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BACKGROUND OF THE INVENTION
1. Field of the Invention
A chemically-structured delivery system for targeting liposomes containing
medication to the tooth structure of the oral cavity.
2. Description of Prior Art
A general background for understanding the chemical process steps that go
into making vesicles and liposomes is set forth clearly in a publication
"Biochemistry" by Lubert Stryer, published by W. H. Freeman and Company,
San Francisco, Calif., U.S.A., copyright 1981.
The repertoire of membrane lipids is extensive, and Stryer states they may
even be bewildering, but they do possess a critical common structural
theme in that membrane lipids contain both a hydrophilic and hydrophobic
moiety.
A space-filling model of a typical lipid has a general shape roughly
rectangular with two fatty acid chains approximately parallel to one
another and a hydrophilic moiety pointing in the opposite direction.
It is common practice to use a short hand illustration which has been
adopted to represent these membrane lipids. The hydrophilic unit called
the polar head group is represented by a circle and the hydrocarbon tails
are represented by lines which may be straight or wavy.
The polar head groups have affinity for water and the hydrocarbon tails
avoid water and seek lipid media. A bi-molecular sheet, known also as a
lipid bi-layer, is the favored structure for most phospholipids and
glycolipids in aqueous media.
The structure of a bi-molecular sheet is inherent in the structure of lipid
molecules. Their formation is a rapid and spontaneous process in water.
Hydrophobic interaction is the major driving force for the formation of
lipid bi-layers. It is important to the final construction of a targeted
liposome that there are van der Waals attactive forces between the
hydrocarbon tails. These van der Waals forces favor close packing of the
hydrocarbon tails, and also will accept the hydrocarbon moiety of target
molecules from an aqueous solution.
Clustering of bipolar lipids is favored by the van der Waals attractive
forces with the significant biological consequence that they will tend to
close on themselves so that there are no ends with exposed hydrocarbon
chains and therefore result in the formation of a compartment which is
normally self sealing because a hole in a bi-layer is energetically
unfavorable.
However, if one of the lipid components of such a closed compartment has
one R-group missing, there will be a fault dislocation which defeats the
self sealing behavior and allows the contents of the liposome to leak from
the inner aqueous compartment.
Therefore, as explained in the prior art and particularly in the Stryer
publication supra, liposomes are aqueous compartments enclosed by a lipid
bi-layer. They can be formed by suspending a suitable lipid, such as
phosphatidyl choline in an aqueous medium. This mixture is then sonicated,
which is an agitation by high frequency sound waves, to give a dispersion
of closed liposomes that are quite uniform in size. There are other
methods of forming such liposomes, and one specific recommended procedure
is set forth in the specification hereinafter.
Molecules, such as sodium fluoride for dental therapy, can be trapped in
the aqueous compartment of liposomes by forming them in the presence of
these substances. For example, if liposomes as small as 500 .ANG. in
diameter are formed in a 0.1M glycine solution, Stryer states that about
2000 molecules of glycine will be trapped in each inner aqueous
compartment. This manner of packaging oral cavity enhancement chemicals is
the first step of the present invention.
The biochemistry of the polyphosphoinositides and the diphosphonates as
noted in the scientific literature demonstrates that these molecules are
capable of participating in chemical reactions that result in the
formation of exceptionally strong coordination complexes with the calcium
ions of the hydroxyapatite crystal over a very broad pH range.
SUMMARY OF THE INVENTION
Lipid vesicles, otherwise known as liposomes, are envelopes having, in
part, a lipophilic membrane. Basically, the vesicle walls are composed of
bipolar molecules having a lipophilic end and a hydrophilic end. These
molecules are intertwined with the hydrophilic ends forming inner and
outer walls with the lipophilic ends sandwiched therebetween.
This invention employes vesicles whose membrane is permeable and contain
entrapped chemicals useful for oral cavity enhancement, such as fluorides,
antiplaque materials and breath fresheners. Permeability is usually
accomplished by the use of lysolecithin as a wall membrane component. A
full teaching of liposomal membranes containing lysolecithin is contained
in the 1976 addition of the Journal of Biochemistry wherein the work of
Takayuki Kitagawi, Keizo Inoue and Shoshichi Nojima, department of
chemistry, National Institute of Health, Kamiosaki, Shinagawa-Ku, Tokyo,
Japan, describing liposomes which have been prepared with lysolecithin,
lecithin, dicetyl phosphate and cholesterol. This report states that
generally, lysolecithin incorporation decreases the effectiveness of the
membranes as a barrier to glucose and made the membranes more "osmotically
fragile". This terminology simply means that by including lysolecithin a
fault dislocation is produced in the membrane wall, allowing the contents
to leak from the vesicle. The amount of the lysolecithin incorporation
will decidedly influence the rate at which the vesicles will leak the
contents. Relatively low concentrations of lysolecithin cause an increase
in the permeability of the liposomes, this report states. These studies
suggested that the induction of a change in the molecular organization by
lysolecithin molecules may cause the permeability change.
Since the work published by the National Institute of Health in Tokyo, the
manufacture of vesicles from totally non-leaking structure to those which
quickly lose their contacts, is now fully developed and well known prior
art.
This invention provides a means whereby the permeable liposome, with its
cargo of oral cavity enhancement material, anchored to tooth structure of
the oral cavity in order that eating, drinking and normal saliva wash will
not dislodge the vesicle. Keeping it in place until the contents are fully
expanded is the touchstone of this invention.
A long chain target molecule is composed having one end lipophilic and the
other end characterized by the ability to chemisorb with the surface of
hydroxyapatite crystals. The lipophilic end is caused to penetrate the
hydrophilic wall of the liposome and form weak van der Waals bonds
characterized as a transient attraction, with the lipophilic membrane. The
hydrophilic end of the target molecule will then project from the
liposome.
The resultant composition when exposed to tooth or bone hydroxyapatite will
cause an attempt by the hydrophilic end of the target molecule to form
strong bident metal ligands with the hydroxyapatite in a chemical bond.
The normal chemical relationship of the chelating hydrophilic end would be
to form a chelate ring with calcium, but because the calcium of tooth
structure is a component of the hydroxyapatite, the attraction which
anchors the hydrophilic end of the target molecule is better characterized
as chemisorption.
The net result of this invention is that a permeable liposome, having a
core volume of an oral cavity enhancement chemical, is attached to the
tooth structure by exposing the tooth to a wash or other carrier
containing the targeted structure of this invention.
DEFINITIONS
Vesicle--Substantially spherical thin walled bladder, usually in a range of
about 250 .ANG. to 1500 .ANG..
Liposome--A larger spherical bladder, often of layered walls, ranging from
about 1000 .ANG. to several micron.
For the purpose of this teaching, a target molecule may be a chemical
structure directly connected to a liposome and having a hydrophilic moiety
capable of chemisorptive bonding to hydroxyapatite, or it may be a
composite (conjugate) molecule with two separate molecules joined by a
bridge, thereby establishing a lipophilic moiety and a hydrophilic moiety.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a structural representation of a unilamellar liposome carrying a
core volume of radioactive trace material for delivery to tooth
hydroxyapatite, and a target anchoring molecule linking the liposome to
the surface of a tooth.
FIG. 2 is a list of the sample codes, lipid constituents, weights in mg.,
sonication and annealing times and temperatures and conditions under which
the various vesicle preparations are made.
FIG. 3 illustrates the results of an experiment designed to study liposome
binding to hydroxyapatite.
FIG. 4 depicts the results of an experiment designed to wash away free or
loosely held liposomes on the hydroxyapatite surface, and
FIG. 5 illustrates that events that occur when liposomes are bound to
hydroxyapatite and allowed to leak their core volume contents over time
into the external media.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The primary object of this invention is to provide a sustained release
mechanism for medical and cosmetic materials in the oral cavity of a
warm-blooded animal.
It is well known that treatment of dental carries and peridontal problems
is carried out in the dental office by application of liquid fluoride
solutions and gels, or by incorporation such medication into toothpaste
and mouthwashes.
The effectiveness of such procedures is the product of concentration and
time in contact with the treated areas. The dentist uses a very high
concentration of the fluoride medication in the liquid or gel and washes
the area free of excess material. The dentist thereafter obtains a high
degree of effectiveness without danger of adverse effect of ingesting the
strong solution. The use of dentifrice is a low concentration application
repeated often and is therefore safe for home application.
This invention addresses the growing needs of extended application time of
the medical and cosmetic materials in contact with hydroxyapatite and
provides a means of effectively treating peridontal disorders as well as
those attendant to dental plaque and dental carries, with the goal of
providing better dental health care. The invention also addresses the
problem of social acceptance by eliminating breath and mouth odors.
The first step in the discovery of this invention was to recognize the
capability of incorporating the medical or cosmetic material into a
liposome, which liposome is permeable to allow the material contained in
the core volume to leak slowly from the vesicle and provide a continuous
supply for an extended period of time.
The manufacture and use of liposomes is now well known by organic chemists
and researchers. Basically, a liposome is created by sonication of polar
lipid material. The liposome will trap a core volume of a water base
environment, or will carry lipid materials in the liposome membrane.
Generally, the components of the liposomes are materials such as
L-distearoyl lecithin and cholesterol. Sonication causes the lipids to
form into spheroidal configuration.
The literature contains much teaching of the actual and proposed uses of
liposomes. One structure germane to this present invention is a "leaky"
membrane made by introducing reagents which cause fault dislocation. The
work of Kitagawa; Inoue, and Nojima, "Properties of Liposomal Membranes
Containing Lysolecithin", J. Biochem., 79: 1123-1133 (1976), is an
example. In this prior work, liposomes were prepared with lysolecithin,
egg lecithin, dicetyl phosphate, and cholesterol. The ability to function
as a barrier to the diffusion of glucose marker and the sensitivities of
the liposomes to hypotonic treatment and other reagents which modifies the
permeability were examined. Generally, lysolecithin incorporation
decreased the effectiveness of the membranes as a barrier to glucose and
made the membranes more "osmotically fragile", i.e. permeable. Cholesterol
incorporation counteracted the effect of incorporated lysolecithin. The
more cholesterol incorporated into liposomes, the more lysolecithin could
be incorporated into the membrane without loss of function as a barrier.
Therefor, it is known how to capture water soluble substances within a
leaky faulted liposome. This invention is directed to attaching a leaky,
or sustained release liposomes to the hydroxyapatite. See Kitagawa, Inoue
and Nojima, supra.
Using this type of vesicle it has been observed objectively that the
treatment materials adhered to the hydroxyapatite for a period of time
longer that could be expected of, for example, a mouthwash deodorant.
The present invention was conceived wherein the properties inherent in the
unique molecular structure of phosphate compounds that belong to the
classes of the polyphosphoinositols and diphosphonates could be employed
to bind the vesicle to the hydroxyapatite for increased time of exposure.
Accordingly, a targeted vesicle delivery system has been developed wherein
selected phosphate compounds and their derivatives are attached at one end
to the lipid vesicle membrane and the other end is available to form
strong bidentate metal ligands which result in the formation of
coordination complexes with the calcium of the hydroxyapatite lattice of
bones and teeth. This attraction is known as chemisorption binding.
One of the important considerations related to the preparation of the
delivery system takes into account the fact that the hydroxyapatite of
tooth enamel is exposed in the oral cavity to the external environment and
thus facilitates the use of a topical vesicle drug delivery system.
According to this invention, the polyphosphoinositides, the diphosphonates
and their derivatives have a moiety held to the lipid membrane of a
liposome for targeting and subsequent binding of the liposomes to the
hydroxyapatite of tooth enamel.
The vesicle delivery system utilizing, for example, the membrane
constituent L-.alpha.phosphatidyl inositol 4,5-diphosphate as a chelating
agent for chemisorption binding to hydroxyapatite, suggests a variety of
new therapeutic uses for this dental delivery system.
Since the polyphosphoinositides are naturally occurring phospholipids with
hydrophilic phospoinositol head groups and hydrophobic fatty acid tail
groups, they are uniquely suited for the incorporation into vesicle
membranes.
FIG. 1 of the drawings illustrates what is considered to be the preferred
embodiment of this invention.
A general definition of the invention is the discovery that an osmotically
fragile, i.e. permeable, liposome may be attached to a tooth surface by
provision of a molecule having a moiety which is lipophilic and therefore
held by van der Waals forces in the lipophilic membrane of the liposome,
and a moiety which is hydrophilic and has an affinity for the
hydroxyapatite of a tooth surface. Such a structure will bind to the tooth
surface for an extended period of time and thereby permit the contents of
the liposome to bathe the tooth surface much more efficiently than any
available technique known prior to this invention.
In the FIG. 1, a complex molecule is shown as the preferred means to target
the liposome to the tooth surface. The connector is bi-polor with one
moiety held by van der Waals attraction in the liposome lipid membrane and
the other end terminating in oxygen ions.
Note, then, that the portion of the molecule labeled "target" also
terminates in oxygen ions which are shown (. . . ) attracted or bonded to
the calcium ion of the hydroxyapatite by chemisorption. The target also
has oxygen ions which are connected by bonding forces to a chromium
bridge. The chromium bridge connects the oxygen ions of the connector and
the target and therefor completes the structure.
It is important to note that the connector L-.alpha.phosphatidyl inositol
4,5-diphosphate could be connected by bonding directly to the
hydroxyapatite without the necessity of the target and bridge illustrated.
As stated herein above, the FIG. 1 is the preferred ideal structure and
the reason is that the selected target N,N,N',N', ethylene diamine tetra
(methylene phosphoric acid), known as Editempa or Dequest produces a
minimum etching of tooth surfaces. Although other molecules, such as the
connector shown, can bond directly to the tooth surface, it is capable of
producing unwanted levels of tooth etching.
Accordingly, those who are skilled in the chemical arts, having this
teaching before them, may select from a class consisting of the
diphosphonates, the class consisting of the polyphosphoinositides, and the
class consisting carboxylic acids, as the preferred general classes of
compounds, those which have a moiety which is lipophilic and a moiety
which has affinity for the hydroxyapatite. In this selection, those
skilled in the art will be able to select various combinations having the
required characteristics, and join them by a chemical bridge if desired as
taught by the FIG. 1. Otherwise, direct binding is acceptable although in
some instances not as desirable as the combination shown in FIG. 1.
To join the moiety by a chromium bridge, the lipid vesicles were collected
and then, with respect to the initial concentration of vesicle connector
molecules, were reacted with a five-fold molar excess of CrCl.sub.3. The
vesicles were then rechromatographed using the same buffer to remove
unreacted CrCl.sub.3. The collected vesicles were then reacted with a
five-fold molar excess of connector molecules. Following this step the
vesicles were then rechromatographed using the same buffer system to
remove unreacted connector molecules. Following the final chromatography,
the vesicles were stored under nitrogen in the refrigerator at 5.degree.
C.
Because there is no known practical means of measuring the extent to which
the present invention effectively delivers and anchors vesicles to the
appetite of the oral cavity in vivo, applicant devised a means for
establishing the extent of the effectiveness of the present invention.
That is, the experiment will demonstrate the affinity of the delivery
system for lipids to the hydroxyapatite.
DENTAL DELIVERY SYSTEM (DDS) PREPARATION
The synthetic procedure for the preparation of the targeted dental and drug
delivery system is described as follows:
28.96 mg of L .alpha.distearoyl lecithin and 1.67 mg of cholesterol, for
the formation of a bipolar vesicle, plus 1.40 mg of
L-.alpha.phosphatidylinositol-4,5-diphosphate, the target molecule, are
solubilized in CHCl.sub.3. MeOH (2:1 v/v) and dried under house vacuum for
15 minutes at 60.degree. C..+-.0.5.degree. C. to form a lipid crust.
Following the drying procedure, 2.0 ml of 40 mM KH.sub.2 PO.sub.4
--K.sub.2 HPO.sub.4, pH 7.4, was added to the lipid crust. The lipid
constituents were then sonicated in the cuphorn at 60.degree.
C..+-.0.5.degree. C. for 15 minutes at setting #4 on the sonicator. The
sample was then annealed with slow turning at 60.degree..+-.0.5.degree. C.
for 15 minutes. Following the annealing step, the sample was centrifuged
in the Triac Clinical Centrifuge on the bloodsetting mode at ambient
temperature for 15 minutes. The supernatant containing the lipid
suspension was chromatographed over a 1.5 cm.times.25 cm Sephadex
G-100-120 column that had been equilibrated with 40 mM phosphate buffer,
pH 7.4. The pooled vesicle fractions collected after chromatography
comprised 4.6 ml and were designated as batch 250-E.
This step by step procedure causes on inherent placing the lipophilic part
of the target substituent in the lipophilic membranes of the liposome with
the hydrophilic head orientated in three-dimensional space extended away
from the membrane surface.
CONTROL MATERIAL
A control preparation, referred to hereinafter as 250C, was prepared as
described for the DDS, except that no target material was supplied, i.e.,
material such as the polyphosphoinositides or diphosphonates.
TEST PROCEDURE
This test procedure was chosen to demonstrate the ability of the DDS to
bind to hydroxyapatite. The vesicle contents will leak out the medication,
breath freshener, of other content as taught by the prior art, and by
fixing these vesicles in place on the tooth surface, will be effective in
bathing the tooth surfaces and gum tissues for any desired time period.
Usually a 24-hour time period will be selected because a fresh supply will
normally be presented through tooth brushing of mouthwash at least once in
each 24-hour period.
Although the DDS will have for its purpose to bind to dental enamel in the
mouth, the laboratory demonstration of the ability of the DDS to bind
hydroxyapatite (HA) utilizes the binding of DDS to a fine aqueous
suspension of HA purchased from Sigma Chemical Company, St. Louis, Miss.
HA is the mineral component of dental enamel. The HA suspension alone will
settle out upon standing and leave a perfectly clear supernatant. The DDS
preparations (250E and 250C) are bipolar lipid vesicles that form
colloidal suspensions. This experiment utilizes both of these attributes:
clear, supernatant for HA alone and cloudy supernatant for DDS alone.
If the HA and DDS are combined the resultant supernatant, after permitting
settling, can be used to indicate whether or not the DDS became bound to
the HA. The two resultant possibilities are:
1. If the resultant supernatant is cloudy, then the HA did not bind the DDS
in a significant way.
2. If the resultant supernatant is clear, then the HA bound all of the DDS.
The experiment was designated to demonstrate the enhanced DDS binding to HA
when the L-.alpha.phosphatidylinositol-4,5-diphosphate was used as a DDS
target molecule (preparation 250E) compared to a vesicle with no target
molecule (250C).
The experiment test was carried out as follows:
Four test tubes numbered 1-4 were used with numbers 1 and 2 for DDS 250E.
Tube numbers 3 and 4 were used for the control vesicles labelled 250C. The
additions were according to the following table:
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DDS-250E Vesicles-250C
1 2 3 4
______________________________________
1.0 ml buffer X X X X
2 drops HA X X
2 drops buffer X X
0.5 ml 250E X X
0.5 ml 250C X X
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The tubes were covered and stirred with small magnetic stirring bars for 70
hours at room temperature (25.degree. C.) to achieve binding equilibrium.
When stirring ceased, the tubes were then allowed to stand overnight to
permit the HA to settle. The supernatants were then described:
______________________________________
Tube #1 Tube #2 Tube #3 Tube #4
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Clear Cloudy; no Cloudy Cloudy; partial
settling of settling of the
DDS control vesicles
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The data of tubes 1-4 are interpreted as showing a complete binding of the
DDS-250E to HA as evidence by the clear supernatant in Tube #1. The lack
of settling in Tube #2 indicates that the DDS 250E, which did not have
hydroxyapatite solution to bind to, is a stable colloid that does not
spontaneously settle. Tube #2 is a control for Tube #1.
Tube #3 had a cloudy supernatant, indicating that the control vesicles of
batch 250C (the vesicles without target molecules) did not bind
efficiently to the HA. Close examination indicated, however, a weak
binding of some vesicles. The partial settling of Tube #4 indicates that
the vesicles without the target molecule is a less stable colloid than the
complete DDS 250E.
The conclusion is that the DDS-250E by virtue of the
L-.alpha.phosphatidylinositol-4,5-diphosphate target molecule does
efficiently bind the HA, which is the mineral component of dental enamel.
The important conclusion to be made from these observations is that a
binding profile can be depicted extending from the weak interaction of the
control vesicles to the strong interaction with hydroxyapatite as
evidenced by the experimental sample. The fact that there is weak vesicle
adherence by hydroxyapatite, as well as strong adherence, introduces the
option of manufacturing vesicles that either bind weakly or strongly to
hydroxyapatite, depending upon the type of vesicle that is needed. This
binding is predicated on the number and character of the functional target
groups on the vesicle surface.
The synthetic processes employed in the manufacture of targeted vesicles
for dental drug delivery systems have been expanded hereafter to include
all other procedural variations that offer an array of targeting
mechanisms which will selectively seek and bind to crystalline
hydroxyapatite surfaces such as are found in tooth enamel and bone.
These methods produce dental delivery systems with maximal, as well as
nominal, hydroxyapatite binding affinities.
The table, FIG. 2, is a list of the sample codes, lipid constituents,
weights in mg., sonication and annealing times and temperatures and
conditions under which the various vesicle preparations are made.
Each of the vesicle preparations outlined in FIG. 2 is treated as follows
to produce the final vesicle product.
The lipid constituents are first solubilized in a solution of
Chloroform-methanol (2:1 v/v) and then dried with slow rotation using a
Buchi rotoevaporator and accompanying waterbath at 60.degree.
C..+-.0.5.degree. C. The lipids are dried under pump vacuum for 15 minutes
before being transferred to a vacuum desiccator and further dried for one
hour at ambient temperature. Following the drying period each lipid crust
is reconstituted with either an aqueous solution of glucose in water at a
concentration of 1 mg/ml or 10 mM phosphate buffer at 7.4.
The lipid constituents are then sonicated in a cuphorn sonicator powered by
a Heat Systems Model W 200R amplifier. The sonication and annealing
procedures then proceed according to the schedule outlined in Table I.
After the annealing procedure the samples are centrifuged in a Triac
Clinical Centrifuge on the bloodsetting mode for 15 minutes at ambient
temperature. The supernatant is then chromatographed over a freshly
prepared 1.5 cm.times.25 cm Sepharose CL-2B-300 column that has been
equilibrated with 10 mM phosphate buffer at pH 7.4. Vesicle fractions are
then evaluated for their lipid concentration based on ultraviolet light
scattering and radiochemical analysis. Ultraviolet light is not absorbed
by lipid vesicle but it is scattered. This refracted light shows up on a
ultraviolet monitor as a light scattered signal which is subsequently
recorded. The extent to which light is scattered is proportional to the
peak height on the recorder. In FIG. 1, the core volume is shown as carbon
14. This material is not to be included in commercial product, but is used
and illustrated for test purposes.
RESULTS
The vesicle sample codes and their lipid constituents are listed in FIG. 2
for easy reference to the following figures:
FIG. 3 illustrates the results of an experiment that was designed to study
vesicle binding to hydroxyapatite (H.A.) (Type III, Sigma). Along the
abscissa of the graph, increasing levels of hydroxyapatite are used to
generate a hydroxyapatite crystal sink that is capable of being saturated
with a given vesicle preparation. The degree of vesicle binding and
subsequent hydroxyapatite saturation is measured by incorporating a
radiolabeled .sup.14 C-DSL constituent into the vesicle membrane at the
time of synthesis and then comparing the amount of radiolabel bound to
hydroxyapatite versus the amount of radiolabel that is free in the
supernatant following the centrifugation of hydroxyapatite crystals. The
results graphed in FIG. 3 are expressed as a percentage of .sup.14 C bound
relative to the hydroxyapatite concentration.
FIG. 3 shows that Sample Code #5, which contains chromium and Dequest in
addition to the L-.alpha.phosphatidyl inositol-4,5-diphosphate group, has
a greater binding affinity at any given concentration of hydroxyapatite
than does the phosphatidyl inositol-4,5-diphosphate or the phosphatidyl
glycerol moiety.
Phosphatidyl glycerol (PG) is also inserted in the vesicle membrane at the
time of sonication, even though (PG) does not in this particular
circumstance function as a connector molecule. However, it occupies the
same spatial or three-dimensional position as a connector molecule.
Phosphatidyl glycerol is an example of a molecule that shows weak binding
affinity to hydroxyapatite at all concentrations of hydroxyapatite tested.
It can be concluded that Sample Code #1 with phosphatidyl glycerol present
in the vesicle membrane is a good control vesicle with insignificant
hydroxyapatite binding affinity.
Intermediate between Sample Code #5 (Chromium-Dequest) and Sample Code #1
(Phosphatidyl glycerol) is Sample Code #2 with L-.alpha.phosphatidyl
inositol-4,5-diphosphate present as the functional binding molecule
occupying the connector molecule position. L-.alpha.phosphatidyl
inositol-4,5-diphosphate has two opposed phosphated groups in positions #4
and #5 on the inositol ring structure that serve to bind to the
crystalline lattice of hydroxyapatite. These phosphate groups can also
bind to chromium ions. Furthermore, phosphatidyl inositol-4,5-diphosphate
is not capable of more than 86% binding capacity as defined by the
parameters of the experimental results shown in FIG. 2. Thus, phosphatidyl
inositol-4,5-diphosphate appears to be intermediate between
chromium-Dequest and phosphatidyl glycerol in its binding affinity for
hydroxyapatite.
FIG. 4 depicts the results of an experiment designed to wash away any free
or loosely held vesicles on the hydroxyapatite surface. Once again, the
percent of .sup.14 C-DSL that is bound is measured in the same manner as
observed for the experiment illustrated in FIG. 3.
The vesicles with the L-.alpha.phosphatidyl inositol-4,5-diphosphate groups
on the vesicle surface bind to hydroxyapatite very well and near 100%
capacity, even after three consecutive equal volume washes with 10 mM
potassium phosphate buffer, pH 7.4. However, the vesicles with simply
phosphatidyl glycerol on their surface are washed off the hydroxyapatite
rather rapidly, as shown with the washout curve in FIG. 4. Only 9% of the
original vesicles remain after three consecutive washes of the
hydroxyapatite.
FIG. 5 demonstrates the events that occur when vesicles which contain
L-.alpha.phosphatidyl inositol-4,5-diphosphate are bound to hydroxyapatite
and allowed to leak their soluble .sup.14 C core volume contents over time
into the external media.
FIG. 3 shows that vesicles with the phosphatidyl inositol-4,5-diphosphate
moiety bind convincingly to hydroxyapatite. Thus, for the experiment shown
in FIG. 5, it can be assumed that the phosphatidyl
inositol-4,5-diphosphate vesicles are bound substantially to the
hydroxyapatite. The physical event which is concomitantly observed after
binding is the continual and cumulative leakage of .sup.14 C-glucose from
the core volume as a function of time.
In a separate experiment, Sample Code #4, with .sup.14 C-glucose-DSL-CHOL
L-.alpha.phosphatedyl inoselot +4,5-diphosphate was observed to bind to a
single human tooth which was immersed in a vesicle suspension for 15
minutes at ambient temperature. In this preliminary experiment, 18.1% of
the available vesicles bound to the crystalline surface of the tooth in 10
mM phosphate buffer, pH 7.4.
Experimentally 50 .mu.l of the stock vesicle preparation from Sample Code
190 4 was added to 650 .mu.l of 10 mM potassium phosphate buffer, pH 7.4,
to form the incubation medium. At the concentration of lipid vesicles used
in this experiment, it is likely that a vesicle monolayer was chemisorbed
to the tooth, signaling that a maximum level of vesicle saturation was
achieved within the parameters of the experiment.
In summary, it can be concluded that maximal binding to hydroxyapatite is
achieved with the Dequest binding molecule, and that by altering the mole
ratio of lipid constituents in the vesicle membrane the core volume
contents can be made to leak at designated and variable rates.
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