetylase histone, 5 mM sodium butyrate was used instead. After incubation at 37 ° C in a cell culture incubator for 18-22 hours, the medium was aspirated and washed with 0.5 ml aliquots of Dulbecco's PBS. The photomicrographs were taken from the samples still on the tissue culture plates with an Olympus IMT-2 inverted microscope using the lOx objective. The PBS was aspirated and 0.5 ml (0.1 ml for the 96-well plates) of 5 μM calcein acetoxymethyl ester (CBAM) in PBS was added to each well and incubated for 40 minutes at room temperature. The cells were washed again with PBS, aspirated and 0.5 ml (0.1 ml for the 96 well plates) of C 2 is added to each well. It is 1% in TE buffer (pH 8.0). The samples were then dissolved in detergent and the readings were taken for corrected total EGFP fluorescence, in terms of the total number of living cells. Fluorescence of the plates was measured in a Cytofluor II fluorescent plate reader (PerSeptive Biosystems, Framingham, MA). The readings for calcein blue loaded on live cells were made at an excitation of 360 nm and emission of 460 nm with a gain of 80. It was verified that these readings were linear with the number of cells originally plated to a level where it was observed confluence. For the data shown in Figure 10, a liposomal package separated from external DNA was used (Example 12). Because the Ca + and Mg + concentrations in RPMI 1640 are significantly lower than in serum, the data in Figure 11 and 12 were obtained after supplementation of serum free medium with Ca + and Mg + to obtain 1.2 and 0.8 mM, respectively, during transfection. An approximate conversion for EGFP fluorescence per unit cell protein can be estimated by measuring the average protein concentrations of 48-hour cultures of OVCAR-3 cells in 24 and 96 well experiments extracted with 1% Triton-100 detergent. An assay with bicinchoninic acid (Pierce Chemical, Rockford, IL) was used with bovine serum albumin as standard. For Figure 9, bar "a", the reading of the fluorescence corrected by the total average background per well was 670 units. From a separate plate, the average total cellular protein per well at the time of transfection (48 hours) was approximately 88.4 μg / well giving 7.6 fluorescence units per μg of the total cellular protein in a volume of 0.5 ml for the experiments in the 24-well plate. In Figure 10, the data for the "a" bar (96 well experiment), represents an EGFP fluorescence corrected by the average background of approximately 420 units per well with an average total cell protein concentration of 27 μg per well. , giving 15.5 units of fluorescence per μg of the total cellular protein in a total volume of 0.1 ml. In Figure 11, (experiment with 96 wells), the fluorescence reading of the "a" bar was 103 fluorescence units per μg of the cellular protein. To model the intraperitoneal delivery (data of Figures 11 and 12), transfection was performed by first adding 50 μl of a cell-free wash fluid, concentrate of the peritoneal cavity of tumor-bearing SCID mice (Example 13) to each well aspirated from a 96-well plate with OVCAR-3 cells grown as previously described. To each well was added 50 μl of a N-C12 DOPE / DOPC-DNA liposome formulation prepared as described in Example 3, and giving rise to a final lipid concentration of approximately 10 mM and a final encapsulated DNA concentration of approximately 7-14 μg / ml (total DNA 67 μg / ml). Incubations were performed as already described. In this case, the peritoneal lavage fluid was adjusted to approximately Ca + and Mg + concentrations in serum (1.2 mM and 0.8 mM, respectively), adding a concentrated standard solution. The liposomal-DNA solution was also adjusted to the same concentrations of Ca + and Mg + by the addition of the concentrated standard solution just before the addition of the liposomes to the cells. The data in Figures 8 and 9 demonstrate that the formulation of the N-C12 DOPE / DOPC (70/30) liposomes encapsulating spermine-condensed plasmid DNA was active in the transfection of OVCAR-3 cells. The data show that the activity was dependent on the presence of the condensing agent spermine and the encapsulation of the plasmid DNA within the liposomes. The data of Figure 10 again demonstrate that the transfection activity is associated with the DNA encapsulated in the lipid and not the free outer DNA. The data of Figures 11 and 12 show that transfection can also occur in the presence of potential interfering substances (e.g., serum proteins) that are found in the intraperitoneal site of OVCAR-3 tumors.
Example 12 - Sedimentation of plasmid DNA and lipid particles
To demonstrate the transfection activity of the encapsulated plasmid DNA it was necessary to separate the free plasmid DNA from the encapsulated DNA in liposomes. The following preparation method was used. The liposomes, prepared by sedimentation to remove the external DNA were used in the experiments, the results of which are shown in Figure 10. For the sedimentation experiments, samples of the N-C12 DOPE / DOPC liposomes were prepared (70:30 ) by the method of Example 3 with spermine, except that 200 mM sucrose was included in the LSB. Also added was rhodamine B-phosphatidylethanolamine (Rh-PE) lisamine labeled in the main group as a lipid probe at a concentration of 10 μg / ml. Then a 500 μl aliquot of the preparation was centrifuged at 16,000 x g for 3 hours. After the supernatant was removed, the package was resuspended in HBSS without Ca2 + / Mg2 +. The suspension was centrifuged at 16,000 x g for 3 hours. The package was resuspended in 500 μl of HBSS without Ca2 + / Mg2 +. Aliquots of 50 μl of each fraction were taken for digestion with DNase I (Example 9). After extraction with phenol / CHCl3 and precipitation with ethanol, the plasmid DNA in each aliquot was measured by the PicoGreen assay
(Example 10) and was used to calculate the percentage of the protected plasmid, and the percentage of the total plasmid DNA, in each fraction. For the measurement of the lipid distribution, an aliquot of 40 μl of each fraction in 0.2% C 2Eg was dissolved in a total volume of 2 ml and the fluorescence was monitored with an excitation wavelength of 560 nm with a band pass filter of 550 ± 20 nm (Melles Griot, Irvine, CA), and an emission wavelength of 590 nm. As a control, empty N-C12 DOPE / DOPC (70:30) liposomes were prepared as in the previous case. After dialysis, 100 μg of the EGFP plasmid was added to 500 μl of the sample. The sample was then centrifuged and quantified for lipids and plasmid DNA. Approximately 80% of the lipid was pelleted under these conditions, while only about 14% of the total DNA was pelleted. The transfection activity of the pelleted material is shown in Figure 10. The transfection activity was clearly associated with the lipid pellet, that is, with the DNA encapsulated in the liposomes.
Example 13 - Washing fluid
To test the effect of the intraperitoneal proteins on the transfection activity of the preparations described herein, a wash fluid was prepared as described hereinafter. 6 ml of washing was taken with cell-free HBSS from a SCID mouse 7 weeks after injection of OVCAR-3 cells and concentrated to 1 ml with a shaker concentrator with a cut-off of 10,000 μm. The recovery of the protein is approximately 60%. This fluid containing approximately 10 mg / ml of protein in HBSS was complemented are Ca + and Mg + up to the normal serum range, added to cultured OVCAR-3 cells and mixed lightly in an equal volume with liposomes in HBSS to the same concentration of Ca / mg, to obtain a final lipid concentration of 10 mM. The results of these transfection experiments are shown in Figures 11 and 12. Despite the known inhibitory effects of serum proteins on the efficiency of transfection, substantial activity remained under these conditions using the formulation prepared by the method of the invention. present invention.
Example 14 - Efficiency of liposome loading
The loading efficiency of the liposomes using the precondensed DNA method was purchased with the method described herein. The liposomes were prepared as described by Ibanez et al., Biochem Cell Biol, 74: 633-643 (1996). Plasmid DNA pEGFP was dissolved in 66 μg / ml in TS buffer (10 mM Tris, 1 mM NaCl, pH 7.0). 2 ml of this solution were mixed with 2 ml of 23 mM spermidine in TS buffer to precondense the DNA producing a very cloudy solution. This was stored overnight at 4 ° C. The next day, a total of 9 μmol of the lipid was dissolved in 1 ml of diethyl ether. To this was added 330 μl of the DNA and spermidine solution with vortexing. The mixture was then immediately sonicated 3 times for 5 seconds each time (Laboratory Supply Sonicator # G112SOI). The diethyl ether was then removed using a rotary evaporator at 37 ° C to form the liposomes. Four of these samples were prepared for each lipid composition. The liposomes were pelleted at 200,000 x g for 30 minutes. The supernatant was removed and 500 μl more of TS buffer was added and the centrifugation was repeated. After three total cycles, the liposomes were extruded through MF membranes with pores of 0.45 μ. The liposomes were used for the determination of the encapsulation at this point or a portion of these was dialyzed against balanced salt solution of Hank without Ca + and Mg +. For comparison the liposomes were also prepared as described in Example 3. DNA encapsulation was measured as described in Examples 9 and 10. All digestions were for 6 hours. The concentration of lipids was measured by HPLC. All components minus cholesterol were quantified using a Waters Sherisorb silica column (3 μ) with a mobile phase of acetonitrile: methanol: H S04, 100: 3: 0.05, and detected by UV absorbance. The cholesterol was measured on a Phenomenex Luna C18 column (5 μ) with a mobile phase of 96: 4 methanol: water and detected by an elastic light scattering detector. The tested lipid compositions are provided in the following table.
Table 1
Na / lipid ratio: (μg / DNA / μmol total lipid) *
* Lipid recovery estimated by HPLC. DNA quantified by extraction and PicoGreen assay.
** Ibanez, M., Gariglio, P., Chavez, P., Santiago, CW and Baeza, I. (1996) "Spermidine-condensed DNA and cone shaped lipids improbé delivery and expression of exogenous DNA transfer by liposomes", Biochem Cell Biol, 74: 633-643 (1996).
Liposomes prepared by condensation of DNA in an emulsion as described herein gave rise to DNA: lipid ratios much higher than liposomes prepared with precondensed DNA.
Example 15 - Determination of lamellarity of liposomes
The liposomes composed of N-C12-DOPE / DOPC 70:30 and encapsulating plasmid DNA were prepared as described in Example 3 and were pelleted and washed as in Example 12. These liposomes also contained a NBD probe at a concentration of 0.5 mole% of total lipids. The liposomes were diluted to a total lipid concentration of 80 μM in phosphate buffered saline in a shaking fluorometer cuvette. The fluorescence of NBD was measured with excitation at 450 nm and emission at 530 nm. A final concentration of 20 mM sodium dithionite was injected into the cuvette with the liposomes to reduce the exposed NBD probe. Figure 13 demonstrates that approximately 50-55% of the NBD signal disappeared indicating that the liposomes in the preparation were mainly unilamellar, ie, approximately half of the lipid probes were exposed to the membrane-impermeable reducing agent, dithionite from sodium.
Example 16 - Transfection of subcutaneous human tumor in SCID mice by intratumoral injection
Human OVCAR-3 cells (2 x 10) were injected subcutaneously into SCID mice and allowed to grow for a few weeks to an average diameter of about 4-7 mm. Liposomes containing spermine, the plasmid pEGFP-Cl and 70% molar of N-C12-DOPE and 30 mole% of DOPC were prepared as in Example 3. The liposomal membranes also contained 0.5 mole% of rhodamine-PE as a fluorescent marker of the liposomes. 0.11 ml of a solution of the liposomes in Hank's balanced salt solution without calcium or magnesium (HBSS) at a total lipid concentration of approximately 40 mM was injected directly into the center of the tumors after adjustment of Ca + concentrations and Mg + at 1.2 and 0.8 mM, respectively. One day later, 0.11 ml of sodium butyrate in 20 mM HBSS was injected into the same sites. After 24 hours, the tumors were excised and frozen. Then, sections of 14-60 μ thickness were obtained on a cryostat instrument at -20 ° C and they were mounted frozen on glass slides and secured with a sliding cover. The samples of frozen tumors were mounted in O.C. T-embedding medium. The transgenic expression of the plasmid pEGFP-Cl was assessed by confocal microscopy of cryosections of 20 μ of frozen tissues, fixed. The frozen sections were examined using the Olympus BX50 / Biorad MRC 1000 confocal microscope with an argon / krypton laser (Ex 488nm, Em 515 for EGFP, Ex 568, Em 585 for rhodamine). The areas of the tissue sections formed an image with a 20x amplification. No improvements were used for the image but a color scale was applied for the preparation of the figure. Figure 14 shows a pair of fluorescent images of a section of tissue taken from a tumor treated with the plasmid pEGFP-Cl. The lower figure shows red fluorescence of the rhodamine-labeled liposomes. The very high lipid signal (yellow) suggests that this section was almost the signal of liposome injection. The upper figure shows green fluorescence due to the transgenic expression. The signal of the expressed plasmid is almost, but does not match, with the lipid signal, and appears to represent actual expression of the plasmid in the tumor. Figure 15 shows another pair of fluorescent images of a different tumor section with expressed EGFP. Figure 16 shows a pair of fluorescent images of a cryosection of the control tumor tissue. The weak green fluorescence, diffuse inherent in the tissue is visible in the upper figure, but not the area of intense fluorescence that was found in any section of tissue control. There was no red fluorescence in any control tumor section.
Example 17 - Transfection of mouse muscle in vivo
The transfection activity of the NC12-DOPE / DOPC liposomes (70:30) encapsulating the plasmid pZeoSVLacZ was tested in vivo in muscle of the mouse paw. The liposomes were prepared as described in Example 3. DBA female mice housed under normal conditions were used for this experiment. 50 μl of liposomes containing the plasmid pZeoSVLacZ were injected directly into the muscle of a hind paw on day one. The injection site was close to the anterior muscle of the leg. The opposite leg received 50 μl of liposomes with the plasmid pEGFP-Cl or without treatment. On day 2, 50 μl of 20 mM sodium butyrate in HBSS was injected into the legs treated with liposomes. The mice were sacrificed on the third day and the leg muscle was excised as four sections: anterior leg, hind leg, anterior thigh and posterior thigh. One half of the tissue of each section was immediately frozen in liquid propane, while the other half was fixed in 4% paraformaldehyde, cryoprotected with 30% sucrose then instantly frozen in propane. The transgenic expression of the pZeoSVLacZ plasmid supplied by the N12-DOPE / DOPC [sic] liposomes (70:30) was evaluated using the Clontech luminescent ß-gal kit. The unfixed muscle was thawed, cut into sections and homogenized as follows. 15 ml of lysis buffer (9.15 ml of K2HP04, 0.85 ml of KH2P04, 20 μl of Triton XlOO, 10 μl of DTT) was added for each 1 mg of wet tissue and homogenized by hand for 15 minutes. Then it was incubated at room temperature for 20 minutes. The samples were then centrifuged for 2 minutes at 14,000 rpm to package the tissue debris. The aliquots of the supernatants were assayed for the β-galactosidase activity as described by Clontech. The light readings were measured after 60 minutes using a Berthold plate luminometer. The readings were averaged for different muscle sections of the same type. The results are shown in Figure 17. A significant increase in the activity of ß-galactosidase over control was found in sections of muscle of the anterior thigh. The slight increases were also observed in the hind leg and the posterior thigh tissue. These results demonstrate in vivo transfection and transgenic expression of the pZeoSVLacZ plasmids when delivered to the mouse muscle using the liposomal vector N12-DOPE / DOPC [sic] (70:30).
Example 18 - Comparison of transfection with condensed, liposomal and cationic lipoplex DNA
Preparation of cationic lipoplex:
The complexes of cationic lipids and helper lipids with plasmid DNA were prepared shortly before use. Lipofectin was purchased from Gibco BRL (Grand Island, N. Y.). For lipofectin the lipid was incubated only in serum-free medium for approximately 45 minutes before DNA complexation, as suggested by the manufacturer (Invitrogen). Equal volumes of 4 μg / ml of DNA and 40 μg / ml of lipid or equal volumes of 20 μg / ml of DNA and 200 μg / ml of lipid, all in serum-free RPMI 1640 medium, were mixed and allowed to incubate for approximately 10-15 minutes before the addition to the wells of the tissue culture plates. The Ca + and Mg + were adjusted to a final concentration of 1.2 mM and 0.8 mM, respectively, by the addition of a concentrated standard solution just before the addition of the lipoplejos to the cells. The lipid / DNA ratio used for lipofectin was based on an optimization comparing some relationships. The DC-cholesterol / DOPE complexes (4/6) were formed practically as already described (Muldoon et al., Biotechniques 22, 162, 167 (1997)) and used for the next 15 minutes. The optimized DNA / lipid ratio was used in all the experiments, that is, 4 μg / ml of DNA were mixed with an equal volume of 20 μg / ml of lipid or 20 μg / ml of the DNA was mixed with an equal volume of 100 μg / ml of the lipid. The other complexes were formed using a series of cationic lipids or lipid mixtures from a single manufacturer (Invitrogen). These were prepared as suggested by the manufacturer at the concentration lx and the suggested lipid / DNA ratios. Transfection assays were performed as described in Example 11.
Comparison with cationic lipoplejos:
The free pelleted liposomes of the external DNA were used for direct comparison of the transfection with the cationic lipoplexes at equal concentrations of the DNA. These data are presented in Table 2 in relation to liposomal treatment (all data after incubation in sodium butyrate, "a non-toxic activator of transgenic expression".
(Tang et al., Human Gene Therapy, 8: 2117-2124 (1997);
Wheeler et al., Biochim Biophys Acta 1280 (1996); Gruner et al., Biochem 24: 2833-2842 (1984)) and physiological concentrations of Ca + and Mg +; all data were normalized in terms of total intracellular esterase activity). In Table 2, cell viability and transfection are taken as 1.0 for the N-C12-D0PE / D0PC liposomes, ie, numbers greater than 1.0 represent the factor by which any of these parameters is greater in the test system . The transfection activity of the N-C12-D0PE-DOPC liposomes (70:30) was, in general, in the range that was found for cationic lipoplexes under these conditions. Some lipoplejos gave considerably less activity and some with considerably greater activity. The lipoplexes containing 3β [N- (dimethylaminoethane) -carbamoyl] cholesterol and dioleoyl phosphatidylethanolamine (DC-chol / DOPE) were particularly active. However, like all cationic lipoplexes, these were considerably more toxic than the liposomes for the particular cells used in these experiments, especially at higher concentrations. This could be observed in fluorescence of lower calcein blue after treatment with lipoplexes (data from Table 2) as well as microscopic observation of rounded and broken cells after treatment (data not shown). In some cases, the efficiency of transfection of cationic lipoplexes actually decreased in relation to liposomes at higher concentration, probably as a result of their toxicity. No toxicity was observed with DNA encapsulated in the liposomes. It is interesting to note that treatment with liposomes commonly caused an increase in the final fluorescence of calcein blue between 10 and 30%, possibly as a result of the protection of the effects of incubation in serum-free medium. The importance of the relatively low toxicity of this liposomal DNA plasmid delivery system is not completely evident in tissue culture systems because the efficiency of transfection reaches saturation at relatively low concentrations of the DNA used in the previous experiments. However, it is expected that the situation in vivo will be very different. The large excess of nonspecific binding sites in vivo may necessitate the use of high DNA concentrations and / or multiple injections for efficient expression in the chosen cells. There may be a limit to the use of cationic lipoplexes in this situation due to its toxicity. OVCAR-3 cells were incubated with washed liposomal pellets (as in Figure 10) or lipid complexes with equal amounts of plasmid DNA pEGFP-Cl for 3 hours in medium without serum. All transfection procedures were as described in Example 11 and include adjustment of Ca + and Mg + concentrations to 1.2 and 0.8 mM, respectively.
a The cationic lipid complexes were prepared with the following lipids: # 1- 1: 1 mixture of Tris- ((2-glutaroyl-4-amino--dioctadecylamine) -4 '- (2,5-diaminopentanoyl- (2", 5"-diaminopropylethyl)) amine, trifluoroacetate and 2-amino- (2 ', 2'-dimethyl) ethyl-methylphosphonic acid-O-octadecyl- (1'-heptadecyl) ester, trifluoroacetate (Pfx-1); 2, 5-diaminopentanoyl-glycyl-glycyl-N-octadecyl- (1 '-heptadecyl) amide, trifluoroacetate (Pfx-2); # 3 - 1: 1 mixture of 2,5-diaminopentanoyl- (2', 3 '- di-3-aminopropyl) -2-aminoacetyl-2-aminoacetyl-N-octadecyl- (1 '-heptadecyl) amide, trifluoroacetate and DOPE (Pfx-3); # 4 - 1: 1 mixture of 0-octadecyl- (1'-heptadecyl) 2-amino- (2 ', 2'-dimethyl) ethyl-methylphosphonic acid ester, trifluoroacetate and 2,5-diaminopentanoyl-2-aminoacetyl- N-dioctadecyl amide, trifluoride (Pfx-4); # 5 - 1: 1 mixture of 2,5-diaminopentanoyl- (2,5-di-3-aminopropyl) -glutaroyl- -octadecyl- (1'-heptadecyl) amide, trifluoroacetate and 2,5-diaminopentanoyl- (2, 2,5, 5-tetra-3-aminopropyl) -glycyl-N-dioctadecyl amine, trifluoroacetate (Pfx-5); # 6 - 1: 1 mixture of 2,5-diaminopentanoyl- (2,5-di-3-aminopropyl) -1,2-diaminoethyl-O-octadecyl- (1'-heptadecyl) carbamic acid, trifluoride and DOPE (Pfx) -7); # 7 - bis- (2,5-diaminopentanoyl- (2, 5-di-3-aminopropyl) cystyl-N-dioctadecyl amine) disulfide, trifluoroacetate (Pfx-8); # 8 Lipofectin; # 9 - DC-cholesterol / DOPE 4/6.
b The data are expressed in relation to the liposomes containing? -acil-PE, taken as 1.0, that is, the numbers represent the factor by which each lipoplex is more or less toxic or active. Data from more than one series of experiments were compared using lipid # 2 as a standard.
c The transfection efficiency was measured by EGFP fluorescence as in Figure 11 and corrected for total cell esterase esterase as reflected in the total calcein blue fluorescence (see Example 11).
One skilled in the art will readily realize that the present invention is well suited to carry out the objectives and obtain the purposes and advantages mentioned, as well as those inherent therein. The compounds, compositions and methods, methods and techniques described herein are presented as representative of the preferred embodiments, or are suggested to be exemplary and are not intended as limitations on the scope of the present invention. Changes in the present and other uses will be apparent to those skilled in the art and will be understood within the spirit of the attached clauses.
Claims (1)
- CLAIMS A method for encapsulating a bioactive complex in a liposome, which comprises the steps of: (a) dissolving at least one unfriendly lipid in one or more organic solvents (b) combining a first aqueous suspension containing a bioactive agent with a lipid containing an organic solution of step (a) to form an emulsion comprising the bioactive agent and the lipid (c) adding a second aqueous suspension comprising a complexing agent to the emulsion of step (b) (d) incubating the emulsion of step (c) to allow the complexing agent to make contact with the bioactive agent thereby forming a complex of the bioactive agent with the complexing agent within the water drops stabilized with lipid, wherein the complex is not greater in diameter than the diameter of the drop, and (e) removing the organic solvent from the suspension of step (d), to form liposomes comprising the complexed bioactive agent and the lipid. A method for encapsulating a bioactive complex in a liposome comprising the steps of: (a) dissolving at least one unfriendly lipid in one or more organic solvents (b) combining a first aqueous suspension containing a complexing agent with the lipid containing an organic solution of step (a) to form an emulsion comprising the complexing agent and the lipid (c) adding a second aqueous suspension comprising the bioactive agent to the emulsion of step (b) (d) incubating the emulsion of step (c) to allow the complexing agent to make contact with the bioactive agent thereby forming a complex of the agent bioactive with the complexing agent within the drops of water stabilized by the lipid, where the complex is no larger in diameter than the diameter of the drops, and (e) removing the organic solvent from the suspension of step (d), to form liposomes comprising the complexed bioactive agent and the lipid. The method of claim 1, wherein the bioactive agent is a nucleic acid. The method of claim 1, wherein the nucleic acid is DNA.
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