Method to increase the stability of nanocapsules during storage thereof

Methods of increasing storage stability of lipidic nucleus nanocapsules comprising adding a monosaccharide cryoprotective agent into the aqueous dispersion of the nanocapsules while maintaining gentle stirring until the same dissolves totally. Afterwards, freezing said dispersion thus obtained at a temperature of not less than -40.degree. C. for about 2 to 4 hours, and the eliminating the water by heating progressively to a temperature of about 35.degree. C. at reduced pressure, whereby a stable lyophilized product is obtained. The obtained product can be conveniently reconstituted by adding water or a more complex aqueous solution thereto. The nanocapsules have use in medicine, pharmacy, cosmetics, chemical industry, agriculture, and veterinary science.

TECHNICAL FIELD OF THE INVENTION 
The present invention is in the technical field of lyophilization and, 
specifically, lyophilization of nano-capsules, submicron spheres formed by 
a lipidic nucleus surrounded by a polymeric membrane or a water insoluble 
substance. Lyophilization of nanocapsules makes it possible to increase 
their stability during storage thereof, in such a way that 
industrialization and subsequent marketing thereof for use in medicine, 
pharmacy, cosmetics, chemical industry, agriculture, veterinary science, 
etc. become possible. 
PRIOR ART OF THE INVENTION 
Colloidal systems of a polymeric nature in the form of nanocapsules and 
nanoparticles have been the object of numerous studies over the last few 
years. This is due to the fact that the use of this type of system for 
vehiculization of biologically active substances has brought about great 
expectations as a medium to reduce the doses of very toxic drugs and even 
to be directed towards a hypothetical target organ, among many other 
potential uses. 
However, the use of biodegradable polymers, which would permit their 
optional use in medicine, veterinary science, etc., poses the problem of 
their degradation in an aqueous medium which would make storage thereof 
impossible in the form of a colloidal suspension for the time needed to 
market them. On the other hand, given the collidal nature of these 
systems, there is a tendency to the instability of the same which gives 
rise to an aggregation of initially individualized nanocapsules, and even 
a loss of the encapsulated substance by diffusion through the polymeric 
membrane during storage thereof. 
Nanovesicular systems in the form of nanocapsules of a size smaller than on 
micrometer, formed by a solid or liquid lipidic nucleus containing one or 
more active substances, and surrounded by a membrane formed by a polymer 
or a water insoluble substance, have been described in different studies 
and inventions. However, suitable methods to increase the physical and 
physicochemical stability of the colloidal suspensions obtained have not 
been described, nor methods to prevent the degradation or dissolving of 
the polymers forming the wall, which would give rise to instabilization of 
the nanocapsules, causing them to break or the substance contained inside 
them from escaping. 
It is obvious, that upon not having the stability of these systems solved 
for long periods of storage time is a very important limitation for their 
potential industrial use and marketing. 
The patents BE-A 869107, FR-A 2504408 and FR-A 2515960 describe the 
preparation and use of biodegradable nanoparticles and nanocapsules 
obtained from the polymerization of alkyl cyanoacrylates and containing a 
biologically active substance. 
European patent EP 480729A1 describes the microencapsulation of drops of 
oil of a size between 1 and 5 micra to orally administer a lyophilized 
product in the form of powder. Microencapsulation is described as a system 
to avoid degradation of unstable drugs in the conditions of the stomach. 
The product object of this patent is used in the form of paste or powder. 
This differentiates it from the present invention which refers to a 
colloidal suspension smaller than 1 micron which is lyophilized and which 
can be rehydrated in order to be reconstituted as individualized 
nanocapsules, without producing aggregation of the same or an increase in 
size. 
U.S. Pat. No. 4,247,411 describes lyophilization of liposomes to increase 
their stability, avoiding oxidation and contamination of the product. 
Conceptually and physically, liposomes are radically different from 
nanocapsules. Liposomes are vesicles formed by one or several bi-layers of 
phospholipids that surround an aqueous nucleus. Nanocapsules are formed by 
a polymeric wall that surrounds a lipidic type nucleus, usually an oil. 
Therefore, the cited patent refers to different products from those that 
are the object of the present invention. 
The above cited patent uses filler substances such as: inorganic salts, 
colloidal silica, starch or aluminosilicates to avoid aggregation of the 
liposomes. Besides, as it is inferred from all the described examples, the 
lyophilization is carried out by freezing with liquid nitrogen, which 
makes industrialization thereof difficult to a large degree. 
Unlike said patent, a monosaccharide is used in the method of the present 
invention as a cryorotective agent and the freezing is done to a 
temperature no lower than -40.degree. C., which can be easily reached by 
any lyophilizer existing on the market. Therefore, this process does not 
need any special adaptation of the processes usually used on an industrial 
level. 
French patent 8618444 describes the preparation and use of nanocapsules 
formed from preformed polymers and with different lipidic substances as 
the nucleus. Elimination of the solvents is done by lyophilization. In the 
single example in which reference is made to lyophilization thereof, use 
of trealose 20% is described, obtaining a product with a very high 
osmolality with regard to biological liquids such as blood, tears, etc. 
It is very important to point out that in the cited patent, lyophilization 
is a process used exclusively to eliminate the solvents used during 
preparation and the purpose of this process is not to improve the 
stability of the systems obtained. 
Subsequently, in the study published by the same authors "Lyophilization de 
vecteurs colloidaux submicroniques," STP Pharma 5(11) 738-744, 1989, M. 
Auvillain, G. Cave, H. Fessi et J. P. Devissaguet, lyophilization of 
nanoparticles and of nanocapsules using different cryoprotective agents 
and various lyophilization conditions is studied. In this study, which 
explicitly refers to the above cited patent, the authors reach the 
conclusion that due to the fragility of the wall of the nanocapsules and 
to the composition thereof, use of approximately trealose 30% is necessary 
and besides, freezing down to temperatures between -70.degree. C. and 
-196.degree. C. using cooling mixtures or liquid nitrogen if one wishes to 
obtain a product with a correct reconstitution, has to be carried out. 
Therefore, the need to reach very low freezing temperatures makes the 
industrial use of the lyophilization process difficult and costly. 
Use of trealose 30% gives rise to a product with an osmolality much higher 
than mOsm/Kg., which limits its use when an isotonic product is needed 
with regard to some biological liquids. Likewise, trealose is a very 
expensive product and its use at high concentrations significantly 
increases the cost of the final product. 
DESCRIPTION OF THE INVENTION 
The present invention proposed a method to increase the stability of 
nanocapsules being smaller that 0.5 micron of a polymeric nature by means 
of lyophilization. The lyophilization process that is proposed overcomes 
the disadvantages of prior ones, and it is useful to preserve nanocapsules 
made from polymers and oils, synthetic as well as natural ones. 
Due to the structure of nanocapsules, formed by a lipidic nucleus, normally 
liquid, and by a fragile polymeric wall with a thickness of a few 
nanometers, it was to be expected that the use of high concentrations of a 
cryoprotective agent was necessary and in combination with a freezing 
temperature much lower than -40.degree. C. to ensure total freezing of the 
system and to prevent the formation of large crystals that would affect 
the integrity of the nanocapsules. 
However, it has been found, in accordance with the present invention, that 
when nanocapsules are lyophilized in the presence of a certain amount of a 
cryoprotective agent, especially a monosaccharide, such as glucose, by 
means of lyophilization it is possible to eliminate water from the 
suspension of nanocapsules and subsequently rehydrate the same without 
modifying the size of the same, without aggregates appearing or even, 
without a loss of the encapsulated substance being produced. 
The freezing temperature required for the correct lyophilization of 
nanocapsules is approximately -40.degree. C. and it is not necessary to 
use a cooling mixture or liquid nitrogen to reach lower temperatures. In 
this way, lyophilization of nanocapsules on an industrial level, and 
therefore, practical use thereof, is made easier to a large degree. 
Use of a monosaccharide such as glucose at low concentrations makes it 
possible not to increase the cost of the product as in case when trealose 
is used and at the same time it makes it possible to obtain a product that 
upon being rehydrated can be hypotonic, isotonic or hypertonic in relation 
to biological liquids such as blood, tears, etc. This is a considerable 
advantage given that it is possible to modulate the final osmolality of 
the rehydrated product in terms of the use or way in which one wishes to 
administer the medicine. 
Therefore, the present invention refers to a method to make it possible to 
increase the stability of nanocapsules during storage thereof in an 
economical easily industrializable way that makes it possible to obtain a 
rehydrated product maintaining its initial characteristics. 
According to the present invention in the method chosen to carry out the 
process, the cryoprotective agent is added to the composition once the 
nanocapsules have been formed and it is kept gently stirred, 250 r.p.m. 
until it totally dissolves. This cryoprotective agent is added to prevent 
the nanocapsules from breaking, from their being crushed or aggregated, 
which would give rise to a heterogeneous product after lyophilization 
thereof; the cryoprotective agent is advantageously a monosaccharide such 
as glucose in concentrations from 3% (w/v) up to 10% (w/v.) 
The product is dosed in vials or else placed on a tray and it is inserted 
in the lyophilizer where freezing is proceeded with for about 2 to 4 
hours. Subsequently, water is eliminated by heating progressively up to 
+35.degree. C. with a vacuum in the neighborhood of 0.2 mbars. The product 
that is obtained is a lyophilized material tablet formed by a fine powder 
of lyophilized nanocapsules. The lyophilized material tablet is 
reconstituted by adding a specific volume of water, buffer solution, 
electrolyte solution, viscosity modifying solution, etc. or any 
combination of the same, obtaining a colloidal suspension of 
characteristics practically identical to the initial ones. 
The present invention also provides the composition of lyophilized 
nanocapsules obtained in the lyophilization process. 
The copolymers that form the wall of the nanocapsules are synthetic or 
natural. In the case of synthetic polymers, for example, they may be 
poly(d,l)lactic acid, a semisynthetic polymer such as, for example, 
ethylcellulose, cellulose acetophthalate, etc.; acrylic acid copolymers 
and acrylic acid polymers (for example: Eudragit.sup.R); lactic acid and 
glycolic acid copolymers; glycolide derivatives (propiolactone, 
butyrolactone, pivalolactone, epsiloncaprolactone derivatives, etc.); 
maleic acid and benzyl maleate copolymers, polysaccharides, etc. In the 
case of natural ones, for example, gelatin, arabic gum, etc. 
The substance forming the lipidic nucleus can be for example a hydrogenated 
oil, a natural oil, natural oil derivatives such as coconut oil, castor 
oil, etc., a synthetic oil, ethoxylated oleic glycerides, diethylene 
glycol monoethyl ether, C.sub.8 -C.sub.10 ethoxylated glycerides, 
phospholipids, petroleum derivatives, etc. 
The substance contained in the nucleus of the nanocapsules can be a 
biologically active substance such as a medicinal active principle, an 
active principle precursor, a contrast substance, a pigment, a dye, an 
adhesive, a lubricant, etc. The substance contained in the nucleus can be 
dissolved or dispersed in the same. 
In the composition that is subjected to the lyophilization process, the 
continuous phase that surrounds the nanocapsules is an aqueous phase that 
contains a natural surface active agent such as lecithins, an anionic 
synthetic surface active agent such as sodium or cationic lauryl sulfate, 
for example a quaternary or non-ionic ammonium such as for example 
ethoxylated sorbitan esters, fatty alcohol esters and polyoxyethylene 
glycol esters, polyoxyethylene polyoxypropylene glycols or else a 
suspension agent such as dextrane, polyvinyl alcohol, etc. The ratio 
between the weight of the nanocapsules and the weight of the aqueous 
continuous phase of the dispersion is generally 0.01 to 0.5 
The final lyophilized product can be rehydrated, compressed, extruded or 
can form part of a more complex composition. 
EMBODIMENTS OF THE INVENTION 
The present invention is additionally illustrated by means of the following 
examples, which must not be considered restrictive of the scope of the 
same which is defined by the attached set of claims.

EXAMPLE 1 
POLYEPSILONCAPROLACTONE-MIGLYOL 840.RTM. NANOCAPSULES 
0.996 g. of Lutrol F68.sup.R are dissolved in 50 ml. of deionized water and 
filtered through 0.22 .mu.m (AQUEOUS PHASE). 0.250 g of 
polyepsiloncaprolactone are dissolved in 25 ml. of acetone using 
ultrasound for 5 minutes and 0.250 ml. of Miglyol 840.RTM. (Dynamit Nobel) 
(ORGANIC PHASE) are added. The organic phase is added to the aqueous phase 
is added to the aqueous phase with gentle stirring. Once it has been 
totally added, the recently formed colloidal suspension is placed in a 
rotavapor where the organic solvent is eliminated under vacuum and the 
suspension is concentrated to a final volume of 30 ml. The pH is adjusted 
to 7 with NaOH 0.01N. 
Glucose is added up to a concentration of 2, 3, 4 or 5%; it is dosed in 
glass vials, it is frozen down to -40.degree. C. and the water is 
eliminated by increasing the temperature up to about 35.degree. C. and 
with reduced pressure of 0.2 to 0.4 mbars for 12-14 hours. The final 
product is a white and compact tablet of lyophilized product. The 
lyophilized product is rehydrated with deionized water obtaining a 
colloidal suspension with the same characteristics as before 
lyophilization thereof. 
The average particle size and the polydispersity are measured by photonic 
correlation spectroscopy and potential Z by electrophoretic mobility 
(Zetasizer 3, Malvern Instruments.) The determinations are made before 
lyophilizing and after rehydrating the lyophilized product. The results 
obtained are given in the following table: 
______________________________________ 
Average (nm) 
size Polydispersity 
Potential Z 
% Glu- 
Before After R Before 
After Before 
After 
cose lyophi. lyophi. Tf/Ti 
lyophi. 
lyophi. 
lyophi. 
lyophi. 
______________________________________ 
2 294.3 326.9 1.11 0.146 0.058 -16.70 
-19.38 
3 297.8 313.7 1.05 0.132 0.097 -16.41 
-15.15 
4 312.8 310.4 0.99 0.084 0.161 -16.38 
-15.55 
5 305.0 308.9 1.01 0.110 0.156 -17.15 
-13.83 
______________________________________ 
Tf/Ti = Average size after lyophilizing/Average size before lyophizing 
EXAMPLE 2 
POLYEPSILONCAPROLACTONE-EDENOR TI5.RTM. NANOCAPSULES 
The technique described in Example 1 is used, but Miglyol 840.RTM. (Dynamit 
Nobel) is replaced by Edenor TiO5.RTM. (Pulcra.) The average particle size 
and polydispersity are measured by photonic correlation spectroscopy and 
potential Z by electrophoretic mobility (Zetasizer 3, Malvern 
Instruments.) The determinations are carried out before lyophilizing and 
after rehydrating the lyophilized product. The results obtained are given 
in the following table: 
______________________________________ 
Average (nm) 
size Polydispersity 
Potential Z 
% Glu- 
Before After R Before 
After Before 
After 
cose lyophi. lyophi. Tf/Ti 
lyophi. 
lyophi. 
lyophi. 
lyophi. 
______________________________________ 
2 281.4 287.7 1.02 0.064 0.149 -23.31 
-22.65 
3 289.8 271.9 0.94 0.126 0.142 -23.34 
-21.89 
4 274.8 260.5 0.95 0.186 0.203 -22.69 
-22.04 
5 278.4 267.0 0.96 0.167 0.155 -23.20 
-21.19 
______________________________________ 
Tf/Ti = Average size after lyophilizing / Average size before lyophilizin 
 
EXAMPLE 3 
POLYEPSILONCAPROLACTONE-EDENOR TiO5.RTM. NANOCAPSULES 
The technique described in Example 1 is used, but Miglyol 840.RTM. (Dynamit 
Nobel) is replaced by Edenor TiO5.RTM. (Pulcra) and 0.750 ml. are used, 
instead of 0.250 ml. Only glucose 6% is used as the cryoprotective agent. 
The average particle size and polydispersity are measured by photonic 
correlation spectroscopy and potential Z by electrophoretic mobility 
(Zetasizer 3, Malvern Instruments.) The determinations are carried out 
before lyophilizing and after rehydrating the lyophilized product. The 
results obtained are given in the following table: 
______________________________________ 
Average (nm) 
size Polydispersity 
Potential Z 
% Glu- 
Before After R Before 
After Before 
After 
cose lyophi. lyophi. Tf/Ti 
lyophi. 
lyophi. 
lyophi. 
lyophi. 
______________________________________ 
6 282.8 270.9 0.96 0.174 0.163 -27.63 
-23.58 
______________________________________ 
Tf/Ti = Average size after lyophilizing / Average size before lyophilizin 
 
EXAMPLE 4 
POLYLACTIC-GLYCOLIC 75:25-MIGLYOL 840.RTM. NANOCAPSULES 
The technique described in Example 1 is used but polyepsiloncaprolactone 
(Sigma-Aldrich) is replaced by the polylactic-glycolic copolymer 75:25 
(Boerhinger Ingelheim.) The average particle size and polydispersity are 
measured by photonic correlation spectroscopy and potential Z by 
electrophoretic mobility (Zetasizer 3, Malvern Instruments.) The 
determinations are carried out before lyophilizing and after rehydrating 
the lyophilized product. The results obtained are given in the following 
table: 
______________________________________ 
Average (nm) 
size Polydispersity 
Potential Z 
% Glu- 
Before After R Before 
After Before 
After 
cose lyophi. lyophi. Tf/Ti 
lyophi. 
lyophi. 
lyophi. 
lyophi. 
______________________________________ 
2 295.8 305.3 1.03 0.185 0.180 -15.58 
-12.56 
3 302.9 296.7 0.98 0.170 0.171 -13.91 
-12.12 
4 288.5 289.3 1.00 0.212 0.171 -13.55 
-10.67 
5 289.5 275.6 0.95 0.196 0.086 -13.48 
-10.45 
______________________________________ 
Tf/Ti = Average size after lyophilizing / Average size before lyophilizin 
 
EXAMPLE 5 
POLYLACTIC-GLICOLIC 75:25 EDENOR TiO5.RTM. NANOCAPSULES 
The technique described in Example is used, but Miglyol 840.RTM. (Dynamit 
Nobel) is replaced by Edenor TiO5.RTM. (Pulcra.) The average particle size 
and polydispersity are measured by photonic correlation spectroscopy and 
potential Z by electrophoretic mobility (Zetasizer 3, Malvern 
Instruments.) The determinations are carried out before lyophilizing and 
after rehydrating the lyophilized product. The results obtained are given 
in the following table: 
______________________________________ 
Average (nm) 
size Polydispersity 
Potential Z 
% Glu- 
Before After R Before 
After Before 
After 
cose lyophi. lyophi. Tf/Ti 
lyophi. 
lyophi. 
lyophi. 
lyophi. 
______________________________________ 
2 249.2 278.4 1.12 0.197 0.199 -22.34 
-19.62 
3 252.5 270.2 1.07 0.127 0.149 -20.80 
-19.39 
4 249.8 247.8 0.99 0.094 0.093 -20.87 
-18.95 
5 239.2 239.0 0.99 0.168 0.122 -21.20 
-19.96 
______________________________________ 
Tf/Ti = Average size after lyophilizing / Average size before lyophilizin 
 
EXAMPLE 6 
POLYEPSILONCAPROLACTONE-INDOMETHACIN 0.1% NANOCAPSULES 
3.32 g. of Lutrol F68 are dissolved in 200 ml. of deionized water and 
filtered through 0.22.mu. (AQUEOUS PHASE) 0.415 g. of 
polyepsiloncaprolactone are dissolved in 100 ml. of acetone using 
ultrasound for 5 minutes. 0.101 g. of indomethacin are dissolved in 0.830 
ml. of Miglyol 812.sup.R and are added to the previous acetone solution 
(ORGANIC PHASE:) The organic phase is added to the aqueous phase with 
stirring. Once it has been totally added, the recently formed colloidal 
suspension is placed in a rotavapor where the organic solvent (acetone) is 
eliminated under vacuum and the product is concentrated to a final volume 
of 100 ml. The final colloidal suspension has its ph adjusted to 5.5 with 
NaOH 0.01N. The resulting concentrations are: 
______________________________________ 
Lutrol F68 .RTM. 3.32% (w/v) 
Poly-E-caprolactone 0.415% (w/v) 
Miglyol 812 .RTM. 0.83% (w/v) 
Indomethacin 0.10% (w/v) 
______________________________________ 
Glucose is added up to a concentration of 3, 4 or 5%; it is dosed in glass 
vials and it is frozen down to -40.degree. C. It is lyophilized with a 
vacuum between 0.2-0.4 mbar approximately for 12-16 hours until a final 
temperature of about 30.degree. C. is reached. The final product is a 
slightly yellow compact tablet. After reconstituting with 2 ml. of 
purified water, a colloidal suspension with the same characteristics as 
the initial one (before lyophilizing) is obtained. 
The average particle size and polydispersity are measured by photonic 
correlation spectroscopy and potential Z by electrophoretic mobility 
(Zetasizer 3, Malvern Instruments.) The determinations are carried out 
before lyophilizing and after rehydrating the lyophilized product. The 
results obtained are given in the following table: 
______________________________________ 
Average (nm) 
size Polydispersity 
Potential Z 
% Glu- 
Before After R Before 
After Before 
After 
cose lyophi. lyophi. Tf/Ti 
lyophi. 
lyophi. 
lyophi. 
lyophi. 
______________________________________ 
3 219.95 217.8 0.99 0.072 0.140 -12.71 
-10.44 
4 218.85 218.1 0.99 0.088 0.116 -14.42 
-10.12 
5 221.45 210.9 0.95 0.124 0.159 -14.20 
-10.25 
______________________________________ 
Tf/Ti = Average size after lyophilizing / Average size before lyophilizin 
 
After ultrafiltration in a centrifuge at 2500 rpm, the amount of 
indomethacinin the filtrate is determined. The amount of indomethacin in 
the total formula less the amount of indomethacin in the filtrate, 
determined by high resolution liquid chromatography corresponds to the 
amount of indomethacin included in the nanocapsules that is kept at the 
same level before and after lyophilization, just as it is shown in the 
following table: 
______________________________________ 
% ENCAPSULATION INDOMETHACIN 
% Glucose Before lyophilizing 
After lyophilizing 
______________________________________ 
3 91.05 89.30 
4 91.05 88.95 
5 91.05 89.10 
______________________________________ 
EXAMPLE 7 
1.992 g. of Lutrol F68.RTM. are dissolved in 100 ml. of deionized water and 
filtered through 0.22.mu. (AQUEOUS PHASE.) 0.498 g. of 
polyepsiloncaprolactone are dissolved in 50 ml. of acetone using 
ultrasound for 5 minutes. 0.1217 g. of carteolol base are dissolved in 
0.96 ml. of Edenor TiO5.RTM. and are added to the previous acetone 
solution (ORGANIC PHASE.) 
The organic phase is added to the aqueous phase with stirring. Once it has 
been totally added, the recently formed colloidal suspension is placed in 
a rotavapor where the organic solvent (acetone) is eliminated under vacuum 
and the product is concentrated to a final volume of 60 ml. The final 
colloidal suspension has its pH adjusted to 7 with HCl 0.01N. The 
resulting concentrations are: 
______________________________________ 
Lutrol F68 .RTM. 3.32% (w/v) 
Poly-E-caprolactone 0.83% (w/v) 
Edenor TiO.sub.5 .RTM. 
1.6% (w/v) 
Carteolol base 0.2% (w/v) 
______________________________________ 
Glucose is added to a concentration of 3, 4, 5, 6 or 7%; it is dosed in 
vials and it is frozen down to -40.degree. C. It is lyophilized with a 
vacuum between 0.2-0.4 mbar approximately for 12-16 hours until a final 
temperature of about 30.degree. C. is reached. The final product is a 
white compact tablet in all cases. After reconstituting with 2 ml. of 
purified water a colloidal suspension with the same characteristics as the 
initial one (before lyophilizing) is obtained. 
The average size and polydispersity of the nanocapsules obtained are 
measured by photonic correlation spectroscopy and potential Z by 
electrophoretic mobility (Zetasizer 3) and, just like the other 
physicochemical controls, they are determined before lyophilizing and once 
the product has been reconstituted. 
______________________________________ 
Average (nm) 
size Polydispersity 
Potential Z 
% Glu- 
Before After R Before 
After Before 
After 
cose lyophi. lyophi. Tf/Ti 
lyophi. 
lyophi. 
lyophi. 
lyophi. 
______________________________________ 
3 245.7 262.7 1.07 0.264 0.182 -26.91 
-21.64 
4 259.9 242.9 0.93 0.135 0.187 -21.505 
-21.87 
5 256.9 246.7 0.96 0.162 0.146 -21.905 
-19.91 
6 259.9 241.4 0.93 0.107 0.177 -19.33 
-19.96 
7 258.8 241.9 0.93 0.177 0.150 -20.11 
-20.01 
______________________________________ 
Tf/Ti = Average size after lyophilizing / Average size before lyophilizin 
 
After ultrafiltration in a centrafuge at 2500 rpm, the amount of carteolol 
base in the filtrate is determined. The amount of carteolol in the total 
formula less the amount of carteolol in the filtrate, determined by high 
resolution liquid chromatography, corresponds to the amount of carteolol 
base included in the nanocapsules which is kept at the same level before 
and after lyophilization, just as it is shown in the following table: 
______________________________________ 
% ENCAPSULATION CARTEOLOL BASE 
% Glucose Before lyophilizing 
After lyophilizing 
______________________________________ 
3 82 83 
4 82 82.5 
5 82 83 
6 82 83 
7 82 82.5 
______________________________________