WO2014108191A1

WO2014108191A1 - Electronic device unit comprising a polymer coating

Info

Publication number: WO2014108191A1
Application number: PCT/EP2013/050392
Authority: WO
Inventors: Anne BENEDIKT; Benedikt Hartwig; Jan Hendrik Schattka; Norbert Windhab
Original assignee: Evonik Industries Ag
Priority date: 2013-01-10
Filing date: 2013-01-10
Publication date: 2014-07-17

Abstract

The invention refers to an electronic device unit being equipped on the outside with a coating layer comprising a lactic acid polymer or copolymer.

Description

Electronic device unit comprising a polymer coating

Field of the invention

The invention refers to an Electronic device unit being equipped on the outside with a coating layer comprising a lactic acid polymer or copolymer which causes a cell proliferation rate of additional 10 to 40 % higher than an untreated control in a cell proliferation assay with 3T3 fibroblast cells.

Technical Background

US 2008/0042849 describes a bioimplant Radio Frequency Identification (RFID) tag to be implanted in a living body of an animal to conduct information management for the animal through wireless communication. A quite small bioimplant RFID tag capable of being hypodermically implanted in a small animal such as a mouse is disclosed. The bioimplant RFID tag having a diameter of 1 mm and a length of 7 mm includes a very small antenna having a length of 6 mm and a width of 1 .5 mm and IC chip (μ chip®) having a shape of 0.5 mm by 0.5 mm rectangle. The tag is mounted on the antenna. The antenna is wound on a resin rod made of a flexible material. In this connection a slit is disposed in the antenna for impedance matching. Therefore the antenna is very small. A predetermined communication distance can be secured. For example the radio wave frequency is 2.45 Gigahertz (GHz) to be used for communication distance of several millimetres is securable. The input output impedance of the IC chip may be for instance 50 ohm. Several insertion tools are disclosed. For instance a syringe with a piston may be used to force the RFID tag directly under the skin. A curved needle with a suture, that carries the RFID tag at its end, may be used to drag the chip under the animal skin. A knot in the suture may be used to mark the insertion place and to prevent movement of the tag.

Morra, M. (Expert review medical devices 4 (3), 2007, 361 - 372, Biomolecular modification of implant surfaces) describes that the surfaces of soft-tissue prostheses may be modified by the addition of collagen or hyaluronan in order to effect a decrease by fribrosis and prevention of post surgery adhesion. By modifying the surfaces of soft-tissue prostheses with synthetic peptides, like arginine-glycine- aspartic acid (RGD), an increased cell adhesion may be effected.

It is generally known to implant electronic device units like for instance RFID chip units under the skin of various small animals, pets, domestic animals or animals for production (farm animals like pigs, sheep, cattle or horses). The most common insertion tool for implanting the electronic device unit under the skin of an animal is a syringe with a hollow plastic or steel needle. The syringe is provided with a piston to press the electronic device unit through the channel of hollow needle. After penetration of the animal skin with by the tip of the needle, the electronic device unit is forced by the piston through the channel of the hollow needle under the animal skin.

Description of the figures

Fig 1/1 : Graph showing the results of the cell proliferation test with 3T3 cells.

HA=Hyaluronic acid; R203=RESOMER® R 203 H; μg/ml is the concentration of the HA, R203 or HA-R203 mixtures filled in the wells of the well plate before drying and over laying with 3T3 cells in cell culture medium for the cell proliferation test.

Examples 1 - 20:

1 -9 : 0 (untreated control)/ 10/ 50/ 100/ 200/ 400/ 600/ 800/ 1000μg/mL hyaluronic acid

10: 500μg/ml hyaluronic acid+ Omg/ml RESOMER® R203

1 1 : 0 μg/ml hyaluronic acid+100mg/ml RESOMER® R203

12-14: 500μg/ml hyaluronic acid+55/75/100mg/ml RESOMER®R203

15-17: 250μg/ml hyaluronic acid+55/75/100mg/ml RESOMER®R203

18-20: 100μg/ml hyaluronic acid+55/75/100mg/ml RESOMER®R203 Object of the invention

The most common way implanting an electronic device unit, for instance a RFID chip unit, under the skin of an animal is a syringe with a hollow steel needle. The syringe is provided with a piston to press the electronic device unit through the channel of hollow needle. After penetration of the animal skin by the tip of the needle to a certain depth and by pressing the piston the electronic device unit is forced through the channel of the hollow needle out of the needle under the animal skin where it remains after removal of the needle out of the skin.

However in many cases the injected or implanted electronic device unit dislocates by migration through the tissues of the animal. Thus the implanted electronic device unit may be found in places of the animal far away from the place of injection. This may impede the correct reading of the chip unit data or the function of the electronic device unit and/or therefore impedes its intended use for instance an electronic identification of production animals.

It was seen as an object of the present invention to provide an electronic device that can be injected or implanted under the skin of an animal without or with at least remarkably reduced tendency of migration in the animal tissue.

The object was solved by an electronic device unit being equipped on the outside with a coating layer comprising a lactic acid polymer or copolymer. The coating may effect a cell proliferation rate of additional 10 to 20 % higher than an untreated control in a cell proliferation assay with 3T3 fibroblast cells. Detailed description of the invention

The invention refers to an electronic device unit being equipped on the outside with a coating layer comprising a polylactic acid polymer or copolymer.

Release assay

Each coated RFID chip containing RESOMER^® and/or HA was added into a glass vessel, filled up with 3 mL of the buffer Dulbecco's Phosphate Buffered Saline (D- PBS) and incubated at 37 °C. Samples of 500 μΙ_ per vessel with RFID chip were taken after an incubation period of 5 and 24 hours and used for further ELISA or MTT analysis.

Cell proliferation assay with 3T3 fibroblast cells

Cell proliferation with 3T3 fibroblast cells (MTT assay).

As a first step of the MTT assay 3x10³ 3T3 fibroblast cells were seeded in each well of a 96 well plate; 0.2 mL DMEM cell culture medium was applied to the wells prior to the cells. After 24 h of cell growth in the cell culture plate, the 0.2 mL cell culture medium was changed to 0.2 mL containing 20 μL of the corresponding samples of the release assay. Beside the samples of the release assay different concentrations of the hyaluronic acid in medium (10/50/100/200/400/600/800/1000 pg/mL) were used as a reference to proof their positive effect on the cell proliferation of fibroblasts. Control samples contained only cell culture medium without hyaluronic acid or a release sample (Untreated Control). To allow further cell growth an incubation step of 3 days at 37°C follows.

Afterwards the liquid on the cells is again removed and substituted by 100 μL of a mixture containing MTT reagent in a ratio 1 :5 with cell culture medium lacking FBS. After 1 -2 h incubation at 37 °C the colorimetric reaction can be monitored by measuring the absorption at 405 nm with a multiplate reader. A dried film of the coating layer may effect a cell proliferation rate of additional 10 to 40 %, preferably 10 to 20 % or 20 to 40 % higher than an untreated control, set as 100 % value, in a cell proliferation assay with 3T3 fibroblast cells.

Electronic device unit

The electronic device unit is a unit which is typically intended to be implanted, preferably intended to be implanted under the skin of an animal. The electronic device unit may have a rod-like form and size, maybe 5 to 50 mm in length with a diameter of 1 to 10 mm. In some cases the electronic device unit may also have an irregular form which is suitable as long as the form and size allows it to be implanted, preferably to be implanted under the skin of an animal. The suitable size depends of on the size of the animal where the electronic device shall be implanted. In general comparatively small electronic devices are suitable for small animals while larger ones are suitable for bigger animals.

The electronic device unit may be a Radio Frequency Identification (RFID) chip unit, a sensor device unit, for instance for drug monitoring or for the measurement of vital functions or data, an implant containing a source for radiotherapy, a drug pump, for instance for insulin.

The electronic device unit a sensor platform for instance for a continuous long-term monitoring unit for the monitoring of several vital functions such as blood pressure, oxygen saturation, pulse, and perfusion. The electronic device unit may be a wireless readout unit which is electrical re-charging from the body temperature, a pH sensor, for instance intended to be part of a closed loop system inside of a patient which is able to react to pre-defined deviations of readings by neurostimulation or drug application. The electronic device unit may be a drug pump for instance for insuline and/or a drug monitoring device for instance for the blood glucose level. The electronic device unit may be combining microsystem technology, for instance devices used during cardiovascular surgery. The electronic device unit may be a RFID cross identification unit. Polymer Coating

The Electronic device unit comprises a polymer coating.

The polymer coating may be selected from lactic acid polymers (polylactic acid) or copolymers. The term "lactic acid polymers or copolymers" shall mean polymers comprising polymerized monomer units, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70 % by weight or up to 100 % of polymerized lactic acid or lactide units. A lactide is a cyclic diester of lactic acid. The term lactide shall mean L-lactide, D-lactide, D,L-lactide or meso-lactide. Suitable comonomers that may be polymerized with the lactic acid or lactide respectively are glycolide, epsilon-caprolactone, trimethylene carbonate or dioxonane. Lactic acid polymers or copolymers may include also a AB- or ABA-blockcopolymer containing an A-Block selected from polylactic acid polymers or copolymers and a B- Block selected from a polyethylenglycol polymer.

The polymer coating may preferably be selected from lactic acid polymers or copolymers synthesized from monomer components or from a mixture of monomer components selected from the group consisting of a) to I): a) D- and L-lactide,

b) L-lactide and glycolide,

c) D, L-lactide and glycolide,

d) L-lactide and epsilon-caprolactone,

e) L-lactide and dioxanone,

f) L-lactide and trimethylene carbonate,

g) L-lactide, D-lactide, meso-lactide or D, L-lactide,

h) L-lactide,

i) DL-lactide,

j) statistically distributed monomer units of L-lactide, D-lactide, meso-lactide or DL-lactide and epsilon caprolactone,

k) statistically distributed monomer units of L-lactide, D-lactide, meso-lactide or DL-lactide and dioxanone, I) statistically distributed monomer units of L-lactide, D-lactide, meso-lactide or DL-lactide and trimethylene carbonate.

These kind of lactic acid polymers or copolymers are biodegradable polymers and well known in the art for example from EP1468035, US6706854,

WO2007/009919A2, EP1907023A, EP2263707A, EP2147036, EP0427185 or US5610266.

A preferred lactic acid polymer is Poly(D,L-Lactide). The inherent viscosity ([dL/g] 0.1 % in CHCI₃, 25 °C) may be in the range 0.2 to 0.4, most preferred from 0.25 to 0.35 dL/g (RESOMER® R 203 H).

Additives to the polymer coating

The polymer coating may comprise hyaluronic acid. The polymer coating may comprise collagen.

The polymer coating may comprise the tri-peptide arginine-glycine-aspartic acid (RGD) or a peptide consisting of 4 and 7 amino acids including the sequence RDG.

Preferred peptids consisting of 4 and 7 amino acids including the sequence RDG are:

SEQ.ID.No.1 GRGD

SEQ.ID.No.2 GRGDS

SEQ.ID.No.3 GRGDY

SEQ.ID.No.4 GRGDSP

SEQ.ID.No.5 GRGDSY

SEQ.ID.No.6 CGRGDSY Coating layer thickness

The polymer coating may have a mean thickness in the range of about 10 to 200, preferably in the range of about 20 to 80 μιτι.

Process

A suitable process for preparing an electronic device unit comprising a polymer coating may be by spray coating, dip coating or fluidized bed coating of the electronic device unit by a polymer solution or dispersion.

Use

The polymer coated electronic device unit as described in here is suitable to be implanted in an animal or can be used to be for implanting in an animal.

Thus the invention discloses the use of the polymer coated electronic device unit as described herein as an implant for an animal.

The invention discloses the use of the polymer coated electronic device unit as described herein as an implant with anti migration properties for an animal.

Examples

Fig. 1/1 :

10: 500 μg/ml hyaluronic acid+ 0mg/ml RESOMER® R203

1 1 : 0 μg/ml hyaluronic acid+100mg/ml RESOMER® R203

12-14: 500μg/ml hyaluronic acid +55/75/100mg/m I RESOMER®R203

15-17: 250μg/ml hyaluronic acid +55/75/100mg/m I RESOMER®R203

18-20: 100μg/ml hyaluronic acid+55/75/100mg/ml RESOMER®R203

Materials, Equipment and Methods

The following chapter gives an overview about the materials, equipment and methods used in the different parts of this study.

Materials

Equipment

Dipping for in vitro trials: RESOMER^® 203 H - HA coating

For he dipping process the RFID chip was pushed in a plastic pipette tip and afterwards attached on a metal stick which was fixed into an overhead stirrer. After dipping the tip/RFID chip combination under clockwise rotation (50 rpm) into the prepared dipping mixture, the drying process with cold air was carried out till dried coating layer. The dipping step was performed three times (every step 2 minutes) for each RFID chip to reach sufficient coating thickness due to low concentration of the final dipping mixture.

The dipping process with RESOMER^® as biodegradable polymer was performed in three concentrations (55/75/100 mg/mL) which all contain 100/250/500 g/mL hyaluronic acid. For the preparation of the heterogeneous RESOMER -HA mixture the specific amount of polymer was weighed into a 5 mL screw-cap vial using a top-loading balance before complete dissolving in acetone. Afterwards the needed volume of aqueous HA solution (2 mg/mL) was added to the organic polymer solution and mixed by manual shaking.

In the following tables the specific composition of each dipping trial is documented.

Subsequently the thickness of the coating layer on the RFID chips was determined before starting release assay. Release assay

Each coated RFID chip containing RESOMER^® and/or HA was added into a glass vessel, filled up with 3 mL of the buffer Dulbecco's Phosphate Buffered Saline (D- PBS) and incubated at 37 °C. Samples of 500 μL per vessel with RFID chip were taken after an incubation period of 5 and 24 hours and used for further ELISA or MTT analysis.

ELISA

For the detection of the hyaluronic acid in the samples of the release assay a commercially available kit is used and performed according to the manual of the manufacturer. 100 μL of each sample as well as 1 :10 and 1 :50 dilutions of the samples are used in the ELISA assay in parallel to a concentration series of the hyaluronic acid (1600/800/400/200/100/50 ng/mL) as a standard curve. The measurement of the absorption at 405 nm is done by the means of a multiplate reader. The calculation of the hyaluronic acid concentration in the samples can be performed due to the internal standard curve.

MTT assay 3T3

In order to verify the effect of released hyaluronic acid on the cell growth of 3T3 fibroblast cells aliquots of the release samples were used for a cell proliferation / MTT assay.

As a first step of the MTT assay 3x10³ 3T3 fibroblast cells were seeded in each well of a 96 well plate; 0.2 mL DMEM cell culture medium was applied to the wells prior to the cells. After 24 h of cell growth in the cell culture plate, the 0.2 mL cell culture medium was changed to 0.2 mL containing 20 μL of the corresponding samples of the release assay. Beside the samples of the release assay different concentrations of the hyaluronic acid in medium (10/50/100/200/400/600/800/1000 μg/mL) were used as a reference to proof their positive effect on the cell proliferation of fibroblasts. To allow further cell growth an incubation step of 3 days at 37°C follows.

Afterwards the liquid on the cells is again removed and substituted by 100 μL of a mixture containing MTT reagent in a ratio 1 :5 with cell culture medium lacking FBS. After 1 -2 h incubation at 37 °C the colo metric reaction can be monitored by measuring the absorption at 405 nm with a multiplate reader.

RESOMER® 203 H - HA coating Dipping process

During the preliminary dipping step using RESOMER^® containing hyaluronic acid as model substance it was observed that only a heterogeneous mixture (with low viscosity) of them is possible to use as dipping mixture. After modification of excipients and concentrations a suitable composition between organic RESOMER^® solution and aqueous HA solution was obtained therefore.

Also it can be mentioned that no significant increasing of viscosity was reached by advanced polymer concentration. As result of both RESOMER^® dipping mixtures thereof a dipping cycle of three times in the whole process is necessary to enable sufficient layer thickness on surface of RFID chip.

Determination of the coating thickness

All prepared dipping samples (in vitro / in vivo) were analyzed according to the method description.

Coated RFID chips from the preliminary dipping step using RESOMER^® alone and in combination with hyaluronic acid: Their specific results of layer thickness are documented in Table 9; differences according to the coating layer thickness are based on variation of used polymer concentrations.

ELISA of release samples

To evaluate the release of hyaluronic acid (HA) out of a RESOMER^® R 203 H coating, RFID chips were coated with several mixtures of different RESOMER^® and HA concentrations and incubated in a release assay for 5-24 h in D-PBS (Dulbecco's Phosphate-Buffered Saline) at 37 °C. Using a commercially available ELISA kit the obtained samples were analysed according to the presence of hyaluronic acid that should be released out of the RESOMER^® coating.

The ELISA assay with samples (and their dilutions) of different coated chips displayed a hyaluronic acid concentration of ~ 3-1 1 μg/mL (values of the 1 :10 dilutions multiplied with the dilution factor) for almost every sample.

Due to internal mistakes of the assay procedure during the time period of this study, no further results related to the hyaluronic acid concentrations are available.

MTT assays of release samples

In order to verify the effect of the released hyaluronic acid on the cell growth of 3T3 fibroblast cells (3T3 fibroblast cells (No.: ACC 173 from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany)). 20 μL of the samples of the release assays were used for a cell proliferation assay / MTT assay.

Beside the samples of the release assay different concentrations of the hyaluronic acid were used as a reference to proof their positive effect on the cell proliferation of fibroblasts. Results

Cell proliferation assay with 3T3 fibroblast cells

The standard concentration series of hyaluronic acid (10 μg/mL - 1 img/mL) confirmed the effect on the cell growth of fibroblast cell in general. The results showed a significant increase of 20 - 30 % of the cell growth for all release assay samples in comparison to the 100 % untreated reference of example 1 (100 % line marked as bold black line in Fig 1/1 ).

Claims

Claims 1 . Electronic device unit being equipped on the outside with a coating layer

comprising a lactic acid polymer or copolymer.

2. Electronic device unit comprising a polymer coating according to Claim 1 , wherein the electronic device unit is a Radio Frequency Identification (RFID) chip unit, a sensor device unit, an implant containing a source for radiotherapy or a drug pump.

3. Electronic device unit according to Claim 1 or 2, wherein the lactic acid

polymer or copolymer coating is selected from polymers is synthesized from monomer components or a mixture of monomer components selected from the group consisting of a) D- and L-lactide,

b) L-lactide and glycolide,

c) D, L-lactide and glycolide,

d) L-lactide and epsilon-caprolactone,

e) L-lactide and dioxanone,

f) L-lactide and trimethylene carbonate,

g) L-lactide, D-lactide, meso-lactide or DL-lactide,

h) L-lactide,

i) DL-lactide,

j) statistically distributed monomer units of L-lactide, D-lactide, meso- lactide or DL-lactide and epsilon caprolactone,

k) statistically distributed monomer units of L-lactide, D-lactide, meso- lactide or DL-lactide and dioxanone,

I) statistically distributed monomer units of L-lactide, D-lactide, meso- lactide or DL-lactide and trimethylene carbonate.

4. Electronic device unit according to one or more of Claims 1 or 3, wherein the polymer coating comprises an AB- or ABA-blockcopolymer containing an A- Block selected from polylactic acid polymers or copolymers and a B- Block selected from a polyethylenglycol polymer.

5. Electronic device unit according to one or more of Claims 1 or 4, wherein the polymer coating comprises a Poly(D,L-Lactide) with an inherent viscosity in the range from 0.2 to 0.4.

6. Electronic device unit according to one or more of Claims 1 or 5, wherein the polymer coating may comprise the tri-peptide arginine-glycine-aspartic acid (RGD) or a peptide consisting of 4 and 7 amino acids including the sequence RDG.

7. Electronic device unit according to one or more of Claims 1 or 6, where the polymer coating comprises hyaluronic acid.

8. Electronic device unit according to one or more of Claims 1 or 7, where the polymer coating comprises collagen.

9. Electronic device unit according to one or more of Claims 1 to 8, where a dried film of the coating layer effects a cell proliferation rate of additional 10 to 40 % in contrast to an untreated control in a cell proliferation assay with 3T3 fibroblast cells 10. Process for preparing an electronic device unit comprising a polymer coating according to one or more of Claims 1 to 9, by spray coating, dip coating or fluidized bed coating of the electronic device unit by a polymer solution or dispersion. 1 1 . Use of a polymer coated electronic device according to one or more of Claims 1 to 9 as an implant for an animal.