U.S. patent application number 11/215199 was filed with the patent office on 2006-03-23 for lipid microtubules with contolled bilayer numbers.
Invention is credited to Ronald R. Price, Banahalli R. Ratna, JoelM Schnur, Mark S. Spector.
Application Number | 20060062840 11/215199 |
Document ID | / |
Family ID | 34860619 |
Filed Date | 2006-03-23 |
United States Patent
Application |
20060062840 |
Kind Code |
A1 |
Price; Ronald R. ; et
al. |
March 23, 2006 |
Lipid microtubules with contolled bilayer numbers
Abstract
The wall thickness of lipid microtubules are controlled by
selecting a methanol/water system and determining the required
amount of a lipid to form the desired wall thickness. The lipid is
dissolved in a small portion of the heated methanol and that clear
solution is added to the remaining amount of the heated
methanol/water system. By slowly cooling the solution, microtubules
are formed which have the desired wall thickness. Preferred
microtubules have a wall thickness of just 2 bilayers and they are
robust so they can be further coated. They can be made with a large
aspect ratio and with lengths of greater than 250 microns. The
process permits production of microtubules in very high yields.
Inventors: |
Price; Ronald R.;
(Stevensville, MD) ; Schnur; JoelM; (Burke,
VA) ; Ratna; Banahalli R.; (Springfield, VA) ;
Spector; Mark S.; (Alexandria, VA) |
Correspondence
Address: |
NAVAL RESEARCH LABORATORY;ASSOCIATE COUNSEL (PATENTS)
CODE 1008.2
4555 OVERLOOK AVENUE, S.W.
WASHINGTON
DC
20375-5320
US
|
Family ID: |
34860619 |
Appl. No.: |
11/215199 |
Filed: |
August 29, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
08703608 |
Aug 27, 1996 |
6936215 |
|
|
11215199 |
Aug 29, 2005 |
|
|
|
Current U.S.
Class: |
424/450 ;
264/349 |
Current CPC
Class: |
C11B 3/00 20130101 |
Class at
Publication: |
424/450 ;
264/349 |
International
Class: |
A61K 9/127 20060101
A61K009/127; B29B 15/00 20060101 B29B015/00 |
Claims
1-31. (canceled)
32. A population of lipid microtubules having a controlled wall
thickness, wherein the median length of at least 50 microns as made
by the method comprising: (a) selecting a methanol-water system to
be used which is characterized by a volume ratio of methanol and
water that totals 100 volume percent, wherein the methanol and
water are first filtered to remove any particulates and wherein the
filter is at least as fine as a 0.22 micron filter, (b) determining
the amount of lipid to be used for the solvent system selected in
step (a) so as to produce the desired wall thickness of the
microtubule; (c) dissolving the determined amount of lipid from
step (b) into a portion of the methanol which has been heated to a
temperature above the transition temperature for the lipid to form
a clear solution; (d) adding the heated methanol lipid solution of
step (c) into a mixture of the remaining methanol and water as
selected in step (a) which has been heated to a comparable
temperature above the transition temperature for the lipid as in
step (c) so that the total amount of methanol and water is in the
desired amount selected in step (a); and (e) cooling the heated
mixture in step (d) slowly to permit the formation of microtubules
with a controlled, uniform number of bilayers.
33. A population of lipid microtubules according to claim 32,
wherein the median length is at least 100 microns.
34. (canceled)
35. The lipid microtubule of claim 32 having a controlled wall
thickness of just 2 bilayers.
36. A population of lipid microtubules according to claim 35,
wherein the median length is at least 250 microns.
37-38. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to the formation of microcylinders or
microtubules from phospholipids with the rational control of wall
thickness (i.e. the number of bilayers) of microcylinders.
[0003] 2. Description of the Previously Published Art
[0004] Multibilayer lipid microstructures have been made by cooling
from multilamellar liposomes in water, and from solutions of
ethanol and other alcohols. The resulting multibilayer tubules have
typically a broad distribution of bilayers with a median of about
10 bilayers. Single bilayer tubules have been prepared by cooling
from solutions of water and methanol. Single bilayer tubules,
however, are extremely fragile.
[0005] U.S. Pat. No. 4,990,291 discloses forming lipid
microcylinders from a thermal cycling process in what is basically
a lipid/water system. After purification and drying of a
diacetylenic phospholipid the tubule is formed by first hydrating
the lipid at about 10.degree. C. above its endothermic transition
point. Then the lipid mixture is cooled slowly at a rate not to
exceed 1.degree. C. per minute, preferably not greater than
0.5.degree. C. per min to a formation temperature 1.degree. to
10.degree. below the lipids' exothermic transition temperature also
known as the gel phase transition. The solution is held at the
formation temperature for between 30 minutes and 2 hours, most
preferably 1 hour. Once the tubule structures are formed they are
stable as long as the tubule structures are not heated above the
endothermic transition temperature. If desired, the tubule
structures can be polymerized by any of the well-known means to a
permanent tubule form. The tubules formed are usually extremely
straight hollow cylinders of approximately 0.5 micrometer diameter
and 5 to 100 micrometers in length. These tubules can be used in a
vast variety of ways. The tubules can be used to hold materials in
a-manner well known for lipid vesicles described in the patent. In
addition, the tubules can be coated with metals as described in
U.S. Pat. No. 4,911,981. This U.S. Pat. No. 4,990,291 patent does
not use methanol or methanol/water solutions, or describe any
process of crystallization from a mixed alcohol water system.
[0006] U.S. Pat. No. 4,887,501 discloses forming lipid
microstructure from a mixed solvent system to produce helix or
cylinder microstructures. The first step is to add a lipid to a
lipid solvating organic solvent. Then a predetermined amount of
water is added to the solvent/lipid mixture and the solution is
allowed to sit for a predetermined amount of time and at a
predetermined temperature. The temperature is preferably maintained
about 10-30.degree. C. below the melting point of the lipid as
defined in excess water. A broad range of solvents are disclosed
and the concentration of the lipid in the organic solvent lipid
solution is typically preselected to be less than about 2
mg/ml.
[0007] The most preferred organic solvents are relatively polar
organic solvents such as tetrahydrofuran, chloroform, and alcohols
and polyols, such as methanol, ethanol, propanol, isopropanol,
butanol, isobutanol, propylene glycol, ethylene glycol and mixtures
of these. In the 14 examples the only alcohols used are ethanol and
isopropanol. There is no discussion of the number of bilayers in
the tubule wall. In Example 4 the diameters range from 0.2 to 3.0
microns. This reference does not describe a method to yield a
uniform microtubule dispersion at high yield. It also does not
teach rational control of bilayer numbers or any method of
obtaining the same. The lipid is first dissolved in the solvent and
then the water added. When practiced as described, the direct
result of the addition of water is the immediate formation of lipid
structures from the dissolved lipid in the presence of local
concentrations of water. This leads to a very large number of
liposomes, bilayer ribbons and sheets as well as micelles formed in
addition to the desired microtubules. Such materials degrade the
sample, decrease the yield of microtubules and thus diminish the
degree of control that may otherwise be realized.
[0008] U.S. Pat. No. 4,887,501 further teaches that a range of
bilayers resulted from formation in a mixed solvent system, and
that variation in the solvent concentrations as well as the
concentration of the lipid in relation to the mixed solvent had
little effect on the number of bilayers, and that in contrast the
number of bilayers were effected to a greater extent by the
hydrocarbon chain length in the lipids used to form the
microtubules.
[0009] To date the yield and morphology of the microtubules has
been difficult to reproduce on a rational basis, and especially
conversion rates have been poor. This problem exists in both
systems of thermal cycling and mixed solvents. It is almost
impossible to obtain conversion rates that are economically viable
with thermal cycling, and in addition there is no correlation
between concentration and aspect ratio or yield. Further the
presence of a very large number of non-cylindrical lipid structures
makes it very difficult if not impossible to process the resultant
structures into a homogenous cylindrical product. In the solvent
methodology it is imperative that thermal and chemical mixing
(including the heat of mixing of the alcohol and water) be
minimized. Any mixing once the tubules have formed leads to
differential shear and thus mechanical disruption of the high
aspect ratio microcylinders and also leads to birdnesting which can
be thought of as a tubule logjam. Such inconsistent morphology
results in microcylinders which may vary from 4 to 20 or more
bilayers in a single batch. The inner diameters remain about 0.5
microns with the outer diameters varying as a result of the number
of bilayers.
[0010] These multiple-walled cylinders have the further problem
that the yield (and thus the costs) of actual microcylinders from
an initial concentration of lipid may be up to 500% lower than if
the same amount of lipid was used to form microcylinders with a
fixed, lower number of bilayers such as a double bilayer wall per
structure.
[0011] Although one might desire to make smaller tubules from an
economic point of view, the literature does not teach one how this
can be done and especially how it can be done efficiently. It also
does not teach the minimum number of bilayers that are needed for a
strong product.
[0012] B. R. Ratna et al in "Effect of alcohol chain length on
tubule formation in
1,2-bis(10,12-tricosadiynol)-sn-glycero-3-phosphocholine," Chem.
Phy. Lipids. 63, 47 (1992) studied the volume fractions of alcohols
from which tubule formation was observed. For the methanol/water
system they found the range to be 65/25-90/10. For lower fractions
the lipid precipitates out in an amorphous form whereas on the
higher alcohol side of the window, the lipid remains in solution
even at room temperature. The authors also measured the number of
bilayers in the tubules grown from different alcohols. They found
the number of bilayers constituting the wall of the tubule is
independent of the alcohol/water ratio. However, the number is
found to be strongly dependent on the chain length of the alcohol.
They studied methanol, ethanol and 1-propanol. For methanol and
using an 85/15 methanol/water system, 95% of the tubules grown were
made of a single bilayer and the remaining 5% being made from two
or three bilayers. There is no teaching that the number of bilayers
could be controlled at two to form a more robust structure, and in
fact teaches away from utilization of methanol to make
multi-layered structures. The paper clearly indicates that there is
no way using its reaction conditions that more than one bilayer
tubules can be formed from methanol-water solutions. When the
alcohol was changed to ethanol or 1-propanol the wall thickness as
well as its variance increase considerable. Samples grown in both
of these longer chain solvents have an average of 6-7 bilayers with
a standard deviation of 3 bilayers. Thus the literature has taught
that a methanol/water system basically produces a microtubule with
a wall having only a single bilayer.
[0013] These tubules produced from methanol, however, which
normally have the single bilayer break very easily and can not be
used for metalization and subsequent commercial applications.
[0014] G. Nounesis et al in "Melting of Phospholipid Tubules," Phy.
Rev. Lett. 76, 3650 (1996) describe the morphological
transformations and the bilayer phase-transformation of
DC.sub.8,9PC tubules in methanol/water solutions. They note that at
a lipid concentration, .rho., less than 2 mg/cm.sup.3 the majority,
.about.95%, of the tubules formed have single bilayer walls. With
.rho.>4 mg/cm.sup.3, most of the tubules have from two to four
bilayers in the walls. This discussion illustrates the common
knowledge that the wall produced in methanol/water system is
generally a single bilayer. Although at a higher lipid
concentration wall is produced having more than a single bilayer,
there is no suggestion that just a two bilayer can be produced
since they report that the microtubules have from 2 to 4
bilayers.
[0015] Previous work with ethanol/water systems in both thermal
cycling process and the mixed solvent system has indicated that the
median number of bilayers in any population of microcylinders is
about 10, and many have been observed having up to 20 or more. For
the case of the smallest median wall size of approximately 10
bilayers for the ethanol system, the number of microstructures that
could possibly form is reduced by at least 500% at a minimum over a
more desired system where the average wall size is only two
bilayers.
OBJECTS OF THE INVENTION
[0016] It is an object of this invention to provide for the
rational control of the number of lipid bilayers that comprise
lipid microstructures such as lipid tubules.
[0017] It is a further object of this invention to provide the
formation of robust microtubules which can be processed for further
treatment without disruption of the microstructure.
[0018] It is a further object of this invention to provide lipid
microstructures which have greater than a single bilayer
microtubule so that they will be sufficiently robust to allow for
continued processing such as metallization, inclusion in polymer or
ceramic matrixes, or exposed to in vivo as well as in vitro
environments.
[0019] It is a further object of this invention to provide lipid
microstructures which have just two bilayers so as to form robust
tubules with the minimum amount of lipid material.
[0020] It is a further object of this invention to form lipid
microstructures with an aspect ratio of the tubules which is
greater obtained than from previous methods.
[0021] It is a further object of this invention to form lipid
microstructures having a much narrower range of diameters, due to
the regularity of the number of bilayers.
[0022] It is a further object of this invention to provide for a
process for the rational control of the number of lipid bilayers
comprising the walls of microcylinders.
[0023] It is a further object of this invention to provide a
process to produce lipid microstructures which have greater than a
single bilayer microtubule so that they will be sufficiently robust
to allow for continued processing.
[0024] It is a further object of this invention to form robust
lipid micro structures where the number of bilayers is less than in
other methods yet at least two bilayers so there are more
individual tubules formed from the same weight of lipid.
[0025] It is a further object of this invention to increase the
yield of individual lipid microstructure from a initial
concentration of lipid monomer.
[0026] It is a further object of this invention to provide a
substantial cost reduction in the process of forming lipid
microstructures due to the use of more efficient production with
increased overall yield.
[0027] It is a further object of this invention to provide greater
conversion of lipid to microcylinders such that the yield is
>90% conversion.
[0028] It is a further object of this invention to provide the
ability to control the number of bilayers when forming lipid
microstructures to be within a narrow range.
[0029] It is a further object of this invention to provide a
process to make lipid microcylinders of predetermined morphology
from a mixed methanol/water system.
[0030] It is a further object of this invention to provide a means
of thermally cycling a lipid/solvent system whereby the aspect
ratio of the microcylinders may be controlled.
[0031] It is an object of this invention to provide a method in
which microcylinders formed from lipids may be produced in bulk by
a rational process, whereby the number of lipid bilayers that
comprise lipid microcylinders may be predetermined to the optimum
number of two, with a conversion rate of >98% of the lipid
utilized, thus offering a very large increase in the numbers of
such structures per volume of lipid and alcohol, and thus reducing
costs up to 100 to 500%.
[0032] These and further objects of the invention will become
apparent as the description of the invention proceeds.
SUMMARY OF THE INVENTION
[0033] The present invention relates to a method for controlling
the wall thickness of lipid microtubules formed by cooling a heated
methanol-water mixture containing the dissolved lipid. The first
step is select a methanol-water system to be used which is
characterized by a volume ratio of methanol and water that totals
100 volume percent. Next the amount of lipid to be used is
determined based on the solvent system selected in the first step
so as to produce the desired wall thickness of the microtubule.
That determined amount of lipid is then dissolved into a portion of
the methanol which has been heated to a temperature above the
transition temperature for the lipid to form a clear solution. This
heated clear solution is then added to a mixture of the remaining
methanol and water as selected in the first step which has been
heated to a comparable temperature above the transition temperature
for the lipid. Thus the total amount of methanol and water is in
the desired ratio amount selected in the first step. This solution
is finally allowed to cool slowly to permit the formation of
microtubules with a controlled, uniform number of bilayers.
[0034] This process permits the production of unique populations of
tubules where the population of lipid microtubules can have from
70% to greater than 95% of the tubules having a wall thickness of
just two bilayers. Similar control can be had with the median
length of the tubules so that populations can be made which vary
from over 50 microns to over 250 microns in length. The tubules
with just two bilayers are robust and can be coated with a large
variety of coatings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a graph showing concentration dependence of the CD
spectra of DC.sub.8,9PC tubules in methanol/water (7:3) at
25.degree. C.
[0036] FIG. 2 is a graph showing the ratio of the spectral
intensities at 205 nm and 195 nm.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] The instant invention relates to the efficient production of
an engineered biochemical product (microtubules) in such a way that
the number of bilayers in an individual microcylinder and thus
control of the diameter and aspect ratio of the resulting
structures may be predetermined in a rational and repeatable
manner. Control of the wall thickness allows for an increase in the
numbers of microcylinders that may be produced from a selected
quantity of lipid as opposed to having no rational control of the
process and subsequent poor yield with respect to the number of
structures formed from a defined solvent system at a specified
lipid concentration. This is absolutely imperative with systems
where the physical characteristics must be defined, within a narrow
tolerance range.
[0038] A surprising result not previously reported in the patent or
scientific literature is that by utilization of specific alcohol
water solvent mixing ratios with narrowly defined concentrations of
lipid it is possible to increase the yield of the formation process
up to 500% and to provide an extremely narrow distribution of
bilayer numbers permitting rational control of the chemical
engineering parameters necessary to scale up lipid microcylinder
production for economically viable production of a defined
product.
[0039] The production of microcylinders of predetermined bilayer
numbers at high yield has not been described in the literature, and
in fact, the published methods point to the alcohol chain length
being the dominating factor as seen in the Chem. Phy. Lipids (1992)
article by B. R. Ratna et al cited above.
[0040] In an especially preferred embodiment, a mixed alcohol/water
bath is utilized such that the alcohol is methanol blended with
water at the volume ratio of 85:15 methanol to water. The methanol
and water should preferably first be filtered to remove any
particulate material. The formation method includes the addition of
a known microcylinder forming polymerizable lipids such as lecithin
at a concentration of about 5 mg/ml such that 15 parts methanol by
volume and 3 parts water by volume are blended and warmed to a
point at least 5.degree. C. greater than the lipids chain melting
transition temperature in the solvent system of choice. As an
example, with the lipid 1,2
bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine
(DC.sub.8,9PC) the temperature would be a minimum of 45.degree. C.,
but not sufficiently high to cause chemical degradation of the
lipid. For a discussion of lipid nomenclature and structures, see
U.S. Pat. Nos. 4,877,501 and 5,290,690 the entire contents of which
are incorporated herein by reference.
[0041] A further two parts by volume of the alcohol (methanol) is
heated as above and an amount of the lipid is added sufficient to
provide a concentration of 5 mg/ml in the final formation bath is
added. This is agitated until all of the material is observed to
have been fully solvated.
[0042] Following this step the mixture is then filtered using a
0.22 micron filter to remove any particulate and bacteria, and the
resultant solution is then added to the alcohol water bath. This
important step will eliminate any particulates that might become
foci for unwanted lipid deposition and removal of microbes that
would contaminate the final tubule suspension.
[0043] This is mixed until uniform and the entire system is then
cooled to just below the transition temperature of 32.degree. C. at
a rate not to exceed 1.degree. C. per hour, following which the
microcylinders are observed to form. When the solution begins to
become turbid the temperature is further lowered to 20.degree. C.
for a period of 72-144 hours.
[0044] The solution is then dialyzed against water to remove the
alcohol and then against an HCl/water solution at pH 1.4 for a
period of time sufficient to lower the pH of the tubules to 1.4. A
3% solution of a commercial palladium tin catalyst in a 0.1 M
solution of HCl is introduced such that it is exactly 6 times the
volume of the microcylinder suspension used. This addition is done
gradually so as not to offer a catastrophic change in osmolarity
within the microtubule suspension. This step is completed when the
solution clears and the microcylinders have settled due to their
increased density. Electroless plating is conducted according to
the manufacturers recommendations. An example of such a process is
given in Example 3.
[0045] Microtubules that are to be coated with a polysaccharide
such as sodium alginate or chitosan glutimate are dialyzed against
water to remove the methanol, diluted 10 times with a mixture of
from 0.25% to 2.0% of a polysaccharide, following this dilution the
suspension is very gently agitated by swirling to prevent
settlement of the microstructures. The reaction is allowed to
continue for up to 24 hours after which the tubules are separated
by centrifugation or filtration and then re-suspended in water to
remove all exogenous polysaccharide. The polysaccharide may be
polymerized by crosslinking with calcium salts in the case of
alginates or with an aldehyde or amine such as spermine as would be
the case with the chitosan. Following coating the chitosan coated
tubules may be further processed for electroless metal deposition
just as the non coated microtubules would or they may be further
decorated with a range of biochemically active materials.
[0046] Best results will be obtained by carefully controlling the
process conditions.
[0047] It is very important that in order to obtain a uniform
product of individual microcylinders of narrowly defined structure
that the lipid be fully solvated in alcohol above the transition
temperature, and that all remnants of impurities and non-soluble
reaction products be removed.
[0048] Following this step the concentrated lipid solution is added
to the majority volume of alcohol and water which has been mixed to
a homogenous consistency and at a homogenous temperature. By
avoiding any high concentrations of water or the chance of
additions at a lower temperature sufficient to instantly form
unwanted microstructures, a more controlled and regular product is
produced at a higher yield. These unwanted microstructures might
provide a foci for further precipitation of non-tubular structures
and thereby reduce the yield.
[0049] Further, it is necessary that the mixture be very evenly
mixed following addition of the lipid so that the dilution is
uniform and that the final mixture is 85:15 throughout the mixture
thus avoiding variation.
[0050] Addition of the water to the alcohol with no temperature
control or protection from excess hydration will allow for
precipitation of the lipid leading to an increase in non-tubular
structures such as liposomes or bilayer ribbons or sheets in the
final product, detracting from rational control of the final
product.
[0051] Finally the rate of cooling is important to obtaining high
aspect ratios and the absence of any unwanted structures. When
formation occurs at a rapid rate there is a greater
variability.
[0052] The utilization of an 85:15 v/v mixture of methanol and
water and the utilization of a specific quantity of lipid (such as
5 mg/ml) results in the desired product formation for the
particular lipid used in the examples. Lipid loadings in excess of
this figure results in highly visco-elastic solutions that are
difficult to process and which contain a large number of liposomes.
Lesser amounts of lipid results in formation of single bilayer
microtubules which are not sufficiently robust to allow for
continued processing. For other lipid materials it is expected that
loadings as low as 2 mg/ml will be acceptable as well as any larger
amount.
[0053] It is also expected that for other lipids the volume ratio
of methanol to water could vary from about 98:2 to 40:60.
[0054] At this time the lipids known to form microstructures in
this manner include phospholipids with diacetylenic moieties in
their acyl chains and having total carbon contents of C-15 to C-27.
There may be others, but investigations on other acceptable
materials have not yet been done.
[0055] A significant advantage of the present invention is the
greater conversion of lipid to microcylinders such that the yield
is greater than 90%, more preferably greater than 95% and most
preferably greater than 98%.
[0056] Since the controlled number of bilayers is less than in
other methods of formation, there are more individual tubules
formed from the same weight of lipid. The ability to form just two
bilayers is very important as it reduces the amount of lipid needed
to form a single tubule which increases the number of tubules
formed per gram of lipid. This leads to the production of tubules
with approximately five times less lipid materials and these
smaller walled tubules provide a 500% cost reduction in the
formation of such tubules over previous methods. As costs of
synthesis for tubule forming lipids is very high, a 5 fold increase
in yield translates into a considerable cost savings. Further,
reduced processing needs required to remove the unreacted lipid
saves processing time and improves economy. Thus, the significant
advantage of the present process is a substantial cost reduction
that accrues from more efficient production and the increase in
overall yield.
[0057] One major advantage is the ability to process these
microtubules for further treatment without disruption of the
microstructure. Previous attempts to process tubules formed in
methanol had failed due to the fact that only single bilayers were
formed in significant numbers and the methods utilized resulted in
the microstructures being destroyed by the processing.
[0058] An unexpected result was the ability to control the number
of bilayers within a narrow range during formation. This ability to
control the number of bilayers is in direct conflict with
previously patented and published results and was not expected
prior to this invention. The inner diameter is regulated by both
the initial exterior diameter during formation and the wall
thickness. Since each bilayer has a thickness of approximately 8-10
nm, by controlling the number of bilayers at two, an average of 8
bilayers is eliminated which results in a considerable reduction in
the outer diameter of up to 200 nm. It has been found that the
ideal number of bilayers is two. This thickness provided sufficient
strength for commercial applications with the optimal amount of
lipid being used. By using this process the same amount of lipid
will produce 5 times more tubules. This will reduce the cost of the
required lipid for a particular application by 500%.
[0059] In addition the aspect ratio, which is the ratio of the
length to the width, of the tubules is greater than from previous
methods. This is due to the regularity of the number of bilayers
they have a much narrower range of diameters. With a length of 250
microns and a diameter of 0.5 micron, an aspect ratio of 500:1 is
easily obtained. Furthermore, previous methods have resulted in
distribution ranges of metallic microtubules that average less than
10 microns in length once metallized. This results in low aspect
ratio. Utilization of the method according to this invention has
resulted in microtubules which have been observed to average 70
microns in length when metallized.
[0060] The number of bilayers in the wall of the tubule was
measured using a transmission electron microscope (Zeiss 100). The
tubule samples were stained with uranyl acetate which adsorbs
preferentially to the polar region of the bilayer. As a result the
bilayers, when viewed under high magnification
(>100,000.times.), appear in the transmission electron
micrograph as alternately dark and bright parallel lines. Then the
number of bilayers in the wall of the tubule can be determined by
simply counting the number of bright lines present. The number can
also be measured by circular dichroism (CD) spectroscopy.
[0061] The tubule formation process has been studied with circular
dichroism (CD). This analytical technique is described by L. Velluz
et al in "Optical Circular Dichroism. Principles, Measurements and
Applications" (Verlag Chemie, Weinheim) 1965. The CD spectra of
DC.sub.8,9PC tubules in methanol/water (7:3) shows an interesting
concentration dependence. The ratio of the ellipticity at 205 nm to
that at 195 nm changes with the lipid concentration is shown in
FIG. 1.
[0062] FIG. 1 shows a comparison of the CD spectra at 25.degree. C.
of methanol/water tubules prepared with different lipid
concentrations. The molar ellipticity of the 4 mg/ml. DC.sub.8,9PC
tubules is almost 50% greater than the 1 mg/ml sample at 205 nm,
while the spectra are nearly identical below 200 nm. The ratio of
the spectral intensity of the 205 nm to the 195 mn band as a
function of the lipid concentration is shown in FIG. 2. Up to about
1 mg/ml the ratio is constant, while it begins to increase above
this concentration. This is attributed to the tubules becoming more
multilamellar as the concentration of lipid is increased. This was
confirmed by electron microscopy. It has been as a result of the
fundamental understanding of the formation process gained by these
circular dichroism studies that it became clear that by varying the
concentration of lipid in a methanol/water solution and then
cooling it that the number of bilayers could be rationally
controlled.
Experimental Conditions
[0063] The lipid 1,2
bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine
(DC.sub.8,9PC) was purchased from Avanti Polar Lipids and
recrystallized in acetone. The samples for UV absorption
measurements were made by dissolving the lipid in spectroscopic
grade solvents (Aldrich). For the CD studies, the tubules were
prepared by dissolving DC.sub.8,9GPC in the appropriate alcohol and
mixing with water at 55.degree. C. On slowly cooling the mixture
through the lipid chain melting temperature (.about.37.degree. C.),
tubules are formed. The UV absorption measurements were performed
using a CARY dual-beam spectrometer operating at room temperature.
The CD studies were performed on a Jasco J-720 spectropolarimeter
operating between 175 and 700 mm. The samples were placed in
water-jacketed quartz cells with path lengths of 0.1, 0.2, or 1.0
mm. Temperature control was provided by a water circulator which
provided thermal stability of about 0.2.degree. C. The spectrometer
was calibrated with ammonium-d-camphorsulfonate
([.THETA.].sub.291=7910 deg cm.sup.2/dmol) and D-pantoyllactone
([.THETA.].sub.291=-16140 in water, [.THETA.].sub.223=-12420 in
methanol). Variations in the placement and orientation of the
sample showed that the CD spectrum was independent of birefringent
and scattering effects. Samples for electron microscopy were
negative stained using uranyl acetate to enhance the contrast.
Observations were made using a transmission electron microscope
(Zeiss EM-010) operating at 60 kV.
[0064] Having described the basic aspects of the invention, the
following examples are given to illustrate specific embodiments
thereof.
EXAMPLE 1
[0065] This example illustrates the production of microtubules
according to the invention.
[0066] Eighty five liters of technical grade methanol is
pre-filtered utilizing a 5 micron porous polymer membrane filter
and then fine filtered utilizing a 0.22 micron pore size membrane
filter to remove any particulate materials as is a further 15
liters of deionized water.
[0067] Five liters of the methanol is then removed to solubilize
the lipid, and the remainder is charged into a stainless steel or
glass temperature controlled vessel and heated with constant
agitation to between 45.degree. C. and 50.degree. C.
[0068] Five liters of the methanol is heated to 50.degree. C. and a
selected quantity of the lipid 1,2
bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine
(DC.sub.8,9PC) is added to the mixture sufficient that the final
ratio of chemically pure lipid is at a ratio of 5 gm/l in the final
mixture. Thus in this example if 100% pure it would be 500 grams
total.
[0069] The lipid is fully solubilized until a clear solution is
obtained. Following this step the resultant mixture is then
filtered through a 0.22 micron membrane filter and added with
agitation to the heated methanol and water solvent system.
[0070] The resulting mixture is allowed to blend by pumping or by
use of a mechanical mixer for a sufficient amount of time to ensure
full and homogenous mixing. A period of 30 minutes, for example,
has been found acceptable.
[0071] Following this step the lipid/solvent is then allowed to
cool in as homogenous a thermal environment as possible to a
uniform temperature of 32.degree. C. at a rate of change not to
exceed 1.degree. C. per hour where it is held until a very slight
haze is observed in the solution. Then the temperature is lowered
to between 15 and 20.degree. C. until the solution is observed to
become opaque and the viscosity increases until the mixture
exhibits a pronounced viscoelastic property. This may take a period
of 24 to 100 hours. When a very high yield of tubules is observed
by microscopic analysis it is possible to then remove the excess
methanol solution by dialysis against distilled water utilizing a
2,500 dalton cut off reverse osmosis membrane.
[0072] Following this step the tubules may be stored at 4.degree.
C. until further processing is possible.
[0073] In an effort to measure the amount of lipid that had not
been consumed in the formation process, a dialysis bag with a
25,000 dalton molecular weight cut off was employed to remove any
remaining alcohol and phospholipid from the tubule sample.
Typically it has been observed that the lipid will rapidly leave
solution when the alcohol is diluted 100:1 in 15-20.degree. C.
water at which time the presence of the lipid may be visually
observed. Following the dialysis for each of 3 samples done to
quantify the yield and structure of the microtubules by this
method, no lipid structures were observed forming in the water
phase. This was taken to confirm a >99% conversion factor for
lipid into microcylinders. Each of the above samples was further
examined by electron microscopy and further processing as in
Example 2 to denote the ability of the sample to withstand the
rigors of further processing.
[0074] Measured lengths were directly observed by light microscopy
to average >250 microns in length.
EXAMPLE 2
[0075] This example illustrates the metallic coating of
microtubules made according to the invention.
[0076] The microtubules may be immediately dialyzed against 0.1N
HCl until it is observed that they have reached a pH of 1.5.
Following this step the microtubules are allowed to settle by
gravity. Excess water is removed and the resulting volume is
determined.
[0077] A commercial palladium-tin catalyst solution is added
(Shipley Co. Cataposit 44) at a rate of 6 volumes of the full
strength catalyst solution to 1 volume of the microtubules. The
catalyst is observed to bind to the microtubules and then the
resulting tubules and bound catalyst is observed to settle as the
resultant specific gravity increases. To remove all unbound
catalyst and acidic salt solution that makes up the commercial
bath, the excess bath is removed and a pH 1.0 water solution is
added and the tubules allowed to resettle. This is followed by a
minimum of 3 more changes of water until the pH of the water/tubule
solution is observed to reach pH 5.5.
[0078] Electroless plating may be conducted with a solution of a
commercial plating bath such as Cuposit or Niposit (Shipley Co.) or
a laboratory formulated bath. The finished electroless metal bath
is added to the catalyzed microstructures at a ratio of 1:1 to 5:1.
A preferred ratio for the Cuposit 324 bath is 3:1. Following the
plating reaction the microcylinders are then rinsed to remove the
excess bath and kept in an oxygen free environment until use is
desired.
EXAMPLE 3
[0079] This example illustrates another process for electroless
plating of the tubules.
[0080] The microcylinders may be pretreated by one of the two
following methods. First a solution of 0.1N HCl is introduced to
the tubule suspension just sufficient to lower the pH to 1.5 pH.
Or, as an alterative, a solution of acidic salts such as Cataposit
444 may be used at the rate of 220 g/liter of distilled water until
a pH of 1.5-2.0 is achieved. Following this pretreatment step a
commercial catalyst system is introduced such as Cataposit 44 made
by the Shipley Co. at the recommended formulation such that the
final amount of catalyst exceeds the amount of lipid suspension by
a ratio of 6:1. The catalyst will be observed to bind to the
microcylinders and increase their specific gravity, at which time
they will settle due to increased density. Following this treatment
the catalyzed microcylinders are rinsed with at least 4 changes of
water until all excess unbound catalyst and acidic salts are
removed from solution. The pH of the resultant suspension in water
is increased to the published value for the electroless deposition
bath such as a pH of 8.0 with NaOH or KOH.
[0081] The electroless deposition bath is then mixed according to
the manufacturer's published methods and added to the catalyzed
microcylinders such that the volumetric ratio of catalyzed tubules
is in a ratio of 6:1.
[0082] When the plating reaction has stopped and the microcylinders
consist of metallic walls deposited over the lipid template they
are removed from solution for processing. If desired the lipid may
be removed for reprocessing with a hot methanol rinse.
EXAMPLE 4
[0083] This example illustrates the manufacture of multiple
coatings of a polysaccharide.
[0084] Electrostatic binding of a polysaccharide such as sodium
alginate or chitosan is possible to the microtubules. This is
accomplished by suspending the fully formed lipid microtubule in a
suspension of the solubilized polysaccharide such that the final
concentration of the polysaccharide is in the range of 0.25 to 2.5%
in solution where the concentration of lipid tubules to water is in
the range of 0.5 to 2.5% by weight. Following incubation of the
lipid tubules with the polysaccharide for at least an hour with
very, very gentle agitation, the tubules are then centrifuged or
filtered to remove the excess unbound polysaccharide. This step may
be repeated. Once this layer is electrostatically bound to the
surface it may be crosslinked with a calcium solution to form a
polymer in the case of alginate or treated with an amine such as
spermine to cross link the chitosan.
[0085] The resulting coating may then be electrostatically bound to
a dissimilar charged polysaccharide such as chitosan over alginate
or alginate over chitosan. This would result in a more robust and
stabilized microtubule which is better able to withstand mechanical
agitation.
[0086] Alternatively, a metal coating could be bound to the
chitosan layer by adding the catalyst as in the example of the
electroless plating the lipid microstructures. This offers the
advantage of being able to remove the lipid that comprises the
bilayer where it has been shielded from reaction with the catalyst.
This recovery of lipid is achieved by warm solvent extraction of
the lipid from the metallic microstructures and is enhanced by the
overcoat of chitosan which will bind the catalytic salt and permit
over coating with metal.
EXAMPLE 5
[0087] This example illustrates a method for producing multiple
bilayer tubules.
[0088] A temperature controlled reaction vessel is filled with a
mixed alcohol (methanol) and water mixed solvent system such that
it contains 75 parts by volume methanol and 15 parts water and
heated to at least 5.degree. C. above the lipid transition
temperature which is typically 45.degree. C. to 50.degree. C. A
further 10 parts alcohol (methanol) is heated as above and a
quantity of lipid is dissolved by stirring to yield 8-10 mg/ml of
the final mixture. This amount is larger than the 5 mg.ml used for
just making 2 bilayers. When dissolved it is filtered through a
0.22 micron filter.
[0089] The temperature is lowered to the transition temperature of
the lipid and held till tubule formation is observed. It is then
lowered at a rate of 1.degree. C. per minute to ambient temperature
or at least 20-25.degree. C. The tubules formed have more than just
2 bilayers. Following formation, the tubules are preferably stored
at a temperature of about 4.degree. C., although they can be stored
at any temperature which is below their transition temperature.
[0090] It is understood that the foregoing detailed description is
given merely by way of illustration and that many variations may be
made therein without departing from the spirit of this
invention.
* * * * *