Note: Descriptions are shown in the official language in which they were submitted.
'; CA 02380490 2002-O1-29
PCT/DE00/02493
Guggenbichler, J. Peter et al.
Our Ref.: E 2185 PCT
Process for preparing antimicrobial plastic bodies having improved long-time
performance
The invention relates to processes for preparing antimicrobial metal-
containing plastic
bodies, in particular articles for medical requirements. These articles are in
particular used in
the form of catheters.
A considerable disadvantage of plastic articles for medical requirements, in
particular of
catheters for long-term and short-term use, is that the plastics used are
easily infected by
germs which are often multi-resistant and form a biofilm on the surface of the
plastic body or
on the outer and interior surface of the catheter. Prophylactic impregnation
of the surfaces by
means of antibiotics has to be ruled out due to the high selection of
resistant microorganisms
involved.
Thus, in the past years numerous attempts have been made to impregnate the
plastic
surfaces with silver ions originating from, e.g., silver nitrate, acetate or
chloride. Among all
heavy metal ions, silver ions have a very broad antimicrobial spectrum and
high toxicity
towards microorganisms in that they, e.g., bind to the cell wall via SH
groups, block the
respiratory chain, stop cell proliferation via DNA binding, but have low
toxicity towards
animal cells. In this context, however, sufficient microbial activity could
not be observed in
various clinical studies. Moreover, the etching effect and the poor water
solubility,
respectively, of silver salts cause further problems in use.
When metal surfaces such as silver are contacted with physiological NaCI
solution,
metal ions (silver ions) will be released depending on the size of the metal
surface. Admixing
a polymer such as polyurethane with a metal powder such as silver powder,
however, will not
be successful since due to the small surface area relatively high
concentrations of metal
powder will be necessary, which causes mechanical problems in the plastic
material. The
critical surface area required for antimicrobial activity, thus, cannot be
obtained by admixing
metal powder.
EP-A-0 711 113 discloses a new technology in which metallic silver is vapor
deposited
on polyurethane films which are compounded in comminuted form. This made it
possible to
achieve uniform distribution of silver particles in polymer material and,
thus, obtain a surface
area sufficiently large for bacteriostatic activity. The antimicrobial
activity of said plastic
bodies has been very well established as regards reduction and prevention of
adherence,
,' CA 02380490 2002-O1-29
2
biofilm formation and long-time performance as well as toxicity and
compatibility. The
applicability of the aforesaid plastic bodies is, however, limited due to the
time-consuming
and costly preparation process, in particular caused by the vapor deposition
of silver.
US-A-5,180,585 furthermore describes an antimicrobial composition comprising
inorganic particles having a first microbicidal layer and a second layer which
protects the
underlying first layer. The preparation process is relatively complex.
Thus, the object underlying the present invention is to provide a process for
preparing
antimicrobially active plastic bodies which do not exhibit the aforesaid
disadvantages, i.e. can
easily be prepared and provide sufficient concentration of silver ions at the
surface.
This problem is solved by means of a process which is characterized in that
prior to
molding the plastic body at least one component of the precursor of the molded
article is
treated with a silver colloid.
Many polymer compounds commonly used in the medical field can be used as the
starting material for the plastic body. Among these are in particular
polyethylene,
polypropylene, crosslinked polysiloxanes, polyurethanes, (meth)acrylate-based
polymers,
cellulose and cellulose derivatives, polycarbonates, ABS, tetrafluoroethylene
polymers, and
polyethylene terephthalates as well as the corresponding copolymers.
Polyurethane,
polyethylene and polypropylene as well as polyethylene-polypropylene
copolymers are
particularly preferred. The metal used is preferably silver, copper, gold,
zinc or cerium.
Among these metals, silver is particularly preferred.
Apart from colloidal metal, one or several polymer materials are used in the
preparation
of the plastic bodies according to the invention. Further additives can also
be added to the
mixture of colloidal metal and plastic(s). These are, in particular, inorganic
particles such as
barium sulfate, calcium sulfate, strontium sulfate, titanium oxide, aluminium
oxide, silicon
oxide, zeolites, mica, talcum, kaolin etc. In this context, barium sulfate
which can
simultaneously act as a X-ray contrast medium for specific fields of
application is particularly
preferred.
Prior to molding, one or several polymer components and/or one or several of
the
inorganic additives are treated with the colloidal metal solution.
After mixing of the starting materials which have (in part) been treated with
a colloidal
metal, the resulting mixture is further processed in order to obtain a molded
plastic article.
This can be done in mixers, kneaders, extruders, injection molding machines or
(hot) presses.
CA 02380490 2002-O1-29
3
The metal colloids with which the plastic materials or inorganic particles are
treated are
suitably prepared by reducing metal salt solutions. In order to stabilize the
resulting colloid,
protective agents such as gelatin, silica or starch may be used.
If the preferred metal silver is used, e.g. ammoniacal silver nitrate solution
in gelatin is
slowly blended with a suitable reducing agent. Apart from aldehydes (e.g.
acetaldehyde),
aldoses (e.g. glucose), quinones (e.g. hydroquinone), inorganic complex
hydrides (sodium or
potassium boranate), reducing nitrogen compounds (hydrazine, polyethylene
imine) as well as
ascorbic acid can be used as the reducing agent.
Plastic precursors such as pellets and/or said inorganic particles such as
barium sulfate
are then treated with said colloidal silver solution, dried and molded into
the respective shape.
Applying said silver colloid onto the starting materials and subsequent drying
can be repeated
several times so that in this way very high silver concentrations can be
introduced into the
plastic material. This is of particular advantage if barium sulfate is coated
with silver since in
this way the plastic pellets do not necessarily have to be coated in advance.
The suspension can also be freed from solvent by filtration and it can
subsequently be
freed from all low-molecular organic compounds by first washing it with about
5% ammonia
solution and then several times with distilled water. As described above,
after drying in air the
filter residue will give a homogeneous material. This process can also be
repeated several
times.
The use of e.g. gelatin, (fumed) silica or starch as a colloidal stabilizer
can be omitted if
silver is adsorbed by the inorganic particles, since the microcrystalline
silver particles
produced during reduction bind to the surface of said inorganic particles via
adsorption and,
thus, the formation of a continuous silver coating on the solid is avoided.
Water soluble
adjuvant chemicals used can be removed with water.
By varying or omitting the colloidal stabilizers as well as the reducing
agents, the
particle size of the silver and, thus, the mobility of the resulting silver
ions can be controlled
over a wide range and, moreover, by using low-molecular aldehydes as the
reducing agents
which partially crosslink gelatin, very strong adhesion to the polymer can be
achieved.
In the following, the process according to the invention will be exemplified
by way of
examples.
Example 1:
Preparation of the silver colloid
CA 02380490 2002-O1-29
4
1.0 g gelatin (DAB) are dissolved in 100 ml distilled water at 40°C
whilst stirring.
Subsequently, 1.0 g (5.88 mmol) AgN03 p.a. are added thereto and the resulting
solution is
blended with 1.0 ml (14.71 mmol) water containing 25% NH3.
For the preparation of the silver colloid, 258.7 mg (5.88 mmol, 330 ~1)
acetaldehyde,
dissolved in 50 ml distilled water, are slowly dripped into the above solution
at 40°C over a
period of time of 30 min.
Example 2:
Coating of polyurethane pellets
min after the dripping according to Example 1 has been stopped, about 50 mg
polyurethane pellets made of Tecothane TT-1085A are added and first vigorously
stirred for 2
h at 40°C and then for 3 h at room temperature so that they are coated
with colloidal silver.
The silver colloid is separated by rapid filtration over a folded filter of a
suitable pore
size, the pellets are once again washed with the filtrate and the still wet
pellets are transferred
into a evaporating dish. After superfluous silver colloid solution which does
not adhere to the
polymer has been removed, the resulting product is dried for 10 h at
70°C.
Example 3:
Adsorption of colloidal silver on barium sulfate
a) 0.666 g gelatin and then 6.66 g AgN03 are subsequently dissolved in 500 ml
distilled
water at 50°C. About 8.5 ml 25% aqueous NH3 solution are added until
the reaction is slightly
alkaline.
A solution of 3.53 g anhydrous a-D-glucose in 150 ml distilled water is slowly
dripped
in at 50°C whilst stirnng vigorously and as soon as about half of the
glucose solution has been
dripped in, the resultant silver colloid is blended with 333 g BaSOa. After
the dripping has
been stopped, the suspension is further turbinated for about 2 h at
50°C and then freed from
its volatile components by evaporation and drying at 70°C. The material
is comminuted in a
hand-held mortar.
b) The procedure is analogous to Example 3a), with the exception that 6.66 g
fumed
silica (Degussa, Aerosil 200) are used instead of gelatin. The particle size
of the colloidal
silver was in the range of from 10 to 50 nm, as determined via a scanning
electron
micrograph.
Example 4:
Alternative adsorption of colloidal silver on barium sulfate
The procedure is analogous to Example 3a), with the exception that 1.21
distilled water,
2 g gelatin, 20 g AgN03 and 26 ml 25% NH3 solution are used. As the reducing
agent, a
CA 02380490 2002-O1-29
solution of 10.59 g glucose in 400 ml distilled water is used and blended with
333 g BaS04 in
analogy with Example 3a). The suspension is then further turbinated for 3 h at
50°C and kept
for about 8 h at 70°C until the reaction is complete. The Ag-colloid
adsorbed on BaS04 is
freed from water and the components soluble therein (gelatin, gluconic acid,
NH4N03 and
NH3) by filtering the suspension which still should be as warm as possible and
subsequently
washing the residue four times with distilled water. Drying takes place at
70°C and
comminution is effected as in Example 3a).
The residual amount of organic material (gelatin, gluconic acid, glucose) of
the material
obtained according to Example 4 was determined by means of two independent
methods with
the proviso that under the conditions used gelatin and gluconic acid have
comparable
solubility in water.
By combustion analysis:
In this context the C and H values are below the measuring tolerance indicated
by the
manufacturer of the apparatus of 0.3%, i.e. with a finished compounded
polyurethane material
comprising 20% BaSOa and 0.8% Ag, the total amount of organic residues can be
calculated
to be theoretically at most 0.182 wt% (lowest value that can be detected by
the apparatus).
Thus, the actual value should be considerably lower.
By thermogravimetry:
When comparing the material obtained according to Example 4 with a reference
sample
prepared in an identical way, but not washed (weight loss about 3.2%) and pure
BaSOa, a total
weight loss of at most 0.28 wt% (gelatin: 0.045 wt%, gluconic acid: 0.235 wt%)
or better can
be observed. Thus, the finished compounded polyurethane comprising 20% BaSOa
and 0.8%
Ag exhibits a total content of organic residues of <_ 0.056 wt% (gelatin: _<
0.009 wt%, gluconic
acid: <_ 0,047 wt%). Due to its considerably higher sensitivity,
thermogravimetry is preferable
over combustion analysis.
Example 5:
Determination of antibacterial activity
In order to determine whether the plastic bodies according to the invention
can be
infected with germs, five cylindrical samples each of the respective plastic
(diameter 3 mm,
length 13 mm) were incubated with a composition containing Staphylococcus
epidermis in a
Trypcase-Soy-Broth nutrient solution at 175°C. The following plastic
bodies were examined
(no. 1 is commercially available and untreated, nos. 2 and 3 are according to
the invention):
Specimen 1: section taken from a PU catheter obtained from the company Arrow
(ES
04701)
CA 02380490 2002-O1-29
6
Specimen 2: according to Example 2 of the present invention
Specimen 3: according to Example 3 of the present invention.
The S specimens were each subjected to four test sequences under the following
conditions:
Test sequence 1: initial concentration of Staphylococcus epidermis 5 x 10'
CFU/ml
Test sequence 2: initial concentration of Staphylococcus epidermis 108 CFU/ml
Test sequence 3: as in test sequence 1, but measured in physiological buffer
solution at
37°C after previous incubation for 5 hours.
Test sequence 4: as in test sequence 1, the plastic bodies having been treated
with
natural urine filtered to be sterile at 37°C for 4 hours.
Table 1 shows the number of infected plastic bodies which was determined by
visual
control.
Table 1
number of
infected
specimens
specimen test sequencetest sequencetest sequencetest sequence
type 1 2 3 4
Comparison1 3 5 4 5
2 0 0 0 0
Invention3 0 3 0 0
After compounding, the catheter materials are not impaired in their mechanical
properties required for therapeutical purposes (roughness, homogeneity and
elasticity). The
process can easily be adapted to varying requirements in the production
process, since
antimicrobial activity is maintained irrespective of whether the silver is
introduced into the
polymer material by coating the polyurethane pellets (Example 2) or via the X-
ray contrast
medium (Examples 3 and 4).
The plastic articles according to the invention show significantly higher
antimicrobial
activity with respect to adherence and biofilm formation as well as
considerably improved
long-time performance as compared with prior art materials at comparably lower
toxicity.
The preparation processes according to the invention can easily be controlled,
are
economical and suited for large scale production. Example 4 additionally
provides a process
for removing all "adjuvant chemicals" from the inorganic contrast medium so
that the grant of
a protective certificate on the process should be possible.
' CA 02380490 2002-O1-29
Example 6:
Dependence of antimicrobial activity on time
Catheters (the amounts are based on the finished compounded material):
1) polyurethane catheters 20% BaS04 + 0.8% Ag, length 1.0 cm (Example 4)
2a) silicone catheters 25% BaS04 +1% Ag, length 1 cm, thickness 1.3 mm and
width 2 mm (Example 4)
2b) silicone catheters 25% BaSOa + 0.33% Ag + 0.33% SiOz (Example 3b)
silicone wall sections, length 1 cm, thickness 1 mm
and width 2 mm
3) control Argen Tec 1 lumen catheter (Sicuris) Extr. 1/99
20% BaSOa + 0.9-1% Ag
Sterilization: Storage in a hot-air cabinet at 90°C for 3 hours.
Previous tests showed that
after this period of time the samples are free of germs. (even before that
time samples are
largely not infected with germs)
Germs: S. epidermidis (ref.: Infection Suppl. 6199)
E. coli "
Nutrient medium: Trypcase Soja
Way of proceeding:
- samples are incubated with 5 x 10' germs at room temperature in a suspension
of
0.45% NaCI with 2.5% glucose for 8 hours
- the germ suspension is subsequently removed by centrifugation
- washing two times (2 min of renewed suspension in physiological sodium
chloride
solution whilst swiveling)
- transferring the samples into sterile sodium chloride solution in a Petri
dish
- sampling every hour, after 6 hours every 2 hours and transferring the
samples into
Trypcase Soja Medium after slight swiveling in physiological sodium chloride
solution
- incubation for 24 to 36 hours
- evaluation of the sample for sterility (turbidity=measurement of the end
point).
Results of tests with S. epidermidis
All samples are tested five times (+~-H-h)
CA 02380490 2002-O1-29
8
Time: Sample 1 Sample 2a Sample 2b Control
h
0 +++++ +++++ +++++ +++++
1 +++++ +++++ +++++ +++++
2 +++++ +++++ +++++ +++++
3 ++++- ++++- +++++ +++++
4 ++++- ++++- -t-h+-~-+ -+-~+-++
++-__ -~-+___ ++++- ~-++++
6 _____ +____ ++___ .+--f--t-~-~-
g _____ _____ _____ +++~-+
_____ _____ _____ +++++
12 _____ _____ _____ ++++_
16 _____ _____ _____ .~--~-.~-__
18 _____ _____ _____ +++__
+ = broth turbid after 36 hours
- = broth clear (sterile) after 36 hours
Discussion:
In this test the antimicrobial activity of solids depending on time could be
examined. It is
shown that silver-filled samples exhibit antimicrobial activity already after
6 hours and a
contaminated catheter can be made sterile again within this period of time
even at a
unphysiologically high inoculum. Lower Ag concentration as in sample 2b will
also have a
positive result.
Results of tests with E. coli
All samples are tested five times (+-++++)
CA 02380490 2002-O1-29
9
Time: Sample 1 Sample 2a Sample 2b Control
h
0 +++++ +++++ +++++
1 ++-+++ +++++ ~-H-h+
2 +++++ +++++ +~--t-++
3 ++++- +++++ +++++
-a--t-++- +++++ -~--~-+~--~
++++- ++++- +++++
-~-++__ ++++- +++++
g -~-+___ +++__ +++++
+____ +____ +++++
12 _____ -____ +++++
16 _____ ____- ++++_
18 _____ _____ ++++_
The results for S. epidermidis are equally good even after the silver has been
eluted in
physiological NaCI solution for 1, 2 and 3 weeks and the results are identical
to those in Table
1.
The examination for cytotoxicity was carned out by the company Toxikon,
Bedford
Mass., USA. It was shown that the samples prepared are not toxic and fulfil
the requirements
of the elution test ISO 10993.
