Method of binding recognizing substances to liposomes

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BACKGROUND OF THE INVENTION

The present invention relates to the preparation of microscopic drug delivery systems (MDDS) utilizing drug-encapsulating bioadhesive liposomes.

Currently, the topical and local administration of a drug can be in its free form, dissolved or dispersed in a suitable diluent, or in a vehicle such as a cream, gel or ointment. By definition, "topical" administration includes non-invasive drug administration, while "local" includes invasive, i.e., through a localized injection or infusion. Examples of therapeutic or designated targets for topical or local drug administration include burns; wounds; bone injuries; ocular, skin, intranasal and buccal infections; ocular chronic situations such as glaucoma; intraperitoneal infections, tumors and metastasis; and topically and locally accessed tumors. Several difficulties exist with either the topical or local administration of a drug in its free form. For example, short retention of the drug at the designated site of administration reduces the efficacy of the treatment and requires frequent dosing. Exposure of the free form drug to the biological environment in the topical or local region can result in drug degradation, transformation into inactive entities and nondiscriminating and uncontrollable distribution of the drug. Such degradation and uncontrollable distribution of the drug can result in toxicity issues, undesirable side effects and loss of efficacy.

Microscopic drug delivery systems (MDDS) have been developed for improved drug administration relative to administration of drugs in their free form. Drug-loaded MDDS can perform as sustained or controlled release drug depots. By providing a mutual protection of the drug and the biological environment, MDDS reduces drug degradation or inactivation. As a system for controlled release of a drug, MDDS improves drug efficacy and allows reduction in the frequency of dosing. Since the pharmacokinetics of free drug release from depots of MDDS are different than from directly-administered drug, MDDS provides an additional measure to reduce toxicity and undesirable side effects.

MDDS is divided into two basic classes: particulate systems, such as cells, microspheres, viral envelopes and liposomes; or nonparticulate systems which are macromolecules such as proteins or synthetic polymers. Liposomes have been studied as drug carriers and offer a range of advantages relative to other MDDS systems. Composed of naturally-occurring materials which are biocompatible and biodegradable, liposomes are used to encapsulate biologically active materials for a variety of purposes. Having a variety of layers, sizes, surface charges and compositions, numerous procedures for liposomal preparation and for drug encapsulation within them have been developed, some of which have been scaled up to industrial levels.

Liposomes can be designed to act as sustained release drug depots and, in certain applications, aid drug access across cell membranes. Their ability to protect encapsulated drugs and various other characteristics make liposomes a popular choice in developing MDDS, with respect to the previous practices of free drug administration.

Despite the advantages offered, utilization of drug-encapsulating liposomes does pose some difficulties. For example, liposomes as MDDS have limited targeting abilities, limited retention and stability in circulation, potential toxicity upon chronic administration and inability to extravasate. Binding of chymotrypsin to liposomes has been studied as a model for binding substances to liposomal surfaces. Recognizing substances, including antibodies, glycoproteins and lectins have been bound to liposomal surfaces in an attempt to confer target specificity to the liposomes. Concentrating on systemic applications and in vivo studies, these previous efforts discuss methods of binding recognizing substances with liposomes and the effectiveness of such modified liposomes. Although the bonding of these recognizing substances to liposomes occurred, the resulting modified liposomes did not perform as hoped, particularly during in vivo studies. Other difficulties are presented when utilizing these recognizing substances. For example, antibodies can be patient specific and, therefore, add cost to the drug therapy.

In addition to the problems outlined above, the prior art has failed to disclose an efficient and effective method of making bioadhesive liposomes useful for scaling-up to an industrial level. In "Preparation of EGF Labeled Liposomes and Their Uptake by Hepatocytes," Ishii et al., Biochemical and Biophysical Research Communications, Vol. 160, pp. 732-36, 1989 ("Ishii et al."), the authors describe uptake of EGF-bearing liposomes by liver cells in suspension. In the preparation of their liposomes, Ishii et al., disclose a procedure involving at least four different steps, each individually involving at least two more sub-steps. These steps include further purification by column chromatography, which can be difficult to scale-up to an industrial level. Furthermore, not only is this process cumbersome, but each additional step contributes to a loss of material or possible inactivation of the EGF. It has been reported that the biological activity of EGF is dependent upon the conservation of the native conformation of EGF, to which the disulfide bonds are critical. In binding EGF to liposomes, Ishii et al. exploited the existence of the disulfide bonds. Specifically, EGF was bound to the liposomal surface by the disulfide bridge linkage using a heterobifunctional crosslinking reagent, N-hydroxysuccinimidyl-3-(2-pyridyldithio) propionate. The complex chemistry of this process results in byproducts whose effect on drug delivery and toxicity are unknown, possibly resulting in inactivation of the EGF. Further, the complex process described by Ishii et al. would be virtually impossible to accomplish in an aseptic environment, as required in a liposome process.

Prior to the development of the present invention, a need existed for a liposome having targeting and retention abilities to a target organ or tissue. Specifically, there remains a need for the development of a "bioadhesive" liposome comprising a liposome having an effective recognizing substance attached thereto. Prior to the present invention, a need also existed for an efficient method for binding recognizing substances to a liposome thereby producing a bioadhesive liposome, using fewer steps than those described in the prior art.

SUMMARY OF INVENTION

According to the present invention, efficient methodologies have been developed to effectively bind various recognizing substances. These include, and are not limited to, collagen, gelatin, hyaluronic acid and epidermal growth factor to liposomal surfaces thereby forming bioadhesive liposomes. Further, the methods of the present invention employ fewer steps than known in the art, thus making such methods more efficient and cost-effective on a commercial scale. Further, the process described in the present invention avoids the risk of inactivating the recognizing substance during creation of the bioadhesive liposome. The bioadhesive liposomes of the present invention have specificity for and the ability to adhere to the designated target area for sustained release of the liposome's therapeutic contents.

The improved process of the present invention includes adding a recognizing substance to a liposome; adding a crosslinking reagent to the mixture of the liposome and recognizing substance; and, allowing the mixture to incubate for a period of time to form the bioadhesive liposome. By modifying regular liposomes through covalent bonding of certain recognizing substance to the liposomal surface, the recognizing substances can be utilized as an adhesive or glue to attach and retain the modified liposome onto a target area despite cellular and fluid dynamics. These "bioadhesive" liposomes offer potential advantages as a MDDS for the administration of drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of bioadhesive liposomes (EGF-modified; open double triangle) and regular liposomes (asterisk) of the LUVET type to A431 cells in culture (in monolayers), as dependent upon liposome concentration. Bound liposomes, denoted as B, are in units of ng EGF per 10.sup.6 cells. Free ligand concentration, denoted as L, are in units of ng EGF per 10.sup.6 cells for bioadhesive liposome (first row of L values) and in units of umoles lipid per 10.sup.6 cells for the regular liposomes (second row of L values).

FIG. 2 shows a time course of the binding of bioadhesive liposomes (collagen-modified) of the MLV type to A431 cells in culture (in monolayers). Collagen is tritium-labeled. The fraction of liposomes relative to the amount present in the initial reaction mixture at zero-time which is cell-associated is determined over time.

FIG. 3 shows the binding of bioadhesive liposomes (collagen-modified) and regular liposomes of the MLV type to A431 cells in culture (in monolayers). Collagen is tritium-labeled (.sup.3 -H) and liposomes are .sup.14 -C labeled. Bound liposomes, denoted as B, are in units of .sup.3 -H DPM per 10.sup.5 cells (left scale) and in units of .sup.14 -C DPM per 10.sup.5 cells (right scale). Free ligand concentration, denoted as L, are in units of .sup.3 -H or .sup.14 -C DPM per 10.sup.5 cells. Bioadhesive liposome with collagen labeled is depicted with open double triangles; bioadhesive liposome with the liposome labeled is depicted with crosses; and, regular liposome is depicted with asterisks.

FIG. 4 shows a schematic drawing of the experimental setup for studying the effects of fluid dynamics on cultures of adherent cells having bioadhesive liposomes attached thereto.

DETAILED DESCRIPTION

According to the present invention, various recognizing substances have been covalently bound to liposomal surfaces through the crosslinking of amine residues. Liposomes, in particular, multilamellar vesicles (MLV) or unilamellar vesicles such as microemulsified liposomes (MEL) and large unilamellar liposomes (LUVET), each containing phosphatidylethanolamine (PE), have been prepared by established procedures. The inclusion of PE in the liposome provides an active functional residue, a primary amine, on the liposomal surface for crosslinking purposes.

Recognizing substances have been successfully linked with PE-liposomes. Recognizing substances useful in the present invention include collagen, gelatin, hyaluronic acid (HA) and epidermal growth factor (EGF). Using commercially available gelatin and collagen, these protein-recognizing substances were linked to the liposomes through amine residues. Hyaluronic acid is a natural polymer with alternating units of N-acetyl glucoseamine and glucoronic acid. Using a crosslinking reagent, hyaluronic acid offers carboxylic acid residues as functional groups for covalent binding. The N-acetyl-glucoseamine contains hydroxyl units of the type --CH.sub.2 --OH which can be oxidized to aldehydes, thereby offering an additional method of crosslinking hyaluronic acid to the liposomal surface in the absence of a crosslinking reagent. EGF is a polypeptide. Although urogastrone and EGF are recognized as biological equivalents, both purified urogastrone or EGF mouse were used as recognizing substances. When used in the specification and claims, the term "EGF" means either urogastrone or epidermal growth factor regardless of the source.

EGF stimulates cell growth and proliferation through interaction with an EGF receptor. EGF receptors are distributed on the cell surface of various organs and are present in burns, wounds, and other designated targets of MDDS such as ocular, dermal and tumors. Accordingly, EGF-modified liposomes potentially offer efficiency as drug carriers to target sites, i.e., organs or tissues, expressing the EGF receptors.

Recognizing substances are bound covalently to discrete sites on the liposome surfaces. The number and surface density of these sites will be dictated by the liposome formulation and the liposome type. The liposomal surfaces may also have sites for noncovalent association. Covalent binding is essential as noncovalent binding might result in dissociation of recognizing substances from the liposomes at the site of administration since the liposomes and the bioadhesive counterparts of the target site (the bioadhesive matter) compete for the recognizing substances. Such dissociation would reverse the administered modified liposomes into regular, non-modified liposomes, thereby defeating the purpose of administration of the modified liposomes.

To form covalent conjugates of recognizing substances and liposomes, crosslinking reagents have been studied for effectiveness and biocompatibility. Crosslinking reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and a water soluble carbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Through the complex chemistry of crosslinking, linkage of the amine residues of the recognizing substance and liposomes is established.

An important feature of the present invention is the binding between the newly-created bioadhesive liposome and potential biological target sites. Biological target sites are divided into two classes. The first class encompasses components of the extracellular matrix (ECM). The ECM can be visualized as a network made of a variety of components, which is not cast loose in a living system, but is connected at some of its points to cells. ECM is found underneath cells, above cells, in between layers of cells, and in between cells in a layer. The second class of potential targets are membrane-embedded receptors.

A complete accounting of binding entities has been determined by the previously known multi-term Langmuir Isotherm equation, as applied for the quantitative description of the relationship between the free and dependent variables: ##EQU1## where n is the number of different types of binding entities that a cellular or an ECM system target site has for a specific recognizing substance; [L] is the concentration of free ligand, which can be recognizing substance, free liposomes or bioadhesive liposomes; B is the total quantity of bound recognizing substance per given number of cells or quantity of ECM, at a given [L]; and, Bmax.sub.i and Kd.sub.i are the total number of sites of a given entity and the corresponding equilibrium dissociation constant. B and B.sub.max are normalized for the same number of cells or quantity of ECM.

For cases in which receptors and non-receptor cell membrane components participate in the recognizing substance binding and in which the dissociation constant of the non-specific binding is sufficiently large with respect to the free ligand concentration, equation 1 can take the form: ##EQU2## where the last term, K.sub.ns [L], is the contribution of the non-specific binding to B and K.sub.ns is the ratio of Bmax to Kd corresponding to the non-specific binding.

"Best-fit" values for parameters n, Bmax.sub.i and Kd.sub.i are obtained by computer-aided data analysis, according to equations (1) and/or (2) above, applying procedures of nonlinear regression analysis.

The interaction of the bioadhesive liposomes with potential biological targets has been established through the use of cultures of A431 cells, in monolayers, as a biological model. This well-established cell line, originating from human epidermoid carcinoma, is enriched with EGF receptors, and as a monolayer, also provides ECM. A431 cells have been repeatedly used for study of the interaction of free EGF and its receptor. A431 cells have been shown to have three classes of EGF receptors, differing in their affinities and populations. The first of these classes is the ultra-high affinity sites with an equilibrium dissociation constant of 0.07 nM and a population of 150-4000 sites per cell. The next class is the high affinity sites with an equilibrium dissociation constant of 0.7 nM and a population of 1.5.times.10.sup.5 sites per cell. The final class is the low affinity sites with an equilibrium dissociation constant of 5.9 nM and a population of 2.times.10.sup.6 sites per cell. Because of their affinity for EGF, A431 is particularly useful for demonstrating the targeting ability of EGF-modified liposomes. However, targeting of bioadhesive liposomes having other types of recognizing substances has also been demonstrated with this cell line, as shown in the following Examples.

The "level of covalent binding" as reported in the Examples below is defined as the quantity of bioadhesive ligand, such as collagen, gelatin, hyaluronic acid or EGF bound to a given quantity of lipid in the final product since the most accurate quantitative measure of liposomes is in terms of lipid quantities. For a given lipid quantity, different liposome types will yield different quantities of liposome. Therefore, similar initial ratios of EGF to lipid for different liposome types should not be expected to yield the same level of binding. Another factor which would yield different results for different liposomes even under the same initial EGF to lipid ratios, is the differences in particle size, therefore in curvature, number and accessibility of PE sites on the surface of the liposome. Therefore, comparisons among liposome types should be avoided.

The effects of the increase in the EGF/lipid ratios in the presence of a crosslinking reagent are shown below in Tables 1 and 2. Generally, an increase in the level of binding occurs with the increase in initial EGF/lipid ratios regardless of which crosslinking reagent is used. At the lower end of the EGF/lipid ratios, the level of covalent binding increases significantly. Beyond initial concentration ratios of 25 ng EGF/uMoles lipid, the increase of binding is less significant. Noncovalently bound product is removed as excess unreacted material and does not appear in the reported results.

EXAMPLES

All of the following Examples using collagen, gelatin and EGF as the recognizing substances, were prepared according to the method described in Example One. Slight modifications, as described in the Examples, were required for the Examples using hyaluronic acid as the recognizing substance. The recognizing substances are assayed by traces of radioactive or fluorescent labels. Alternatively, the lipids are assayed by colorimetric methods. Determination of the protein recognizing substances can be done by the Lowry procedure, while free HA and liposome-bound HA can be determined by the Alcian Blue method.

Example One

EGF is added to a PE-liposome sample and the mixture is buffered by a phosphate buffer saline solution (PBS) to pH of 7.2. For drug-containing liposomes, drug encapsulation was performed in a swelling solution also of PBS. Concentration ratios of EGF to lipid are shown in Table 1. Aliquots from a 25% solution of the crosslinking reagent glutaraldehyde (GAD) are added at a ratio of 10 ul per 1 ml EGF/PE-liposome mixture. Incubation for a desired period (24-72 hours) is completed at either room temperature without stirring or at 37.degree. C. with stirring. Depending upon the liposome used, excess unreacted material was removed, preferably through high speed centrifugation for one hour at 4.degree. C. and 27000 xg or ultrahigh centrifugation for one to two hours, at 4.degree. C. and 250000 xg followed by several repeated washings with EGF-free PBS. Column chromatography or dialysis against PBS may also be used in place of the centrifugation.

TABLE 1 ______________________________________ EGF-LIPOSOME CROSSLINKING BY GAD ngEGF/uMOLE LIPID (a) LIPOSOME TYPE INITIAL FINAL ______________________________________ MLV 0.080 0.009 MLV 0.309 0.006 MLV 0.347 0.016 MEL 0.071 0.004 MEL 0.106 0.009 MEL 0.141 0.025 LUVET 0.016 0.003 ______________________________________ (a) EGF assayed by a radioactive tracer.

Example Two

EGF is crosslinked with PE-liposome samples following the same procedure as in Example 1. Concentration ratios of labeled EGF to lipid are shown in Table 2.

TABLE 2 ______________________________________ EGF-LIPOSOME CROSSLINKING BY GAD ng EGF/uMOLES LIPID (a) LIPOSOME TYPE INITIAL FINAL ______________________________________ MLV 0.26 0.07 MLV 0.78 0.16 MLV 1.60 0.21 MLV 6.00 0.31 MLV 24.70 0.35 ______________________________________ (a) EGF assayed by a fluorescent tracer.

Example Three

Reaction mixtures of EGF and PE-liposomes were prepared as above and buffered by PBS to pH 7 or by 0.5N carbonate buffer to pH 9. Concentrations ratios of EGF to lipid are shown in Table 3. The crosslinking reagent EGDE was added in 0.2-1.0 ml volumes to buffered reaction mixtures of 2.5-3.0 ml volumes. Incubation periods were completed for 10-24 hours at 37.degree. C. with stirring. Depending upon the liposome used, excess unreacted material was removed through high speed centrifugations and washings or dialysis against PBS.

TABLE 3 ______________________________________ EGF-LIPOSOME CROSSLINKING BY EGDE ng/EGF/uMOLE LIPID (a) mg LIPOSOME TYPE INITIAL FINAL pH EGDE ______________________________________ MLV (b) 0.45 0.0078 9 500 MLV 3.72 0.90 9 500 MEL 0.10 0.012 9 500 MEL 0.10 0.0098 9 1000 MEL (a) 0.12 0.0022 7 200 MEL 1.78 0.47 9 500 ______________________________________ (a) EGF assayed by a radioactive tracer. (b) Initial ratios were increased by decreasing lipid concentration.

From these results, the preferred pH of 9 and quantity of crosslinking reagent of 500 mg has been determined.

Example Four

Gelatin was crosslinked to PE-liposomes following the same procedure as in Example One.

TABLE 4 ______________________________________ GELATIN-LIPOSOME CROSSLINKING BY GAD Liposome ugGelatin/uMole Lipid Incubation Type Initial Final Period (a) ______________________________________ MEL 21 0.02 Short MEL 63 0.24 Short MEL 127 0.26 Short MEL 21 15 Long MEL 23 14 Long MEL 25 18 Long MEL 63 43 Long MEL 187 208 Long MLV 18 0.24 Long MLV 66 0.67 Long MLV 281 2.6 Long MLV 556 6.4 Long MLV 1140 13 Long MLV 2350 13 Long MLV 3440 24 Long MLV 5830 26 Long ______________________________________ (a) Incubation Periods: "Short" is 5 minutes; "Long" is 24-48 hours.

Example Five

Collagen is crosslinked to PE-MLV samples with GAD following the same procedure as in Example 1 except incubation was at 4.degree. C., at "Long" incubation periods.

TABLE 5 ______________________________________ COLLAGEN-LIPOSOME CROSSLINKING BY GAD Liposome ugCollagen/uMole Lipid Type Initial Final ______________________________________ MLV 1.64 0.90 MLV 2.06 1.18 MLV 5.01 2.20 MLV 8.96 5.07 MLV 9.83 6.78 MLV 9.86 6.02 MLV 10.68 8.20 MLV 18.79 11.55 MLV 20.00 14.14 ______________________________________

Example Six

Aqueous solutions of HA was pre-activated by incubation with water-soluble carbodiimide, EDC. The components were mixed to yield a preparation system of HA and EDC each at final concentrations of 1.7 mg/ml. The pH of the preparation system was adjusted to 3 by titration with 1N HCl. The preparation system was incubated for a variety of time periods at 37.degree. C. with stirring. Table 6 sho