System for delayed and pulsed release of biologically active substances

A system for controlled release both in vivo and in vitro of entrapped substances, either at a constant rate over a period of time or in discrete pulses, is disclosed. Biologically active substances, such as drugs, hormones, enzymes, genetic material, antigens including viruses, vaccines, or inorganic material such as dyes and nutrients, are entrapped in liposomes which are protected from the biological environment by encapsulation within semi-permeable microcapsules or a permeable polymeric matrix. Release of the entrapped substance into the surrounding environment is governed by the permeability of both the liposome and surrounding matrix to the substance. Permeability of the liposome is engineered by modifying the composition and method for making the liposomes, thereby producing liposomes which are sensitive to a specific stimuli such as temperature, pH, or light; or by including a phospholipase within some or all of the liposomes or the surrounding matrix; or by destabilizing the liposome to break down over a period of time; or by any combination of these features.

DETAILED DESCRIPTION OF THE INVENTION 
The method for controlled release of biologically active substances 
comprises: entrapping at least one substance within liposomes wherein the 
composition and method of preparation are selected to produce liposomes 
with a predetermined rigidity, stability and permeability, wherein the 
substance is contained within the aqueous compartment, either in solution, 
bound to other molecules, or bound to another solid medium, or the 
substance is incorporated into the membrane of the liposome; encapsulating 
the liposomes in a permeable polymeric matrix wherein the composition and 
method of preparation of the matrix are selected to produce material of a 
desired thickness, permeability, non-immunogenicity, and compatibility 
with the liposomes, and wherein additional or other substances may be 
encapsulated within the matrix, either in solution, bound to the matrix, 
or bound to another solid medium; and locating the encapsulated liposomes 
at the desired release site. 
The liposomes are formed primarily of a mixture of phospholipids. 
Cholesterol may be added to decrease membrane thickness, increase 
fluidity, and enhance solute entrapment. Charged phospholipids increase 
the volume of aqueous spaces within the liposomes and delay diffusion of 
entrapped ions of homologous charge. A number of possible lipid 
compositions is summarized on p. 293-306 of "Liposomes" by Gregory 
Gregoriadis. The lipid vesicles should have the following desirable 
properties: (i) the ability to entrap a large percentage of the aqueous 
material, even large macromolecular assemblies; (ii) a high aqueous 
space-to-lipid ratio; and (iii) a widely variable chemistry of the lipid 
components. 
The liposomes may be prepared by any of a number of techniques known to 
those skilled in the art. The preferred method is by reverse-phase 
evaporation, described in "Procedure for Preparation of Liposomes with 
large internal aqueous space and high capture by reverse-phase 
evaporation" by Szoka and Papahadjopoulos in Proc. Natl. Acad. Sci. 
U.S.A., 75(9), 4194-4198 (1978). Using this method, large unilamellar and 
oligolamellar vesicles are formed when an aqueous buffer is introduced 
into a mixture of phospholipid and organic solvent and the organic solvent 
is subsequently removed by evaporation under reduced pressure. 
A mixture of several phospholipids, either pure or mixed with other lipids 
such as cholesterol, long-chain alcohols, etc., may be used with similar 
results in the preparation of reverse-phase evaporation vesicles (REV). As 
described by Szoka et al., the lipid mixture is added to a 50-ml 
round-bottom flask and the solvent removed under reduced pressure by a 
rotary evaporator. The system is then purged with nitrogen and the lipids 
redissolved in the organic phase to form the reverse phase vesicles. 
Useful solvents include diethyl ether, isopropyl ether, halothane and 
trifluorotrichloroethane. When the lipid has low solubility in ether, 
chloroform or methanol can be added to increase its solubility. At this 
point, the aqueous phase is added under nitrogen and the resulting 
two-phase system sonicated briefly (2-5 min. at 0.degree.-5.degree. C. for 
most lipids) until the mixture becomes either a clear one-phase dispersion 
or a homogeneous opalescent dispersion that does not separate for at least 
30 min. after sonication. The degree of opalescence of the preparation at 
this point depends upon the solvent, phospholipid, and amount of aqueous 
phase in the preparation. The mixture is then placed on a rotary 
evaporator with a long extension neck, and the organic solvent removed 
under reduced pressure at 20.degree.-25.degree. C., rotating at 
approximately 200 rpm. 
The system froths during evaporation of the solvent. As the majority of the 
solvent is removed, the material first forms a viscous gel and 
subsequently, within 5-10 min., becomes an aqueous suspension. At this 
point, excess water or buffer can be added and the suspension evaporated 
for an additional 15 min. at 20.degree. C. to remove traces of solvent. 
When lipid mixtures lacking cholesterol are used at low concentrations of 
&lt;7.5 umol of lipid per ml of aqueous phase, the gel phase may not be 
apparent since the system rapidly reverts to a lipid-in-water suspension. 
To remove non-encapsulated material and residual organic solvent, the 
preparation is then either dialyzed, passed through a Sepharose 4B.TM. 
(Pharmacia Fine Chemicals, Piscataway, N.J.) column, or centrifuged 
(100,000.times.g for 30 min.). 
The vesicles may be filtered or passed through a molecular sieve column to 
give a more uniform vesicle size. For example, filtration through a 200 nm 
Unipore filter yields a vesicle size of 120-300 nm. A large number of 
variables may be responsible for determining the final product in terms of 
vesicle size. These include the type of phospholipid and its solubility in 
the organic solvent, the interfacial tension between aqueous buffer and 
organic solvent, and the relative amounts of water phase, organic solvent, 
and phospholipids. 
The entrapment of a particular molecule within the vesicles is dependent on 
several variables, including the ionic strength of the buffer. As the 
ionic strength is increased, there is a decrease in both the percent 
entrapment and the volume of entrapped aqueous space per mole of 
phospholipid. Varying lipid concentration can increase the amount of 
capture up to about 45% in 0.15M NaCl. The percentage of molecules 
entrapped decreases with decreasing total lipid but the ratio of aqueous 
volume within the vesicle/mole of phospholipid increases. Different lipid 
compositions entrapment different amounts of liquid. The addition of 
cholesterol significantly increases the volume of entrapped liquid. 
Reverse-phase evaporation vesicles (REV) tend to capture a larger volume 
of aqueous space than do multilamella (MLV) and sonicated unilamella 
vesicles (SUV). 
The proposed formation of lipid vesicles with simultaneous entrapment of 
suspended molecules is diagrammed in FIG. 1. In panel 1, lipids 10 are 
dissolved in the appropriate solvent 12. In panel 2, lipids 10 form a 
bilayer membrane 14 structure as the compound 16 to be entrapped is added. 
In panel 3, the initial sonication of the buffered aqueous phase 18 in the 
organic solvent 12 in the presence of amphiphatic phospholipid molecules 
produces small water droplets 20 stabilized by a phospholipid monolayer 
22. In panel 4, the droplets or "inverted micelles" 20 are collapsed to 
form a viscous gel-like material 23 when the organic phase 12 is removed 
by evaporation. In panel 5, the gelled material 23 collapses. Very little 
additional material 16 is entrapped by the vesicles 20 at this point. In 
panel 6, some of the inverted micelles 20 disintegrate, releasing their 
entrapped material 16. The excess lipid contributes to a complete bilayer 
24 around the remaining micelles 20, resulting in the formation of 
vesicles 26. 
Other methods for the entrapment of molecules by formation of liposomes may 
be used in the present invention. A method for the preparation of large 
unilamella vesicles (LUVs) is described in "A Simple Method for the 
Preparation of Homogeneous Phospholipid Vesicles" by Barenholz et al. in 
Biochemistry, 16(12), 2806-2810 (1977). This method involves differential 
high-speed ultracentrifugation of sonicated aqueous lipid suspensions. 
A method for "Preparation of Homogeneous, Single-Walled Phosphatidyl 
Choline Vesicles" by Huang and Thompson in Methods in Enzymol. 32, p. 
485-489 (1974) yields spherical, homogeneous vesicles each comprised of a 
single continuous lipid bilayer. The method consists of suspending the 
lipid in an aqueous salt solution, sonicating the suspension at 2.degree. 
C. under an inert gas to prevent oxidation, centrifuging, concentrating by 
ultra-filtration, and sizing by column chromatography. Sonication at 
higher temperatures or in air causes degradation of the phospholipid by 
oxidation of the unsaturated acetyl chains or hydrolysis of the ester 
bonds. The resulting instability of the liposomes can be manipulated to 
advantage in the present invention. 
"The Preparation of Large Single Bilayer Liposomes by A Fast and Controlled 
Dialysis" by Milsmann et al. in Biochem. Biophys. Acta, 512, 147-155 
(1978) discloses another method for preparing single bilayer phospholipid 
vesicles. This method is based on a fast and controlled dialysis of sodium 
cholate from phosphatidyl choline/cholate mixed micelles. 
Yet another method for preparation of liposomes is taught by "Large Volume 
Liposomes by an Ether Vaporization Method" by Deamer and Bangham in 
Biochem. Biophys. Acta, 443, 629-634 (1976). This method forms unilamellar 
liposomes by injecting ether solutions of various lipids into a warm 
aqueous solution. 
Still another method for preparation of liposomes with entrapped molecules 
is described by Gregoriadis in FEBS Letters, 36(3), 292-296 (1973). This 
method is similar to the preferred method, consisting of dissolving the 
lipids in chloroform and evaporating the solvent under vacuum along with 
aqueous solution of the substances to be entrapped. 
The liposomes may be designed and prepared to respond to a specific 
stimulus, or combination of stimuli, as well as to have a particular 
stability, rigidity, and permeability. All liposomes are inherently 
unstable due to interactions between the particular lipids they are 
comprised of and their environment. However, other stimuli such as pH, 
temperature, or light can be used to trigger release of entrapped material 
at a more specific time. 
"pH-triggering of phosphatidyl choline membrane properties via complexation 
with synthetic poly(carboxylic acid)s" by Seki et al in "Polym. Materials 
Sciences and Eng.", Proc. of Acs. Div. of Polym. Materials Meeting in 
Philadelphia, PA., ACS, 51, 216-219 (1984) describes the use of a 
synthetic poly(carboxylic acid), poly(alphaethylacrylic acid) PEAA to 
effect a pH-dependent release of the contents of vesicles formed from egg 
yolk phosphatidyl choline. Phosphatidyl choline vesicles are unaffected by 
PEAA at high pH but are rendered unstable at pH 7 or below. Since the pH 
of lysosomes is approximately 4.6, liposomes which are intact when they 
circulate in the bloodstream at physiological pH may be stimulated to 
release their contents when they are taken up by the lysosome-containing 
cells, usually by endocytosis. 
Liposomes which undergo dramatic increases in permeability when irradiated 
with light are known. Two photosensitive phospholipids, 
1,2-diretinoyl-Sn-glycero-3-phosphocholine and 
1-palmitoyl,2-retinoyl-Sn-glycero-3-phosphocholine, are described by 
Pidgeon and Hunt in "Light Sensitive Liposomes" in Photochem and 
Photobiol. 37, 491-494 (1983). The permeability of liposomes formed from 
either or both of these phospholipids is directly proportional to 
temperature. Upon exposure to 30 to 120 seconds of 360 nm light, the 
permeability of the liposomes increases dramatically, from approximately 
20% to almost 90%. 
Another photosensitive system is described by Kano et al. in Photochem. 
Photobiol. 34, 323-325 (1981) and Chem. Lett. 421-424 (1981). Kano et al 
showed that incorporation of light isomerizable azobenzene lipids into 
liposome membranes produces vesicles with increased membrane permeability 
upon exposure to light. 
The sensitivity of liposomes to temperature is also well known. Specific 
lipid compositions may be formulated so that their transition temperature 
is above the temperature at which the liposomes are to store the entrapped 
substances yet low enough to allow for release when the temperature is 
raised slightly. In vivo, this may be done as easily as subcutaneously 
injecting encapsulated liposomes, then applying a heating pad to stimulate 
release. 
The liposomes may also be destabilized using phospholipase enzymes. These 
may be entrapped within the liposomes or packaged within the surrounding 
matrix. To prevent diffusion or release of the enzyme from the liposome or 
matrix, the molecular weight of the enzyme may be increased by any of a 
number of methods, thereby trapping the enzyme. For example the 
phospholipase may be linked chemically or ionically to solid media such as 
Sephadex.TM., CM-Sephadex.TM. or DEAE-Sephadex.TM. (Pharmacia Fine 
Chemicals, Piscataway, N.J.) or crosslinked between enzyme molecules using 
bifunctional reagents such as glutaraldehyde, possibly in the presence of 
substrate to protect the active site. Also, the enzyme could be covalently 
linked to a soluble polymer such as poly-L-lysine. The phospholipase is 
selected for ability to cleave one or more of the phospholipids making up 
the liposome, and non-toxicity to the organism or surrounding cells if for 
in vivo use. 
For in vitro systems, other agents may be added to the solution to 
stimulate release of the entrapped material. For example, detergents such 
as Triton X100 or high salt solutions can diffuse into the matrix with 
subsequent disruption of the liposomes. Non-ionic detergents such as octyl 
glucoside can be used, and have the advantage of being less damaging to 
proteins compared to ionic detergents, as shown by M. L. Jackson et al, 
Biochem. 21, 4576-4582 (1982). Bivalent metals have also been shown by D. 
Papahadjopoulos and J. C. Watkins in Biochem. Biophys. Acta. 135, 639-652 
(1967) to increase the permeability of liposomal bilayers. 
Other stimuli and methods for preparing liposomes which are responsive to 
these stimuli and characterized by a particular rigidity, permeability, 
and stability are known to those skilled in the art. Essentially, any 
method for preparing liposomes which become unstable after a predetermined 
period of time or whose permeability can be significantly altered by 
manipulation of the immediate environment, either in vivo or in vitro, may 
be used in the present invention. 
A variety of substances can be entrapped within the liposomes. Examples of 
biologically active substances are proteins (such as enzymes, hormones, 
and globulins), polyamino acids, nucleic acids, drugs, vitamins, small 
virus particles, and other small molecules, including inorganic materials 
such as pesticides and nutrients. Other substances such as dyes may also 
be entrapped or located within the permeable matrix. 
As shown in FIG. 2, any water soluble 30 or suspendable molecule can be 
entrapped in the aqueous portion 32 of a liposome 26. Lipid soluble 
molecules 34 can be incorporated into the lipid bilayers 24. Hydrophobic 
ends 36 of water soluble molecules can also be inserted into the lipid 
layers 24. 
In the method of the present invention, the liposomes are encapsulated 
within a permeable polymeric matrix consisting of any non-toxic polymer or 
mixture of polymers which can be polymerized using a method which does not 
harm the liposomes and which results in a matrix of the desired thickness, 
rigidity, permeability, and stability. 
In one embodiment, the permeable matrix consists of microcapsules formed 
around the liposomes. The preferred method for making capsules is taught 
by U.S. Pat. No. 4,352,883 to Lim. Using this method, 500 micron diameter 
(sized by gel filtration) capsules with a permeability of approximately 
6,000 to 40,000 m.w. are formed with a core of alginate cross-linked with 
calcium ions selectively coated with a polycationic skin using polymers 
such as poly-L-lysine and poly-vinyl amine. 
The process is as follows: the liposomes, containing the entrapped 
substances, are encapsulated in a physiologically compatible medium 
containing a water soluble substance that can be made insoluble (gelled). 
The medium is then formed into droplets around the liposomes and gelled by 
changing temperature, pH or ionic strength. The gelled droplets are then 
treated to produce membranes of a controlled permeability about the gelled 
droplets. The presently preferred material for forming the temporary 
capsules is polysaccharide gums, either natural or synthetic, of the type 
which can be gelled to form a shape retaining mass by exposure to a 
different pH or multivalent cations such as Ca++ and ionically crosslinked 
by polymers containing reactive groups such as amine or imine groups which 
can react with acidic polysaccharide constituents. Gelatin or agar may be 
used in place of the polysaccharide gums. 
As shown in FIG. 3, the preferred method of formation of the droplets is to 
use a syringe pump 37 to force the sodium alginate-liposome suspension 38 
through a capillary tube or 22 gauge needle 39 around which flows a 
coaxial stream of air. Droplets 41 ejected from the tip of the needle 39 
immediately contact a 1.5% CaCl.sub.2 solution 43 and gel as sphere-shaped 
bodies 45. 
The preferred method of forming a permanent semipermeable membrane about 
the temporary capsules is to "crosslink" surface layers of the gelled 
alginate using a dilute solution of polyamino acids such as poly-L-lysine 
and poly-vinyl amine. Generally, the lower the molecular weight of the 
polymer, the greater the penetration into the surface of the temporary 
capsule and the less permeable the resulting membrane. Ionic crosslinks 
are produced as a consequence of salt formation between the acid reactive 
groups of the crosslinking polymer and the acid groups of the 
polysaccharide gum. Within limits, permeability can be controlled by 
setting the molecular weight of the crosslinking polymer, its 
concentration, and the duration of reaction. 
The in vivo life of the capsules is a function of the cross-linking 
polymer. For example, proteins or polypeptide crosslinkers, such as 
polylysine, are enzymatically attacked in vivo. Crosslinkers not readily 
digestible in mammalian bodies, such as polyethyleneimine, produce longer 
lasting membranes. 
It is possible to improve mass transfer within the capsule after formation 
of the permanent membrane by re-establishing the conditions under which 
the material forming the temporary capsule is liquid, for example, by 
removing the multivalent cation by ion exchange in phosphate buffered 
saline containing citrate. 
Other methods and materials for encapsulating liposomes include the method 
of Wheatley and Phillips in Adv. in Biotechnol. II, 47-54 (1980) which 
uses encapsulation in polyacrylamide gels. The gels are polymerized by 
free radical polymerization using potassium persulfate as the initiator 
and beta-dimethylaminopropionitrile as the promotor. In this case, the 
reaction results in a sheet of polymer which may be cut into fragments 
after the polymerization reaction is complete. 
Thermal gelation of agarose gels, such as the method of Howel et al in 
Proc. Physiol. Soc., 20p-21p (November 1981) is another possible method. 
The agarose, Seaprep 15/45.TM. (FMC Corporation, Marine Colloids Division, 
Maine), can be loaded at 22.degree. C. with the gels in their sol form. 
Cooling to 15.degree. C. allows the gel to pass through the sol-gel 
transition and the agarose remains intact up to temperatures of 45.degree. 
C. before it melts. 
In general, the requirements for the method and materials for encapsulation 
within a permeable polymeric matrix are as follows: 
The method must not destroy the integrity of the liposomes. 
The matrix must retain the liposomes. 
The matrix must allow for diffusion of the entrapped substance out of the 
matrix after its release from the liposome. 
All materials for in vivo use must be non-toxic to the body. 
There must be no chemical interaction between the components of the matrix 
and both the liposomes and the entrapped substance. 
In one example of the present invention, shown in FIG. 4, myoglobin 40 
containing-liposomes 42, prepared by reverse phase evaporation using the 
method of Szoka et al, Proc. Natl. Acad. Sci. U.S.A., 75(9), 4194-4198 
(1978) and comprising phosphatidyl choline, phosphatidyl glycerol, and 
cholesterol in a molar ratio of 9:1:8, were encapsulated in 500 micron 
diameter capsules 44, prepared with a core 46 of calcium ion-crosslinked 
alginate selectively coated with poly-L-lysine and poly-vinyl amine and 
having a permeability of 6,000 to 40,000 mw. 
One ml. equivalents of encapsulated liposomes 48 were placed in vials 50, 
shown in FIG. 5. A 105 micron polypropylene mesh tube 54, sealed at the 
base with a wax plug 52, was used to retain the encapsulated liposomes 48. 
The vials 50 were then filled with 10 ml. of physiological saline and 
placed on a shaker. The temperature was maintained at 37.degree. C. The 
physiological saline in the vials 50 was changed frequently to mimic the 
infinite sink conditions of the body. 
Release of myoglobin 40 under various conditions was determined over time 
by absorbance at 410 nm. The results are graphed in FIG. 6. The experiment 
was conducted over a 1200 hour period. The untreated encapsulated 
liposomes 62 showed little release of entrapped myoglobin for the first 
200 hours and then released approximately 90% over the next 500 hours. 
Encapsulated liposomes treated with Triton X100 64 displayed an initial 
release of approximately 30% at 100 hours, and a second burst of another 
50% after 500 to 600 hours. Treatment involved brief contact with 10 ml of 
0.1% Triton X100 in physiological saline, pH 7.4. After 5 minutes contact 
time the encapsulated liposomes were transferred to 10 mls of regular 
physiological saline and the experiment was continued in the normal 
manner. Sonicated liposomes 66 also showed an initial release of 
approximately 30% after 100 hours with a subsequent release of an 
additional 45% after another 500 to 600 hours. Sonication involved 
subjecting the encapsulated liposomes, contained within the polypropylene 
tube in a vial filled with 10 ml of physiological saline, to a 5 minute 
burst of sonic energy. This was achieved by immersing the vial in cold 
(4.degree. C.) water in a Laboratory Supplies Sonic bath G112SPIT. With 
the "untreated" 62, Triton X100 64, and sonicated 66 liposomes, almost 
100% of the myoglobin had been released into the surrounding medium by 
1000 hours. With the treated capsules, increased treatment time led to 
greater release of entrapped material at the first pulse. 
The results show that by varying the conditions to which the encapsulated 
liposomes are exposed, the entrapped material can be released immediately, 
slowly over a period of time, or in discrete pulses. 
Liposomes may also be encapsulated within a permeable polymeric matrix. 
Sheets of the liposome-matrix are useful in a number of situations. One 
example is to provide a means for release of nutrients or drugs into a 
cell culture wherein the liposome-matrix is placed in the bottom of the 
cell culture for controlled release into the media. The liposome-matrix 
can be formed and used in sheet form or cut into multiple discrete pieces. 
Another use is in the release of inorganic compounds, such as a dye whose 
release is indicative of the passage of a predetermined time period, or 
fertilizer or pesticide into a plant medium. 
As diagrammed in FIG. 7, a transdermal drug transfer device 70 may be 
constructed utilizing drug-containing liposomes 72 encapsulated within a 
permeable polymeric matrix 74 to deliver a controlled drug dosage. As 
depicted, the device 70 is comprised of a backing material or support 
material 74, adhesive edges 76 for securing the device to the skin, the 
drug-containing liposomes 72 in matrix 74, and a protective covering 78. 
Any of the previously discussed means can be used to provide controlled 
drug delivery, specifically, alteration of the composition and method of 
manufacture of the liposomes and the surrounding matrix. In addition, 
removal of the protective covering can be used to disrupt the 
microcapsules or the surrounding matrix, thereby increasing contact of the 
liposomes with the skin, facilitating delivery of the drug. Also, 
microcapsules can be disrupted by fingertip pressure over the entire 
patch. 
As reported by M. G. Ganesan, N. D. Weiner, G. L. Flynn and N. F. H. Ho at 
Int. J. Pharm. 20, 139-154 (1984), lipophilic drugs such as progesterone 
and hydrocortisone, which are intercalated within the bilayer structure of 
the phospholipid in multilamellar liposomes, directly pass through skin 
from the liposomes. Due to increased soluble payloads of lipophilic drugs 
through liposomal incorporation, more total drug may be delivered through 
skin via liposomes relative to simple aqueous solutions. The liposomes do 
not actually pass through the skin, but provide the means for the enhanced 
delivery of the drug. 
In many situations it is advantageous to provide for a sustained, uniform 
release of a drug over a predetermined period of time. Particularly with 
ill or elderly patients, remembering when a drug-containing device or 
"skin patch" has been placed on the skin is difficult, especially when the 
time period is an odd number of days rather than once daily or once 
weekly. The present invention has another advantage since this problem may 
be overcome by entrapping a colored marker or dye along with the drug in 
the liposomes constructed to release the last portion of the drug. Release 
of the marker provides a way of readily ascertaining that it is time to 
change the skin patch. 
There are a number of advantages to the liposome skin patch for transdermal 
drug transfer over presently available skin patches or liposomes randomly 
distributed in an ointment: the amount and rate of release of the drug is 
controlled, and may be either continuous or in discrete pulses, wherein 
the composition, method of manufacture, and loading of the liposomes as 
well as the loading and available surface area of the skin patch are the 
determining factors; the liposomes are protected and uniformly distributed 
in the matrix, unlike in an ointment; and the liposomes do not contact the 
skin patch or adhesive, as would unencapsulated liposomes. 
The present invention may be embodied in other specific forms without 
departing from the spirit and scope thereof. These and other modifications 
of the invention will occur to those skilled in the art, keeping in mind 
the key feature of the invention is to provide a method and apparatus for 
controlled release, either continuously or in discrete pulses, of an 
entrapped substance wherein the active control is provided by selection of 
the composition and method of manufacture of the liposomes and protective 
matrix around the liposomes, whether microcapsules or a permeable 
polymeric matrix; and then entrapping at least one substance within the 
liposomes and surrounding matrix. Such other embodiments and modifications 
are intended to fall within the scope of the appended claims.