Method of making polymeric gas or air filled microballoons for ultrasonic echography

Method of making gas or air filled microballoons having a mean size in the range of 0.5 to 1000 microns bounded by a 50 to 500 nm thick biodegradable, interfacially deposited, synthetic polymer membrane which is deformable and resilient for use in ultrasonic contrast compositions are described.

BACKGROUND OF THE INVENTION 
The present invention concerns air or gas filled microcapsules or 
microballoons enclosed by an organic polymer envelope which can be 
dispersed or suspended in aqueous media and used in this form for oral, 
rectal and urethral applications or for injection into living beings, for 
instance for the purpose of ultrasonic echography and other medical 
applications. 
FIELD OF THE INVENTION 
The invention also comprises a method for making said microballoons in the 
dry state, the latter being instantly dispersible in an aqueous liquid 
carrier to give suspensions with improved properties over existing similar 
products. Hence, suspensions of the microballoons in a carrier liquid 
ready for administration are also part of the invention. 
RELATED ART 
It is well known that microbodies or microglobules of air or a gas, e.g. 
microspheres like microbubbles or microballoons, suspended in a liquid are 
exceptionally efficient ultrasound reflectors for echography. In this 
disclosure the term of "microbubble" specifically designates air or gas 
microspheres in suspension in a carrier liquid which generally result from 
the introduction therein of air or a gas in divided form, the liquid 
preferably also containing surfactants or tensides to control the surface 
properties and the stability of the bubbles. In the microbubbles, the gas 
to liquid interface essentially comprises loosely bound molecules of the 
carrier liquid. The term of "microcapsule" or "microballoon" designates 
preferably air or gas bodies with a material boundary or envelope of 
molecules other than that of the carrier liquid, i.e. a polymer membrane 
wall. Both microbubbles and microballoons are useful as ultrasonic 
contrast agents. For instance Injecting into the bloodstream of living 
bodies suspensions of gas microbubbles or microballoons (in the range of 
0.5 to 10 .mu.m) in a carrier liquid will strongly reinforce ultrasonic 
echography imaging, thus aiding in the visualization of internal organs. 
Imaging of vessels and internal organs can strongly help in medical 
diagnosis, for instance for the detection of cardiovascular and other 
diseases. 
The formation of suspensions of microbubbles in an injectable liquid 
carrier suitable for echography can be produced by the release of a gas 
dissolved under pressure in this liquid, or by a chemical reaction 
generating gaseous products, or by admixing with the liquid soluble or 
insoluble solids containing air or gas trapped or adsorbed therein. 
For instance, in U.S. Pat. No. 4,446,442 (Schering), there are disclosed a 
series of different techniques for producing suspensions of gas 
microbubbles in a sterilized injectable liquid carrier using (a) a 
solution of a tenside (surfactant) in a carrier liquid (aqueous) and (b) a 
solution of a viscosity enhancer as stabilizer. For generating the 
bubbles, the techniques disclosed there include forcing at high velocity a 
mixture of (a), (b) and air through a small aperture; or injecting (a) 
into (b) shortly before use together with a physiologically acceptable 
gas: or adding an acid to (a) and a carbonate to (b), both components 
being mixed together Just before use and the acid reacting with the 
carbonate to generate CO.sub.2 bubbles; or adding an over-pressurized gas 
to a mixture of (a) and (b) under storage, said gas being released into 
microbubbles at the time when the mixture is used for injection 
One problem with microbubbles is that they are generally short-lived even 
in the presence of stabilizers. Thus, in EP-A-131.540 (Schering), there is 
disclosed the preparation of microbubble suspensions in which a stabilized 
injectable carrier liquid, e.g. a physiological aqueous solution of salt, 
or a solution of a sugar like maltose, dextrose, lactose or galactose, is 
mixed with solid microparticles (in the 0.1 to 1 .mu.m range) of the same 
sugars containing entrapped air. In order to develop the suspension of 
bubbles in the liquid carrier, both liquid and solid components are 
agitated together under sterile conditions for a few seconds and, once 
made, the suspension must then be used immediately, i.e. it should be 
injected within 5-10 minutes for echographic measurements; indeed, because 
the bubbles are evanescent, the concentration thereof becomes too low for 
being practical after that period. 
Another problem with microbubbles for echography after injection is size. 
As commonly admitted, microbubbles of useful size for allowing easy 
transfer through small blood vessels range from about 0.5 to 10 .mu.m; 
with larger bubbles, there are risks of clots and consecutive emboly. For 
instance, in the bubble suspensions disclosed in U.S. Pat. No. 4,446,442 
(Schering) in which aqueous solutions of surfactants such as lecithin, 
esters and ethers of fatty acids and fatty alcohols with polyoxyethylene 
and polyoxyethylated polyols like sorbitol, glycols and glycerol, 
cholesterol, or polyoxy-ethylenepolyoxypropylene polymers, are vigorously 
shaken with solutions of viscosity raising and stabilizing compounds such 
as mono- and polysaccharides (glucose, lactose, sucrose, dextran, 
sorbitol); polyols, e.g. glycerol, polyglycols; and polypeptides like 
proteins, gelatin, oxypolygelatin and plasma protein, only about 50% of 
the microbubbles are below 40-50 .mu.m which makes such suspensions 
unsuitable in many echographic application. 
In contrast, microcapsules or microballoons have been developed in an 
attempt to cure some or the foregoing deficiencies. As said before, while 
the microbubbles only have an immaterial or evanescent envelope, i.e. they 
are only surrounded by a wall of liquid whose surface tension is being 
modified by the presence of a surfactant, the microballoons or 
microcapsules have a tangible envelope made of substantive material other 
than the carrier itself, e.g. a polymeric membrane with definite 
mechanical strength. In other terms, they are microspheres of solid 
material in which the air or gas is more or less tightly encapsulated. 
For instance, U.S. Pat. No. 4,276,885 (Tickner et al.) discloses using 
surface membrane microcapsules containing a gas for enhancing ultrasonic 
images, the membrane including a multiplicity of non-toxic and 
non-antigenic organic molecules. In a disclosed embodiment, these 
microbubbles have a gelatin membrane which resists coalescence and their 
preferred size is 5-10 .mu.m. The membrane of these microbubbles is said 
to be sufficiently stable for making echographic measurements; however It 
is also said that after a period of time the gas entrapped therein will 
dissolve in the blood-stream and the bubbles will gradually disappear, 
this being probably due to slow dissolution of the gelatin. Before use, 
the microcapsules are Kept in gelatin solutions in which they are storage 
stable, but the gelatin needs to be heated and melted to become liquid at 
the time the suspension is used for making injection. 
Microspheres of improved storage stability although without gelatin are 
disclosed in U.S. Pat. No. 4,718,433 (Feinstein). These microspheres are 
made by sonication (5 to 30 KHz) of viscous protein solutions like 5% 
serum albumin and have diameters in the 2-20 .mu.m range, mainly 2-4 
.mu.m. The microspheres are stabilized by denaturation of the membrane 
forming protein after sonication, for instance by using heat or by 
chemical means, e.g. by reaction with formaldehyde or glutaraldehyde. The 
concentration of stable microspheres obtained by this technique is said to 
be about 8.times.10.sup.6 /ml in the 2-4 .mu.m range, about 10.sup.6 /ml 
in the 4-5 .mu.m range and less than 5.times.10.sup.5 in the 5-6 .mu.m 
range. The stability time of these microspheres is said to be 48 hrs or 
longer and they permit convenient left heart imaging after intravenous 
injection. For instance, the sonicated albumin microbubbles when injected 
into a peripheral vein are capable of transpulmonary passage. This results 
in echocardiographic opacification of the left ventricle cavity as well as 
myocardial tissues. 
Recently still further improved microballoons for injection ultrasonic 
echography have been reported in EP-A-324.938 (Widder). In this document 
there are disclosed high concentrations (more than 10.sup.8) or air-filled 
protein-bounded microspheres of less than 10 .mu.m which have life-times 
of several months or more. Aqueous suspensions of these microballoons are 
produced by ultrasonic cavitation of solutions of denaturable proteins, 
e.g. human serum albumin, which operation also leads to a degree of 
foaming of the membrane-forming protein and its subsequent hardening by 
heat. Other proteins such as hemoglobin and collagen are said to be 
convenient also. 
Still more recently M. A. Wheatley et al., Biomaterials 11 (1990), 713-717, 
have reported the preparation of polymer-coated microspheres by ionotropic 
gelation of alginate. The reference mentions several techniques to 
generate the microcapsules; in one case an alginate solution was forced 
through a needle in an air jet which produced a spray of nascent air 
filled capsules which were hardened in a bath of 1.2% aqueous CaCl.sub.2. 
In a second case involving co-extrusion of gas and liquid, gas bubbles 
were introduced into nascent capsules by means of a triple-barelled head, 
i.e. air was injected into a central capillary tube while an alginate 
solution was forced through a larger tube arranged coaxially with the 
capillary tube, and sterile air was flown around it through a mantle 
surrounding the second tube. Also in a third case, gas was trapped in the 
alginate solution before spraying either by using a homogeneizer or by 
sonication. The microballoons thus obtained had diameters in the range 
30-100 .mu.m, however still oversized for easily passing through lung 
capillaries. 
The high storage stability of the suspensions of microballoons disclosed in 
EP-A-324.938 enables them to be marketed as such, i.e. with the liquid 
carrier phase, which is a strong commercial asset since preparation before 
use is no longer necessary. However, the protein material used in this 
document may cause allergenic reactions with sensitive patients and, 
moreover, the extreme strength and stability of the membrane material has 
some drawbacks: for instance, because of their rigidity, the membranes 
cannot sustain sudden pressure variations to which the microspheres can be 
subjected, for instance during travel through the blood-stream, these 
variations of pressure being due to heart pulsations. Thus, under 
practical ultrasonic tests, a proportion of the microspheres will be 
ruptured which makes imaging reproducibility awkward; also, these 
microballoons are not suitable for oral application as they will not 
resist the digestive enzymes present in the gastrointestinal tract. 
Moreover, it is known that microspheres with flexible walls are more 
echogenic than corresponding microspheres with rigid walls. 
Furthermore, in the case of injections, excessive stability of the material 
forming the walls of the microspheres will slow down its biodegradation by 
the organism under test and may result into metabolization problems. Hence 
it is much preferable to develop pressure sustaining microballoons bounded 
by a soft and elastic membrane which can temporarily deform under 
variations of pressure and endowed with enhanced echogenicity; also it 
might be visualized that micro-balloons with controllable 
biodegradability, for instance made of semi-permeable biodegradable 
polymers with controlled micro-porosity for allowing slow penetration of 
biological liquids, would be highly advantageous. 
BRIEF SUMMARY OF THE INVENTION 
These desirable features have now been achieved with the microballoons of 
the present invention as defined in the claims. Moreover, although the 
present microspheres can generally be made relatively short-lived, i.e. 
susceptible to biodegradation to cope with the foregoing metabolization 
problems by using selected types of polymers, this feature (which is 
actually controlled by the fabrication parameters) is not a commercial 
drawback because either the microballoons can be stored and shipped dry, a 
condition in which they are stable indefinitely, or the membrane can be 
made substantially impervious to the carrier liquid, degradation starting 
to occur only after Injection. In the first case, the microballoons 
supplied in dry powder form are simply admixed with a proportion of an 
aqueous phase carrier before use, this proportion being selected depending 
on the needs. Note that this is an additional advantage over the prior art 
products because the concentration can be chosen at will and initial 
values far exceeding the aforementioned 10.sup.8 /ml, i.e. in the range 
10.sup.5 to 10.sup.10, are readily accessible. It should be noted that the 
method of the invention (to be disclosed hereafter) enables to control 
porosity to a wide extent; hence microballoons with a substantially 
impervious membrane can be made easily which are stable in the form of 
suspensions in aqueous liquids and which can be marketed as such also.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
Microspheres with membranes of interfacially deposited polymers, although 
in the state where they are filled with liquid, are well known in the art. 
They may normally result From the emulsification into droplets (the size 
of which is controllable in function to the emulsification parameters) of 
a first aqueous phase in an organic solution of polymer followed by 
dispersion of this emulsion into a second water phase and subsequent 
evaporation of the organic solvent. During evaporation of the volatile 
solvent, the polymer deposits interfacially at the droplets boundary and 
forms a microporous membrane which efficiently bounds the encapsulated 
first aqueous phase from the surrounding second aqueous phase. This 
technique, although possible, is not preferred in the present invention. 
Alternatively, one may emulsify with an emulsifier a hydrophobic phase in 
an aqueous phase (usually containing viscosity increasing agents as 
emulsion stabilizers) thus obtaining an oil-in-water type emulsion of 
droplets of the hydrophobic phase and thereafter adding thereto a membrane 
forming polymer dissolved in a volatile organic solvent not miscible with 
the aqueous phase. 
If the polymer is insoluble in the hydrophobic phase, it will deposit 
interfacially at the boundary between the droplets and the aqueous phase. 
Otherwise, evaporation of the volatile solvent will lead to the formation 
of said interfacially deposited membrane around the droplets of the 
emulsified hydrophobic phase. Subsequent evaporation of the encapsulated 
volatile hydrophobic phase provides water filled microspheres surrounded 
by interfacially deposited polymer membranes. This technique which is 
advantageously used in the present invention is disclosed by K. Uno et al. 
in J. Microencapsulation 1 (1984), 3-8 and K. Makino et al., Chem. Pharm. 
Bull. 33 (1984), 1195-1201. As said before, the size of the droplets can 
be controlled by changing the emulsification parameters, i.e. nature of 
emulsifier (more effective the surfactant, i.e. the larger the hydrophilic 
to lipophilic balance, the smaller the droplets) and the stirring 
conditions (faster and more energetic the agitation, the smaller the 
droplets). 
In another variant, the interfacial wall forming polymer is dissolved in 
the starting hydrophobic phase itself; the latter is emulsified into 
droplets in the aqueous phase and the membrane around the droplets will 
form upon subsequent evaporation of this encapsulated hydrophobic phase. 
An example of this is reported by J. R. Farnand et el., Powder Technology 
22 (1978), 11-16 who emulsify a solution of polymer (e.g. polyethylene) in 
naphthalene in boiling water, then after cooling they recover the 
naphthalene in the form of a suspension of polymer bounded microbeads in 
cold water and, finally, they remove the naphthalene by subjecting the 
microbeads to sublimation, whereby 25 .mu.m microballoons are produced. 
Other examples exist, in which a polymer is dissolved in a mixed 
hydrophobic phase comprising a volatile hydrophobic organic solvent and a 
water-soluble organic solvent, then this polymer solution is emulsified in 
a water phase containing an emulsifier, whereby the water-soluble solvent 
disperses into the water phase, thus aiding in the formation of the 
emulsion of microdroplets of the hydrophobic phase and causing the polymer 
to precipitate at the interface; this is disclosed in EP-A-274,961 (H. 
Fessi), 
The aforementioned techniques can be adapted to the preparation of air or 
gas filled microballoons suited for ultrasonic imaging provided that 
appropriate conditions are found to control sphere size in the desired 
ranges, cell-wall permeability or imperviousness and replacement of the 
encapsulated liquid phase by air or a selected gas. Control of overall 
sphere size is obviously important to adapt the microballoons to use 
purposes, i.e. injection or oral intake. The size conditions for injection 
(about 0.5-10 .mu.m average size) have been discussed previously. For oral 
application, the range can be much wider, being considered that 
echogenicity increases with size; hence microballoons in several size 
ranges between say 1 and 1000 .mu.m can be used depending on the needs and 
provided the membrane is elastic enough not to break during transit in the 
stomach and intestine. Control of cell-wall permeability is important to 
ensure that infiltration by the injectable aqueous carrier phase is absent 
or slow enough not to impair the echographic measurements but, in cases, 
still substantial to ensure relatively fast after-test biodegradability, 
i.e. ready metabolization of the suspension by the organism. Also the 
microporous structure of the microballoons envelope (pores of a few nm to 
a few hundreds of nm or more for microballoons envelopes of thickness 
ranging from 50-500 nm) is a factor of resiliency, i.e. the microspheres 
can readily accept pressure variations without breaking. The preferred 
range of pore sizes is about 50-2000 nm. 
The conditions for achieving these results are met by using the method 
disclosed herein. 
One factor which enables to control the permeability of the microballoons 
membrane is the rate of evaporation of the hydrophobic phase relative to 
that of water in step (4) of the method, e.g. under conditions of freeze 
drying. For instance if the evaporation in is carried out between about 
-40.degree. and 0.degree. C., and hexane is used as the hydrophobic phase, 
polystyrene being the interfacially deposited polymer, beads with 
relatively large pores are obtained; this is so because the vapour 
pressure of the hydrocarbon in the chosen temperature range is 
significantly greater than that of water, which means that the pressure 
difference between the inside and outside of the spheres will tend to 
increase the size of the pores in the spheres membrane through which the 
inside material will be evaporated. In contrast, using cyclooctane as the 
hydrophobic phase (at -17.degree. C. the vapour pressure is the same as 
that of water) will provide beads with very tiny pores because the 
difference of pressures between the inside and outside of the spheres 
during evaporation is minimized. 
Depending on degree of porosity the microballoons of this invention can be 
made stable in an aqueous carrier from several hours to several months and 
give reproducible echographic signals for a long period of time. Actually, 
depending on the polymer selected, the membrane of the microballoons can 
be made substantially impervious when suspended in carrier liquids of 
appropriate osmotic properties, i.e. containing solutes in appropriate 
concentrations. It should be noted that the existence of micropores in the 
envelope of the microballoons of the present invention appears to be also 
related with the echographic response, i.e., all other factors being 
constant, microporous vesicles provide more efficient echographic signal 
than corresponding non-porous vesicles. The reason is not known but it can 
be postulated that when a gas is in resonance in a closed structure, the 
damping properties of the latter may be different if it is porous or 
non-porous. 
Other non water soluble organic solvents which have a vapour pressure of 
the same order of magnitude between about -40.degree. C. and 0.degree. C. 
are convenient as hydrophobic solvents in this invention. These include 
hydrocarbons such as for instance n-octane, cyclooctane, the 
dimethylcyclohexanes, ethyl-cyclohexane, 2-, 3- and 4-methyl-heptane, 
3-ethyl-hexane, toluene, xylene, 2-methyl-2-heptane, 
2,2,3,3-tetramethylbutane and the like. Esters such as propyl and 
isopropyl butyrate and isobutyrate, butyl-formate and the like, are also 
convenient in this range. Another advantage of freeze drying is to operate 
under reduced pressure of a gas instead of air, whereby gas filled 
microballoons will result. Physiologically acceptable gases such as 
CO.sub.2, N.sub.2 O, methane, Freon, helium and other rare gases are 
possible. Gases with radioactive tracer activity can be contemplated. 
As the volatile solvent insoluble in water to be used for dissolving the 
polymer to be precipitated interfacially, one can cite halo-compounds such 
as CCl.sub.4, CH.sub.3 Br, CH.sub.2 Cl.sub.2, chloroform, Freon, low 
boiling esters such as methyl, ethyl and propyl acetate as well as lower 
ethers and ketones of low water solubility. When solvents not totally 
insoluble in water are used, e.g. diethyl-ether, it is advantageous to 
use, as the aqueous phase, a water solution saturated with said solvent 
beforehand. 
The aqueous phase in which the hydrophobic phase Is emulsified as an 
oil-in-water emulsion preferably contains 1-20% by weight of water-soluble 
hydrophilic compounds like sugars and polymers as stabilizers, e.g. 
polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene glycol 
(PEG), gelatin, polyglutamic acid, albumin, and polysaccharides such as 
starch, dextran, agar, xanthan and the like. Similar aqueous phases can be 
used as the carrier liquid in which the microballoons are suspended before 
use. 
Part of this water-soluble polymer can remain in the envelope of the 
microballoons or it can be removed by washing the beads before subjecting 
them to final evaporation of the encapsulated hydrophobic core phase. 
The emulsifiers to be used (0.1-5% by weight) to provide the oil-in-water 
emulsion of the hydrophobic phase in the aqueous phase include most 
physiologically acceptable emulsifiers, for instance egg lecithin or soya 
bean lecithin, or synthetic lecithins such as saturated synthetic 
lecithins, for example, dimyristoyl phosphatidyl choline, dipalmitoyl 
phosphatidyl choline or distearoyl phosphatidyl choline or unsaturated 
synthetic lecithins, such as dioleyl phosphatidyl choline or dilinoleyl 
phosphatidyl choline. Emulsifiers also include surfactants such as free 
fatty acids, esters of fatty acids with polyoxyalkylene compounds like 
polyoxypropylene glycol and polyoxyethylene glycol; ethers of fatty 
alcohols with polyoxyalkylene glycols; esters of fatty acids with 
polyoxyalkylated sorbitan; soaps: glycerol-polyalkylene stearate; 
glycerol-polyoxyethylene ricinoleate; homo- and copolymers of polyalkylene 
glycols; polyethoxylated soya-oil and castor oil as well as hydrogenated 
derivatives; ethers and esters of sucrose or other carbohydrates with 
fatty acids, fatty alcohols, these being optionally polyoxyalkylated; 
mono-, di- and triglycerides of saturated or unsaturated fatty acids; 
glycerides or soya-oil and sucrose. 
The polymer which constitutes the envelope or bounding membrane of the 
injectable microballoons can be selected from most hydrophilic, 
biodegradable physiologically compatible polymers. Among such polymers one 
can cite polysaccharides of low water solubility, polylactides and 
polyglycolides and their copolymers, copolymers of lactides and lactones 
such as .epsilon.-caprolactone, .delta.-valerolactone, polypeptides, and 
proteins such as gelatin, collagen, globulins and albumins. The great 
versatility in the selection of Synthetic polymers is another advantage of 
the present invention since, as with allergic patients, one may wish to 
avoid using microballoons made of natural proteins (albumin, gelatin) like 
in U.S. Pat. No. 4,276,885 or EP-A-324.938. Other suitable polymers 
include poly-(ortho)esters (see for instance U.S. Pat. No. 4,093,709; U.S. 
Pat. No. 4,131,648; U.S. Pat. No. 4,138,344; U.S. Pat. No. 4,180,646); 
polylactic and polyglycolic acid and their copolymers, for instance DEXON 
(see J. Heller, Biomaterials 1 (1980), 51; 
poly(DL-lactide-co-.delta.-caprolactone), 
poly(DL-lactide-co-.delta.-valerolactone), 
poly(DL-lactide-co-.delta.butyrolactone), polyalkylcyanoacrylates; 
polyamides, polyhydroxybutyrate; polydioxanone; poly-.beta.-aminoketones 
(Polymer 23 (1982), 1693); polyphosphazenes (Science 193 (1976), 1214); 
and polyanhydrides. References on biodegradable polymers can be found in 
R. Langer et al., Macromol. Chem. Phys. C23 (1983), 61-126. 
Polyamino-acids such as polyglutamic and polyaspartic acids can also be 
used as well as their derivatives, i.e. partial esters with lower alcohols 
or glycols. One useful example of such polymers is 
poly-(t.butyl-glutamate). Copolymers with other amino-acids such as 
methionine, leucine, valine, proline, glycine, alamine, etc. are also 
possible. Recently some novel derivatives of polyglutamic and polyaspartic 
acid with controlled biodegradability have been reported (see W087/03891; 
U.S. Pat. No. 4,888,398 and EP-130.935 incorporated here by reference). 
These polymers (and copolymers with other amino-acids) have formulae of 
the following type: 
EQU --(NH--CHA--CO).sub.x (NH--CHX--CO).sub.y 
where X designates the side chain of an amino-acid residue and A is a group 
of formula --(CH.sub.2).sub.n COOR.sup.1 R.sup.2 --OCOR (II), with R.sup.1 
and R.sup.2 being H or lower alkyls, and R being alkyl or aryl; or R and 
R.sup.1 are connected together by a substituted or unsubstituted linking 
member to provide 5- or 6- membered rings. 
A can also represent groups of formulae: 
EQU --(CH.sub.2).sub.n COO--CHR.sup.1 COOR (I) 
and 
EQU --(CH.sub.2).sub.n CO(NH--CHX--CO).sub.m NH--CH(COOH)--(CH.sub.2).sub.p 
COOH(III) 
and corresponding anhydrides. In all these formulae n, m and p are lower 
integers (not exceeding 5) and x and y are also integers selected for 
having molecular weights not below 5000. 
The aforementioned polymers are suitable for making the microballoons 
according to the invention and, depending on the nature of substituents R, 
R.sup.1, R.sup.2 and X, the properties of the membrane can be controlled, 
for instance, strength, elasticity and biodegradability. For instance X 
can be methyl (alanine), isopropyl (valine), isobutyl (leucine and 
isoleucine), benzyl (phenylalanine). 
Additives can he incorporated into the polymer wall of the microballoons to 
modify the physical properties such as dispersibility, elasticity and 
water permeability. For incorporation in the polymer, the additives can be 
dissolved in the polymer carrying phase, e.g. the hydrophobic phase to be 
emulsified in the water phase, whereby they will co-precipitate with the 
polymer during inter-facial membrane formation. 
Among the useful additives, one may cite compounds which can "hydrophobize" 
the microballoons membrane in order to decrease water permeability, such 
as fats, waxes and high molecular-weight hydrocarbons. Additives which 
improve dispersibility of the microballoons in the injectable 
liquid-carrier are amphipatic compounds like the phospholipids; they also 
increase water permeability and rate of biodegradability. 
Non-biodegradable polymers for making microballoons to be used in the 
digestive tract can be selected from most water-insoluble, physiologically 
acceptable, bioresistant polymers including polyolefins (polystyrene), 
acrylic resins (polyacrylates, polyacrylonitrile), polyesters 
(polycarbonate), polyurethanes, polyurea and their copolymers. ABS 
(acryl-butadienestyrene) is a preferred copolymer. 
Additives which increase membrane elasticity are the plasticisers like 
isopropyl myristate and the like. Also, very useful additives are 
constituted by polymers akin to that of the membrane itself but with 
relatively low molecular weight. For instance when using copolymers of 
polylactic/polyglycolic type as the membrane forming material, the 
properties of the membrane can be modified advantageously (enhanced 
softness and biodegradability) by incorporating, as additives, low 
molecular weight (1000 to 15,000 Dalton) polyglycolides or polylactides. 
Also polyethylene glycol of moderate to low M.sub.w (e.g. PEG 2000) is a 
useful softening additive. 
Preferably the plasticizers include isopropyl myristate, glyceryl 
monostearate and the like to control flexibility, the amphipatic 
substances include surfactants and phospholipids like the lecithins to 
control permeability by increasing porosity while the hydrophobic 
compounds include high molecular weight hydrocarbon like the 
paraffin-waxes to reduce porosity. 
The quantity of additives to be incorporated in the polymer forming the 
inter-facially deposited membrane of the present microballoons is 
extremely variable and depends on the needs. In some cases no additive is 
used at all; in other cases amounts of additives which may reach about 20% 
by weight of the polymer are possible. 
The injectable microballoons of the present invention can be stored dry in 
the presence or in the absence of additives to improve conservation and 
prevent coalescence. As additives, one may select from 0.1 to 25% by 
weight of water-soluble physiologically acceptable compounds such as 
mannitol, galactose, lactose or sucrose or hydrophilic polymers like 
dextran, xanthan, agar, starch, PVP, polyglutamic acid, polyvinylalcohol 
(PVA), albumin and gelatin. The useful life-time of the microballoons in 
the injectable liquid carrier phase, i.e. the period during which useful 
echographic signals are observed, can be controlled to last from a few 
minutes to several months depending on the needs; this can be done by 
controlling the porosity of the membrane from substantial imperviousness 
toward carrier liquids to porosities having pores of a few nanometers to 
several hundreds of nanometers. This degree of porosity can be controlled, 
in addition to properly selecting the membrane forming polymer and polymer 
additives, by adjusting the evaporation rate and temperature in step (4) 
of the method and properly selecting the nature of the compound (or 
mixture of compounds) constituting the hydrophobic phase, i.e. the greater 
the differences in its partial pressure of evaporation with that of the 
water phase, the coatset the pores in the microballoons membrane will be. 
Of course, this control by selection of the hydrophobic phase can be 
further refined by the choice of stabilizers and by adjusting the 
concentration thereof in order to control the rate of water evaporation 
during the forming of the microballoons. All these changes can easily be 
made by skilled persons without exercizing inventiveness and need not be 
further discussed. 
It should be remarked that although the microballoons of this invention can 
be marketed in the dry state, more particularly when they are designed 
with a limited life time after injection, it may be desirable to also sell 
ready preparations, i.e. suspensions of microballoons in an aqueous liquid 
carrier ready for injection or oral administration. This requires that the 
membrane of the microballoons be substantially impervious (at least for 
several months or more) to the carrier liquid. It has been shown in this 
description that such conditions can be easily achieved with the present 
method by properly selecting the nature of the polymer and the interfacial 
deposition parameters. Actually parameters have been found (for instance 
using the polyglutamic polymer (where A is the group of formula II) and 
cyclooctane as the hydrophobic phase) such that the porosity of the 
membrane after evaporation of the hydrophobic phase is so tenuous that the 
microballoons are substantially impervious to the aqueous carrier liquid 
in which they are suspended. 
A preferred administrable preparation for diagnostic purposes comprises a 
suspension in buffered or unbuffered saline (0.9% aqueous NaCl; buffer 10 
mM tris-HCl) containing 10.sup.8 -10.sup.10 vesicles/mi. This can be 
prepared mainly according to the directions of the Examples below, 
preferably Examples 3 and 4, using poly-(DL-lactide) polymers from the 
Company Boehringer, Ingelheim, Germany. 
The following Examples illustrate the invention practically. 
EXAMPLE 1 
One gram of polystyrene was dissolved in 19 g of liquid naphthalene at 
100.degree. C. This naphthalene solution was emulsified at 
90.degree.-95.degree. C. into 200 ml of a water solution of polyvinyl 
alcohol (PVA) (4% by weight) containing 0.1% of Tween-40 emulsifier. The 
emulsifying head was a Polytron PT-3000 at about 10,000 rpm. Then the 
emulsion was diluted under agitation with 500 ml of the same aqueous phase 
at 15.degree. C. whereby the naphthalene droplets solidified into beads of 
less than 50 .mu.m as ascertained by passing through a 50 .mu.m mesh 
screen. The suspension was centrifugated under 1000 g and the beads washed 
with water and recentrifugated. This step was repeated twice. 
The beads were resuspended in 100 ml of water with 0.8 g of dissolved 
lactose and the suspension was frozen into a block at -30.degree. C. The 
block was thereafter evaporated under 0.5-2 Torr between about -20.degree. 
and -10.degree. C. Air filled microballoons of average size 5-10 .mu.m and 
controlled porosity were thus obtained which gave an echographic signal at 
2.25 and 7.5 MHz after being dispersed in water (3% dispersion by weight). 
The stability of the microballoons in the dry state was effective for an 
indefinite period of time; once suspended in an aqueous carrier liquid the 
useful life-time for echography was about 30 min or more. Polystyrene 
being non-biodegradable, this material was not favored for injection 
echography but was useful for digestive tract investigations. This Example 
clearly establishes the feasibility of the method of the invention. 
EXAMPLE 2 
A 50:50 copolymer mixture (0.3 g) of DL-lactide and glycollde (Du Pont 
Medisorb) and 16 mg of egg-lecithin were dissolved in 7.5 ml of CHCl.sub.3 
to give solution (1). 
A solution (2) containing 20 mg of paraffin-wax (M.P. 54.degree.-56.degree. 
C.) in 10 ml of cyclooctane (M.P. 10-13.degree.) was prepared and 
emulsified in 150 ml of a water solution (0.13% by weight) of 0.13% by 
weight) of Pluronic F-108 (a block copolymer of ethylene oxide and 
propylene oxide) containing also 1.2 g of CHCl.sub.3. Emulsification was 
carried out at room temperature for 1 min with a Polytron head at 7000 
rpm. Then solution (1) was added under agitation (7000 rpm) and, after 
about 30-60 sec, the emulsifier head was replaced by a helical agitator 
(500 rpm) and stirring was continued for about 3 hrs at room temperature 
(22.degree. C.). The suspension was passed through a 50 .mu.m screen and 
frozen to a block which was subsequently evaporated between -20.degree. 
and 0.degree. C. under high-vacuum (catching trap -60.degree. to 
-80.degree. C.). There were thus obtained 0.264 g (88%) of air-filled 
microballoons stable in the dry state. 
Suspensions of said microballoons in water (no stabilizers) gave a strong 
echographic signal for at least one hour. After injection in the organism, 
they Diodegraded in a few days. 
EXAMPLE 3 
A solution was made using 200 ml of tetrahydrofuran (THF), 0.8 g of a 50:50 
DL-lactide/glycolide copolymer (Boehringer AG), 80 mg of egg-lecithin, 64 
mg of paraffin-wax and 4 ml of octane. This solution was emulsified by 
adding slowly into 400 ml of a 0.1% aqueous solution of Pluronic F-108 
under helical agitation (500 r.p.m.). After stirring for 15 min, the milky 
dispersion was evaporated under 10-12 Torr 25.degree. C. in a rotavapor 
until its volume was reduced to about 400 ml. The dispersion was sieved on 
a 50 .mu.m grating, then it was frozen to -40.degree. C. and freeze-dried 
under about 1 Torr. The residue, 1.32 g of very fine powder, was taken 
with 40 ml of distilled water which provided, after 3 min of manual 
agitation, a very homogeneous dispersion of microballoons of average size 
4.5 .mu.m as measured using a particle analyzer (Mastersizer from 
Malvern). The concentration of microballoons (Coulter Counter) was about 
2.times.10.sup.9 /ml. This suspension gave strong echographic signals 
which persisted for about 1 hr. 
If in the present example, the additives to the membrane polymer are 
omitted, i.e. there is used only 800 mg of the lactide/glycolide copolymer 
in the THF/octane solution, a dramatic decrease in cell-wall permeability 
is observed, the echographic signal of the dispersion in the aqueous 
carrier not being significantly attenuated after 3 days. 
Using intermediate quantities of additives provided beads with controlled 
intermediate porosity and life-time. 
EXAMPLE 4 
There was used in this Example a polymer of formula defined in claim 8 in 
which the side group has formula (II) where R.sup.1 and R.sup.2 are 
hydrogen and R is tert.butyl. The preparation of this polymer (defined as 
poly-POMEG) is described in U.S. Pat. No. 4,888,398. 
The procedure was like in Example 3, using 0.1 g poly-POMEG, 70 ml of THF, 
1 ml of cyclooctane and 100 ml of a 0.1% aqueous solution of Pluronic 
P-108. No lecithin or high-molecular weight hydrocarbon was added. The 
milky emulsion was evaporated at 27.degree. C./10 Torr until the residue 
was about 100 ml, then it was screened on a 50 .mu.m mesh and frozen. 
Evaporation of the frozen block was carried out (0.5-1 Torr) until dry. 
The yield was 0.18 g because of the presence of the surfactant. This was 
dispersed in 10 ml of distilled water and counted with a Coulter Counter. 
The measured concentration was found to be 1,43.times.10.sup.9 
microcapsules/ml, average size 5.21 .mu.m as determined with a particle 
analyzer (Mastersizer from Malvern). The dispersion was diluted 100 x, 
i.e. to give about 1.5.times.10.sup.7 microspheres/ml and measured for 
echogenicity. The amplitude of the echo signal was 5 times greater at 7.5 
MHz than at 2.25 MHz. These signals were reproducible for a long period of 
time. 
Echogenicity measurements were performed with a pulse-echo system 
consisting of a plexiglas specimen holder (diameter 30 mm) with a 20 .mu.m 
thick Mylar acoustic window, a transducer holder immersed In a constant 
temperature water bath, a pulser-receiver (Accutron M3010JS) with an 
external pre-amplifier with a fixed gain of 40 dB and an internal 
amplifier with gain adjustable from -40 to +40 dB and interchangeable 13 
mm unfocused transducers. A 10 MHz low-pass filter was inserted in the 
receiving part to improve the signal to noise ratio. The A/D board in the 
IBM PC was a Sonotek STH 832. Measurements were carried out at 2.25, 3.5, 
5 and 7.5 MHz. 
If in the present Example, the polymer used is replaced by lactic-lactone 
copolymers, the lactones being .delta.-butyrolactone, 
.delta.-valerolactone or e-caprolactone (see Fukuzaki et al., J. 
Biomedical Mater. Res. 25 (1991), 315-328), similar favorable results were 
obtained. Also in a similar context, polyalkylcyano-acrylates and 
particularly a 90:10 copolymer poly(DL-lactide-co-glycolide) gave 
satisfactory results. Finally, a preferred polymer is a poly(DL-lactide) 
from the Company Boehringer-Ingelheim sold under the name "Resomer R-206" 
or Resomer R-207. 
EXAMPLE 5 
Two-dimensional echocardiography was performed using an Acuson-128 
apparatus with the preparation of Example 4 (1.43.times.10.sup.9 /ml) in 
an experimental dog following peripheral vein injection of 0.1-2 ml of the 
dispersion. After normally expected contrast enhancement imaging of the 
right heart, intense and persistent signal enhancement of the left heart 
with clear outlining of the endocardium was observed, thereby confirming 
that the microballoons made with poly-POMEG (or at least a significant 
part of them) were able to cross the pulmonary capillary circulation and 
to remain in the blood-stream for a time sufficient to perform efficient 
echographic analysis. 
In another series of experiments, persistent enhancement of the Doppler 
signal from systemic arteries and the portal vein was observed in the 
rabbit and in the rat following peripheral vein injection of 0.5-2 ml of a 
preparation of microballoons prepared as disclosed in Example 4 but using 
poly(DL-lactic acid) as the polymer phase. The composition used contained 
1.9.times.10.sup.8 vesicles/ml. 
Another composition prepared also according to the directions of Example 4 
was achieved using poly(tert.butylglutamate). This composition (0.5 ml) at 
dilution of 3.4.times.10.sup.8 microballoons/ml was injected in the portal 
vein of rats and gave persistent contrast enhancement of the liver 
parenchyma. 
EXAMPLE 6 
A microballoon suspension (1.1.times.10.sup.9 vesicles/ml) was prepared as 
disclosed in Example i (resin=polystyrene). One ml of this suspension was 
diluted with 100 ml of 300 mM mannitol solution and 7 ml of the resulting 
dilution was administered intragastrically to a laboratory rat. The animal 
was examined with an Acuson-128 apparatus for 2-dimensional echography 
imaging of the digestive tract which clearly showed the single loops of 
the small Intestine and of the colon.