EP2760437A1 - Spray system for production of a matrix formed in situ - Google Patents

Abstract

A spray system for production of a matrix which forms in situ is described, this comprising at least one lipophilic component comprising at least one polymer based on glycolic acid and lactic acid and at least one biocompatible solvent having an XlogP3 value of less than 0.2, and at least one hydrophilic component, the at least two components being present separately from one another prior to use and not being mixed until or in the course of spraying, and the components forming a film when sprayed onto human tissue.

Description

 SPRAY SYSTEM FOR GENERATING A SITU-MADE MATRIX

The invention relates to a spray or application system for use in preventing adhesions, in particular surgical adhesions.

 After injuries and surgical procedures, adhesions often develop, which develop into adhesions and scarring and lead to postoperative complications. Especially in abdominal surgery, up to 94% of patients experience primary postoperative adhesions. The peritoneum, as a serous skin, coats the abdominal cavity and consists of a visceral and a parietal leaf with a serous cleft space filled with 5 to 20 ml of fluid, allowing free movement of the organs. Histologically, the peritoneum consists of a single-layered squamous epithelium, also called the mesothelial layer, and a narrow layer of subserous connective tissue. A breach of the mesothelial layer leads to the formation of solid adhesions between the two leaves and the surrounding tissue within a few days. In addition to injury from surgery, the mesothelial layer can be damaged by the use of swabs, the drying out of the surface during surgery, or by contact with talcum from appropriately powdered gloves. Even with minimally invasive surgery, such as laparoscopy, the activation process is set in motion.

Although the pathophysiology of peritoneal adhesions has been researched for more than a century, the findings are still incomplete. FIG. 1 summarizes the pathogenesis of peritoneal adhesions with therapeutic options. Traumatization of the peritoneal tissue is believed to produce an inflammatory response with exudation of inflammatory cells and soluble fibrin monomers. Within about 3 hours, this results in the formation of the first fibrous structures, which, with sufficient fibrinolytic activity by the serine protease Plasmin can be resolved within the first few days. If this does not happen, it leads to the formation of collagen-rich connective tissue, the permanent adhesion, which then causes the difficulties.

 While neutrophilic leukocytes are involved in the inflammatory process in the first two days after the injury, macrophages and mesothelial cells play a crucial role in the development of permanent adhesion. Both cell types are able to release plasminogen, a precursor of plasmin, into the bloodstream. In the blood capillaries, plasminogen is converted to plasmin by the serine protease plasminogen activator (Tissue / urokinase plasminogen activator, t-PA / u-PA). These proteases are also secreted by the mesothelial layer. Triggered by an increased concentration of inflammatory cytokines such as tumor necrosis factor (TNF), transforming growth factor (TGFβ) or interleukins, there is a fall in the active tPA concentration in the post-traumatic phase. This significantly reduces fibrinolytic activity in the abdominal cavity, leading to an imbalance between fibrinolysis and fibrin formation and promoting the formation of permanent adhesions. In turn, the decrease in active t-PA concentration in the tissue is the result of reduced t-PA production in the mesothelial cells and concomitant overexpression of plasminogen activator inhibitor type 1 (PAI-1), the major inhibitor of tissue plasminogen activator. Similar to primary wound closure, in which platelets secrete more PAI-1 to prevent premature lysis of the fibrin and thus initiate primary wound closure, the plasminogen activator inhibitor is increasingly formed in the post-traumatic phase by mesothelial cells and endothelial cells of submesothelial blood vessels , It is therefore considered that t-PA / PAI-1 balance is the key site for the development of peritoneal adhesions.

 For therapy of permanent adhesion, there are various starting points, such as:

 i) primary prophylaxis by avoiding injury and inflammation,

 ii) preventing coagulation of serum-containing exudate by anticoagulants, iii) dissolving fibrin structures by fibrinolytics,

 iv) use of mechanical barriers to the regeneration of the mesothelium layer by separation and

 v) prevention of fibronization.

The use of fibrinolytics and the use of physical barriers have been considered to be promising for therapy, but none of these approaches has prevailed because of the associated disadvantages in the clinic. It turned out indicates that currently available fibrinolytic agents have inadequate antiadhesive activity, which may be due, inter alia, to their short plasma half-life. Due to the therefore required high dosage severe side effects occur, which prevent the use. Known thrombolytic agents are streptokinase, urokinase and t-PA protein alteplase recombinantly produced (available as Actilyse ^®) and its modified form reteplase (commercially available form Rapilysin ^®). Alteplase has a plasma half-life of only 3 to 6 minutes, which could be increased to 13 to 16 minutes for the modified form of reteplase. For this reason, multiple applications and infusion pumps are necessary to achieve continuous levels of effect that produce high side effects.

 Physical barriers have also been used to reduce adhesions. However, a positive influence on postoperative complications has not yet been demonstrated. Currently available physical barrier systems are limited to the local application field. Above all, resorbable tissues are known for this purpose, such as, for example, oxidized cellulose fibers, a combination of hyaluronic acid and carboxymethyl cellulose or PEG gels.

 For example, from US 201 1/0052712 A1 it is known to use polymers as adhesion barriers which are biodegradable. There is proposed a formulation for forming an adhesion barrier comprising a plurality of particles of a polymer combination of a biodegradable polymer and at least one water-soluble polymer deposited in the form of a film on a fabric to prevent adhesion. The water-soluble polymer is intended to attract water from the fabric after application, to swell and thereby to allow the formation of film and provide water, so that the particles gradually degrade and release the active ingredient optionally contained. As the active ingredient, in these particles, e.g. contain an anti-inflammatory agent. The properties of the films obtained with this formulation, however, depend on the amount of water available at the applied site and are not set to be replicable.

It has also been proposed to apply active substances intended to prevent adhesion at the site of the injury or the procedure. Because liquids do not remain in the desired location for long, systems have been developed in the past where agents, optionally encapsulated in biodegradable polymers, are provided in a matrix that is prefabricated. However, fixed systems are uncomfortable for the patient, especially in the region of the abdomen. It has therefore also been proposed instead to use gels that may contain active ingredients. For example, WO 2004/01 1054 describes a polymeric matrix composition having a polymer matrix of various types of low to high molecular weight polymers, which contains a solvent which is hardly miscible with water, which is intended to improve the plasticity of the polymer. The proposed composition is a complex system of different types of polymers and therefore expensive to manufacture and use.

 A disadvantage of the known systems which use water from the environment for matrix formation, by containing a water-soluble or water-swellable polymer to draw water into the matrix, is that through the water, an active substance contained in the matrix to is released quickly, so that initially too high a concentration of active ingredient is present at the site of action. It is desirable to have a uniform delivery without a so-called burst at the beginning. To achieve this, it has been proposed in US 2009/0004273 to use for encapsulation of proteins and peptides a polymer system which does not form a hydrogel when the system comes into contact with tissue water. In order to produce a continuous linear release of the active ingredient and to prevent a burst initially, two different polymer systems of a hydrophobic component and a hydrophilic component are used, which may for example be provided as a film or coating of devices.

 In order to make the applicability of implants more flexible, it has also been proposed to form films or implants in situ. For example, DE 100 01 863 describes implants that are formed at the intended location by mixing a carrier material and a solvent for this purpose shortly before use, so that at least part of the carrier material dissolves, in order then to form liquid-crystalline phases in the body. The carrier material is provided in powder form and is used e.g. obtained by spray drying. In particular, if even an active ingredient is included, it must be ensured that sufficient mixing takes place in order to distribute the active ingredient evenly in the matrix produced.

Other carrier systems formed in situ have been described for the preparation of implants. Since temperature and pH changes as well as reactive constituents for matrix formation in the body can not be used directly, the most commonly used technology is precipitation via a solvent. The solidification of the implant is therefore carried out in the known methods mostly by the contact of a water-insoluble polymer dissolved in an organic solution, with the tissue fluid. To speed precipitation, some of the known processes, as outlined above, add to the composition water-attracting ingredients, eg, swellable polymers. Depending on the carrier system and the organic solvent used, there is then an increase in viscosity to form a viscous gel, or a true precipitation of the carrier system to form a matrix. Often this is one Copolymer of lactic and glycolic acid used, whose precipitation can be controlled by the choice of the solvent and the polymer. Depending on the molecular size of the pharmacologically active component thus release kinetics and duration can be adjusted as needed. Two technologies that are described in the prior art, the Atrigel ^® technology, the N-methyl-2-pyrrolidone used as the water-miscible solvent, and the Alcamer ^® technology employing poorly water-miscible solvent. As is known, a higher miscibility with water leads to a rapid implant formation and thus to an increased porosity of the matrix, while poorly water-miscible solvents or highly concentrated polymer solutions cause a slower implant formation. The former lead to a rapid release of the embedded components and also to an increased initial release of the active substance, the so-called burst. The latter lead to a long-lasting release, whereby the release occurs only after some time. A product based on Atrigel ^® technology is commercially available in the form of a hormone preparation for the treatment of advanced, hormone-dependent prostate cancer.

 Advantage of such systems is that they can be applied directly to the desired location and that also an active ingredient can be embedded in the matrix during application. However, the biggest problem with the known in situ formed implants is the control of the morphology of the implant and thus the control over drug release. The morphology of the implant is dependent on the conditions at the site of use, which makes a reproducibility almost impossible and prevents a release kinetics can be set in a predetermined manner.

 The previously known systems thus all still have disadvantages and are not yet satisfactory in the application. It was therefore an object of the present invention to provide a system which overcomes these disadvantages, which is easy to use, without complicated or expensive measures in its use and reliably helps to prevent the adhesions after injuries and surgical interventions. Furthermore, the system should also provide the opportunity to administer an active ingredient, wherein the release of the drug should be predictable, adjustable and even without causing a burst at the beginning, but also without too long start-up time.

A further object of the present invention is to provide an application system which can be sprayed directly onto the intended site, which is suitable for taking up and releasing in a controlled manner active ingredients, in particular hydrophilic active ingredients such as nucleic acids, proteins and peptides, which can produce a stable film at the site of application whose release properties can be adjusted and optimized. In addition, an application system should be provided which is physiologically compatible. is not inhibiting the activity of proteins, peptides and nucleic acids and thus allows the release of active products.

 Moreover, it was an object of the present invention to provide an application system that can effectively prevent surgical adhesions and helps to avoid permanent adhesions.

 The above objects are achieved with a sprayable application system as defined in claim 1. The sprayable application system, also referred to below as a spray system, comprises at least one lipophilic component which is formed at least from a polymer dissolved in a solvent, and an aqueous component and optionally at least one active substance. It may include other ingredients.

 It has surprisingly been found that with the specific composition defined according to the invention a carrier material is provided which is easy to use, which is stable, which can be applied at the desired site and to the desired extent and provide an active ingredient for the desired duration and at a desired rate.

 The advantageous properties are achieved with a spray system consisting of two components, one component having at least one polymer dissolved in one solvent and the other component having at least one aqueous solvent by mixing the components directly before or during application and by spraying are applied, wherein from the components of the invention in situ, a matrix is formed, which decomposes after a predetermined period of time and selectively releases the active ingredient optionally contained in this time.

 The film formed by the system according to the invention has a high quality and remains for a predetermined time at the applied location and exerts its effect there. Only by combining the features according to the invention is a carrier material having the desired properties obtained.

 Important features of the present invention are the nature of the polymer and the solvent, as well as the form of application, i. contacting the two components directly before or during spraying or during spraying.

One of the essential features of the system according to the invention is the lipophilic component which contains at least one glycolic acid and lactic acid-based polymer and at least one biocompatible solvent for the polymer, the solvent having a predetermined logP value, as explained in more detail below. This lipophilic component is then mixed with at least one hydrophilic component which is at least containing at least one aqueous solvent, mixed immediately before or during spraying, which by precipitation of the polymer produces a matrix which forms a physical barrier at the sprayed location which can effectively prevent surgical adhesions. In a preferred embodiment, the lipophilic component and / or the hydrophilic component contains at least one active substance which is enclosed in the film during spraying and film formation and released therefrom in a controlled manner, which additionally blocks the surgical adhesions in a physiological or biological manner.

The particular advantage of the present application system is that, by selecting the specially applied ingredients, upon spraying by precipitation of the polymer from the solvent, a polymer matrix is formed which, if the composition contains an active agent, also includes it. By precipitation of the polymer is meant that the solubility limit of the polymer is exceeded and the polymer is no longer or no longer completely dissolved in the solvent. By adjusting the individual components, the precipitation and flame formation rate and thus the porosity of the resulting matrix can be predetermined, which leads to controlled release properties. Due to the variability of these polymers, there are many ways to set the optimal properties, but the optimal solution for the individual case is not always easy to find. In the following, therefore, parameters are described which allow the selection of an optimal system.

 Critical to the system according to the invention are in particular the polymer used, the solvent used to dissolve the polymer, the proportion of polymer, solvent and aqueous component and optionally the proportion of active ingredient and its form.

 The material used to form the matrix should be sterilizable and must allow controlled release of an entrapped drug for a period of time during which adhesions and scarring may form. This is a period in the range of at least two weeks and up to six weeks and preferably from two to four weeks. In addition, the material must have such a quality that it retains its strength long enough to fulfill the desired purpose of preventing adhesions.

An important parameter for the selection of the appropriate polymer is the molecular weight. The selection of the appropriate molecular size is made by the inherent viscosity (natural logarithm of the relative viscosity relative to the concentration C of the solute). In a manner known per se, for PLGA polymers, the inherent viscosity is measured at 0.1% in CHCl ₃ at 25 ° C. The polymers used for the system according to the invention are those having an inherent viscosity in the range from 0.1 to 0.8, in particular 0.15 to 0.7 prefers. If the value is below 0.1, the polymers are often too small to have a sufficiently long effect. If the value becomes too large, sufficient film quality can not be ensured, and the startup time for the release may be too long. In order to achieve optimum properties, it is also possible to use mixtures of polymers of different molar mass.

 The molecular weight of the PLGA polymers can also be determined by conventional methods, e.g. with gel permeation chromatography. It has been found that PLGA polymers with a molecular weight in the range of 10 to 63 kDa are well suited.

 Further factors are helpful for the selection of a polymer suitable for the application system according to the invention. The system of the invention must be sprayable, which implies that it must be soluble or suspendable in a biocompatible solvent.

 A measure of the quality and mechanical strength of the film formed from the application system according to the invention is the matrix or film quality, which can be determined by the method described in the examples. FIG. 2 shows the results of tests on the film quality of some combinations of polymers and solvents described in the examples. The quality is essentially determined by the choice of the solvent and the molecular weight of the polymer. For their determination, the percentage of the polymer in the supernatant (loss) and in the precipitate (matrix grade) based on the total amount of polymer used is determined. The matrix quality of the resulting layer or film is critical to the system of the present invention because the system is expected to perform its function for at least two and up to six weeks. This is only possible if the film formed or the layer formed has sufficient strength and at the same time provides the desired release kinetics. The matrix grade should therefore be in a range of 80 to 100%, preferably 90 to 100% and most preferably 95 to 100%, the value being determined by the method described in the example at room temperature, i. about 25 ° C, is determined.

The matrix quality depends inter alia on the molecular weight of the polymers. It has been found that by using higher molecular weight polymers, a larger amount of polymer could be incorporated into the matrix, while with lower molecular weight polymers, polymer (for film formation) was lost. Thus, it has been found that for a PLGA polymer having a ratio of lactide to glycolide of 75:25 and an inherent viscosity of 0.5 to 0.7, ie, a relatively high molecular weight and using a polymer having esterified end groups, almost 100% of the amount of polymer used formed the matrix. When using a PLGA polymer with a ratio of lactide to glycolide of 50:50 and an inherent viscosity of <0.6 and with free end groups, it was found that polymer was lost during matrix formation, ie, it was not incorporated into the matrix. This effect may be enhanced or enhanced by the solvent.

 As stated above, one of the essential components of the system is the polymer used. According to the invention for the application system based on glycolic acid and lactic acid-based polymers, namely poly (lactide-co-glycolide), usually poly (D, L-lactides-co-glycolide), hereinafter also referred to as PLGA polymers used , Both D, L-lactide based polymers and those based on the enantiomerically pure L-lactide based polymers can be used.

 Milk and / or glycolic acid based polymers have been known for some time, even for controlled release systems. As a rule, PLGA polymers are processed into microparticles or implants, which can then be used in different ways. PLGA polymers are biocompatible and biodegradable and their properties can be tailored to suit their purpose.

 For the present invention, glycolic acid and lactic acid based polymers dissolved in a solvent are used whose release kinetics are adjusted by molecular weight, molecular weight distribution and end groups.

 Thus, it can be selected by appropriate selection of the polymer and the solvent whether the resulting matrix should effect a diffusion-controlled, erosion-controlled or both diffusion- and erosion-controlled release. A rapid formation of the high-quality matrix, as achieved according to the invention, contributes significantly to a linear release kinetics without initial loss of active ingredient.

It has been found that PLGA polymers are better suited for the system according to the invention than pure polylactide (PLA) or pure polyglycolide (PGA). By adjusting the ratio of lactic acid to glycolic acid units, the properties, in particular the degradation properties, can be adjusted very well in a manner known per se. PLGA polymers which have a ratio of lactide to glycolide units in the range from about 75 to about 25 to about 25 to about 75 have proved to be preferred for the application system according to the invention. The term "ratio of lactide to glycolide units" always refers to the molar ratio of the units in a polymer Mixtures of different PLGA polymers can also be used Mixtures of all types of PLGA polymers can be used, for example mixtures of polymers in which molar ratio of lactide to glycolide units and / or molecular weight or inherent viscosity and / or type of lactide units (D / L or L) and / or end groups The most suitable mixture for the particular purpose can be found by routine experimentation. The degradation rates of PLGA polymers depend on the proportion of PGA or PLA, with PLGA copolymers generally having shorter degradation rates than PLA polymers or PGA polymers. For this reason, PLGA polymers are preferred. The shortest degradation times are achieved with polymers in which the ratio of lactide to glycolide is 50:50. An increase in the PLA content, due to the additional methyl group in the lactic acid monomer, hampers the hydrolysis of the polymer and at the same time increases the hydrophobicity, which increases the degradation times. Increasing the PGA content in the polymer or using the pure stereoene enantiomer L-lactic acid over the monomer D- / L-lactic acid also increases the degradation times of the polymers by increasing the crystallinity of the polymer as water diffuses more easily into amorphous regions and thus these regions be broken down faster than crystalline. Thus, during degradation, the crystallinity of the polymer steadily increases. By adjusting the ratio, the crystallinity and the degradation time can thus be predetermined. Furthermore, the degradation rates can be accelerated by shorter polymer chains and free end groups. Free end groups, ie, free hydroxy groups and free carboxy groups increase the hydrophilicity of the polymer, thereby increasing the rate of diffusion of the water and its level in the polymer matrix. In addition, by lowering the pH inside the matrix, free carboxyl groups catalyze the hydrolysis of the polymers. Therefore, according to the invention, preference is given to using polymers which have free end groups.

 Preference is given to using according to the invention PLGA polymers having free end groups which have a ratio of lactide to glycolide units of 40:60 to 60:40, more preferably about 50:50 and / or which have an inherent viscosity of less than 0.6. When PLGA polymers having esterified end groups are used, those having a ratio of lactide to glycolide units of 75:25 are preferred.

Suitable PLGA polymers are commercially available, for example Resomer polymers (available from Evonik Industries AG, Essen, Germany), especially those of Resomer ^® H-Series or Resomer ^® S series. Particularly suitable polymers are, for example, Resomer ^® 502H, 503H and 504H or Resomer ^® RG755S. Table 1 below lists some properties for preferred polymers: Table 1: Properties of the Resomer ^® RG polymers used

 * inherent viscosity (i.v., 0.1% solution in chloroform at 25 ° C)

 ** Number of acid groups (potentiometric titration) are taken from the manufacturer data.

 Molar mass, measured via GPC, and

**** The release data are from the publication by Eliaz and colleagues [44 [Eliaz, 2000 # 257]. The release data refers for Resomer ^® RG 755 S and 503 H to the release of bovine serum albumin [44] and for Resomer ^® RG 502 H and 504 H to the release of thymus DNA [45] of an injectable implant (10% to 20% PLGA (m / v) in tetraglycol).

The degradation of the polymer matrix occurs via ester hydrolysis to the biocompatible monomers lactic acid and glycolic acid, which are then metabolized to C0 ₂ and water through the Krebs cycle. The degradation behavior of PLGA implants is due to bulk erosion, which is characterized by the fact that water diffuses faster into the polymer matrix than the degradation of the polymer takes place. Accordingly, there is a homogeneous mass loss over the entire cross section of the polymer matrix. The degradation process can generally be divided into three sections:

 1. Hydration: The polymer absorbs water and swells, already splitting a small proportion of ester bonds. However, there is still no mass loss.

2. Degradation: The average molecular weight decreases sharply. The carboxylic acid groups resulting from the cleavage of the ester bonds lead to a drop in pH within the matrix and, as a consequence, to the autocatalysis of the ester cleavage. The polymer loses mechanical strength or mechanical strength. 3. Solution: At the end of the degradation, low-molecular fragments and oligomers dissolve in the surrounding medium, whereby the dissolved polymer fragments are in turn hydrolyzed to free carboxylic acids.

 The degradation times are crucial for the release of encapsulated macromolecules and nanoscale carrier materials, since these are mainly released by matrix erosion because of their size and thus a targeted adjustment of the release rates is made possible by the degradation rates. The degradation times of the PLGA polymers can be controlled by their composition and the molecular weight of the polymers, wherein the inherent viscosity is usually given as a measure of the molecular weight for the commercially available PLGA polymers.

 The viscoelastic properties of the system also play a role, as shown in FIG. 3 and in the examples.

 For the application system according to the invention it is essential that a material of high quality is produced which retains its mechanical strength for as long as necessary to prevent adhesions and which is subsequently degraded. If the carrier system formed from the administration system according to the invention is loaded with active ingredient, it is additionally necessary that the active ingredient with the desired release kinetics be released.

 The matrix quality of the film obtained from the application system according to the invention depends on the polymer used, the solvent used for its solution and its water solubility. It has been found that the solvent used for the dissolution of the PLGA polymer has a considerable influence on the quality of the matrix produced therewith. The matrix produced by combining the PLGA dissolved in the solvent with the aqueous phase thus depends on the nature and amount of the solvent, in particular its hydrophilicity.

 On the other hand, the selection of the solvent also depends on the type of polymer used. The more lipophilic the polymer, the more lipophilic the solvent must be. The lipophilicity of the polymer depends, inter alia, on the end groups, since a PLGA with free acid groups is more hydrophilic than a PLGA with esterified end groups.

On the one hand, the solvent must sufficiently dissolve the selected polymer so that the polymer can be sprayed; on the other hand, the solubility of the solvent in water must be sufficiently high so that precipitation can be rapid after spraying the two components. One parameter useful in selecting the appropriate solvent is the logP value. Since, as stated above, the matrix quality changes as a function of the molar mass of the polymer by the solvent, a further essential feature of the invention is the solvent. An important parameter for the selection of the solvent is the miscibility with water. The higher the miscibility with water, the faster the matrix formation, however, the porosity is higher, the lower the miscibility with water, the slower the matrix formation, the higher but also the quality.

 The water miscibility of a solvent can be assessed by the logP value.

The logP value denotes the octanol / water partition coefficient, i. the ratio of the concentration of the solvent in a two-phase system of 1-octanol and water. The definition of the logP value is:

 logP = iog- | - = logc ^ - log cj.

 The calculation or determination of the logP value is known per se. One algorithm suitable for determining the logP value is XlogP3 as described in Cheng et al. (Cheng T., Zhaoy, Lix, Lin F., Xu Y., Zhang X. et al., Computation of Octanol-Water Partition Coefficients by Guiding to Additive Model with Knowledge. J. Chem. Inf. Model ; 47: 2140-2148). The logP value calculated in this way gives positive values for lipophilic substances and negative values for hydrophilic substances. It has been found that suitable solvents for the system according to the invention are those whose lipophilicity is not too high, so that preference is given to those solvents which have a negative or at least very small positive XlogP3 value.

 Solvents having an XlogP3 value of less than 0.2, preferably less than 0 and in particular in the range from -0.2 to -1.5, particularly preferably those having an XlogP3 value between -0.25 and -1.0, the XlogP3 value should be greater the more lipophilic the polymer used.

It has been found that when a polymer solution in which the solvent has a log P of less than 0.2 is mixed with an aqueous component and brought to the site of application by spraying, precipitation occurs in a predictable and reproducible manner produced a polymer film with desired properties.

The more lipophilic the polymer used is, the more lipophilic the solvent used must be and the lower its water miscibility. The greater the difference between the solubility of the polymers in the solvent or water, the stronger the influence on the kinetics of the film formation. Therefore, when used as PLGA polymer with esterified end groups, which thus has a higher lipophilicity, should also used solvents are more lipophilic. A more lipophilic solvent has a poorer water miscibility and therefore leads to a high matrix quality. It has been found that the best results are obtained when the polymer / solvent system is near the solubility limit and the solvent has the best possible water miscibility, so that when the aqueous phase is added, rapid and complete film formation occurs, with a high proportion of the polymer can be found in the matrix.

 The solubility of the polymer in the solvent also plays a role. The better the polymer dissolved in the solvent, the more water is required later to precipitate the polymer out of the film and form a film. On the other hand, the solubility must be such that a sufficient amount of polymer can be dissolved in the solvent. It has been found that a solvent which has a solubility for the PLGA to be used at room temperature of at least 5% (mass / volume) (m / v), preferably from 5 to 60%, and in particular a solubility of 10 to 30%, suitable for forming a high quality film. In selecting the appropriate solvent, the following relationships are to be considered: The solubility of a solvent for a polymer decreases with increasing molecular weight of the polymer. The more lipophilic the polymer is, i. the more strongly the polymer is esterified or the longer the polymer chain is, the more lipophilic the solvent must be. Thus, when a very lipophilic polymer is used with a highly water-soluble solvent, the polymer is only slightly dissolved, while a non-lipophilic polymer, e.g. one with free acid groups and lower molecular weight in a hydrophilic solvent can be solved well. On the other hand, the more water is required for the precipitation, the better solved is the polymer. Very good results can be achieved if the polymer and solvent are chosen such that the solubility is between 5 and 15%, with the solvent having an XlogP3 value in the range of -0.3 to -1.0. For this combination, when water is added, the polymer precipitates very quickly and forms a high-quality film. Solvents having an XlogP3 value in this range are known to those skilled in the art.

A well-suited solvent thus combines good water miscibility with such polymer solubility that, for the desired amount of polymer, the solubility in the solvent at the application temperature, i. is between 30 and 40 ° C, near the saturation limit, wherein the solubility at room temperature must be sufficient in any case, that a stable solution is formed.

Tetraglycol, glycerol formal and dimethyl isosorbitol (DMI) have proven particularly suitable. The solvent tetrahydrofurfuryl alcohol-poly- ethylene glycol, termed tetraglycol or glycofurol, is a solvent that has long been used for paren- teralia concentrations up to 50% are used and at this dilution the solvent shows only low toxicity.

Glycerol formal is an odorless, low toxicity solvent consisting of a mixture of 1,3-dioxan-5-ol and 1,3-dioxolane-4-methanol, which is an excellent solvent for many pharmaceuticals and cosmetics. It is mainly used in veterinary medicine as a solvent for injections. Is commercially available for example as glycerol Ivumec ^® and PTH ^®. Ivumec ™ is approved at 0.27% for subcutaneous application in pigs and is usually used at 0.1 ml / kg.

Dimethyl isosorbide (DMI) is known for topical application. Commercially available preparations which contain DMI are Mykosert ^® and Ibuprop-Gel ^®. DMI is used topically as a penetration enhancer. Low hemolytic activity was observed.

It has been found that the higher the water miscibility of the solvent used, the higher the film quality of the film formed from the application system according to the invention. In the examples, experiments are shown to determine the film quality. Figure 4 shows the film quality for some combinations of PLGA polymer and solvent. The water solubility of the abovementioned solvents decreases in the following order: glycerol formal> DMI> tetraglycol. Glycerol formal is therefore in most cases the most preferred solvent for the administration system according to the invention, as long as it can dissolve sufficient polymer. The following Table 2 gives an overview of the properties of some solvents tested:

 Table 2: Properties of some solvents

Designation η [mPas] * XlogP3 ** PLGA Release Application FAM

 friendliness ***

Glycerol 14.4 -0.8 33% parenterally Ivomec ^®

Peteha ^®

Tetraglycol 16.6 -0.3 k. A. parenterally Phenhydan ^®

 Eusaprim

DMI 8,16 -0,6 41% topical Mykosert ^®

Ibutop Gel ^®

Triacetin 18.75 0.2 35% parenteral k. A.

Ethyl l-lactate 2.74 0.2 46% k. A. k. A. * The kinematic viscosity η was determined on a rotational viscometer (MRC 100, Paar Physics) at 25 ° C,

 ** XlogP3 data are calculated values [32]

 *** Solubility data were taken from a publication by Matschke et al., 2002 [33].

The layer thickness of the matrix formed from the spray system according to the invention plays a role in the rate of diffusion of the water. Thus, for PLGA systems with a layer thickness of 150-300 μm, in which the diffusion rate of the water is limited, surface erosion could additionally be detected. Regardless of the solvent 502 H layer thicknesses were measured from about 300 μηι during the viscoelastic examinations for films based on Resomer ^® RG. In contrast, the layer thickness of the films for the longer-chain polymer increased analogously to the observed release kinetics of DMI via glycerol formal to tetra glycol.

Another very important feature of the solvent to be used for the application system according to the invention is biocompatibility or tissue compatibility. In the present application, the tissue compatibility is determined by the influence of the solvent on the metabolic cell stability over a period of 11 hours. A method of determination is described in the examples. The observed LD ₅ o value is the measure of the toxicity. The LD 50 must be at least 1, preferably at least 10 mg / ml in order to consider a solvent for the present application system. The above-mentioned particularly preferred solvents fulfill this requirement. Glycerol formal, which has an LD ₅₀ value of about 1 g / ml at an incubation time of less than 6 hours, has proven particularly suitable in this context. Glycerolformal is therefore a particularly preferred solvent for the inventive system. Figure 5 shows LD ₅ o values for preferred solvents depending on the incubation time.

 In one embodiment, the application system according to the invention consists only of a lipophilic component with polymer and solvent, as described above, and water as the second component. With these ingredients, if they meet the above conditions, mixing and spraying can be used to generate a film in situ which can effectively prevent surgical adhesions.

It is essential for the invention that the spray system according to the invention contains the lipophilic component and the aqueous component separated from one another until they are sprayed on. Only at or just before spraying or during spraying may the Components are mixed. It has been found that even the addition of comparatively small amounts of water leads to a precipitation of polymer. If this precipitation took place too early, the film formation could be disturbed and, if necessary, the spraying device could be blocked by deposition of polymer. Preference should therefore be given to mixing directly during the spraying, for example by the respectively intended amounts of the two components are fed into a mixing chamber and sprayed directly therefrom during mixing from this. Mixing and spraying should thus preferably take place essentially simultaneously.

 In another Ausführangsform an application system is provided, which additionally contains an active ingredient. As active ingredients, all substances useful for the intended application site are considered. The application system according to the invention is particularly suitable for the release of nucleic acids, proteins and peptides. Thus, both proteins and peptides can be released directly as well as the nucleic acids encoding them or even a mixture thereof. It has been found that the application system according to the invention and the film formed therefrom release the nucleic acids in such a form that their subsequent expression is possible. Since the system according to the invention is intended for the prevention of adhesions, fibrinolytic proteins and peptides or the corresponding nucleic acids coding for them are preferably used as active ingredients.

 The active ingredient may be dissolved or dispersed in one of the two components. It has been found that an excessively high proportion of aqueous phase can influence the quality of the film formed. Therefore, if it is desired to add an active ingredient whose solubility in water is not sufficiently high to produce highly concentrated solutions, it is preferable to use the active ingredient in already precipitated form, e.g. to be added in dried form. Particularly suitable are lyophilisates or polyplexes in finely divided solid form, which can be dispersed in the lipophilic component. This has the further advantage that the active ingredient in solid form is more stable for storage.

As stated above, tissue-specific plasminogen activators and their inhibitors in particular play a role in the formation of adhesions. According to the invention, therefore, in one embodiment, a "gene-activated" film formed in situ is applied locally by spraying for the treatment of peritoneal adhesions, since, as described above, after an operation in the abdominal cavity in a time window of 2-3 weeks permanent adhesions can occur and there If it is assumed that the trigger for this is an imbalance between the tissue-specific plasminogen activator (tPA) and its inhibitor (PAI-1), this imbalance is modified according to the invention by providing tPA and / or inhibiting PAI-1 the film formed in situ, the tPA and / or PAI-1 inhibitor and / or this coding nucleic acid contains acids. It has been found that when using the Sprühsy- tems invention containing a tPA-encoding plasmid when a film is incorporated into the film matrix, it is released gradually and at least two weeks to increase the tPA level in the physiological environment. The tPA level in the physiological environment of the sprayed film can also be increased by bringing PAI-1 inhibitor into the environment or by a combination of both. The Examples and Figures 10 and 11 describe properties and results with such films.

It was shown that the incorporation of a gene encoding tPA plasmid into a film according to the invention was able to increase based on glycerol with Resomer ^® RG 504 H in the cell culture experiment the tPA levels over a period of 16 days to 2 ng / ml. This corresponds to a 4-fold increase in tPA concentration over the control. Since in tissue that has been subjected to a surgical procedure, as well as in tissue that is inflamed, the tPA concentration can drop to one fifth of the normal values and more, a therapeutically relevant substance can thus be used for a longer time with a film produced from the spray system according to the invention Increase in the tPA level can not be achieved with the preparations of the prior art.

Furthermore, it has been found that the level of the inhibitor in the stressed and / or inflamed tissue can be increased up to 17 times, which causes a considerable reduction of the tPA level. As a result, the tPA / PAI-1 ratio can vary from 3.5 in the normal state to 0.4 in the inflamed tissue. Therefore, in order to influence the events even more effectively after a surgical procedure and to combat adhesions even more successfully, the spraying system according to the invention particularly preferably contains both at least one tissue-specific plasminogen activator or a nucleic acid encoding it and at least one plasminogen activator inhibitor inhibitor or a nucleic acid encoding same. As shown in the examples, it is possible to effectively restore the tPA / PAI-1 balance by using the inventive spraying system to produce a film which effects cotransfection of a tPA-encoding plasmid DNA and a siRNA against PAI-1. It could be shown that this cotransfection of a tPA-encoding plasmid DNA and a siRNA against PAI-1 leads to a 8.3-fold increase in the tPA / PAI-1 ratio 48 h after transfection, whereas the application of the plasmid alone increased by a factor of 4.5. Depending on the desired effect, the spraying system according to the invention can therefore contain either at least one tissue-specific plasminogen activator or at least one PAI-1 inhibitor or a combination of both or in each case the corresponding nucleic acids. In this way, the desired effect can be set very variably with the system provided according to the invention. In the embodiments described above, the nucleic acid may be RNA, DNA, mRNA, siRNA, miRNA, piRNA, shRNA, antisense nucleic acid, aptamer, ribozyme, catalytic DNA, and / or a mixture thereof. The term DNA includes all suitable forms of DNA such as cDNA, ssDNA, dsDNA, etc., the term RNA includes all suitable forms of RNA such as mRNA, siRNA, miRNA, piRNA, shRNA, etc.

The nucleic acid may be linear or circular, it may be single-stranded or double-stranded. The term "nucleic acid" is also understood to mean a mixture of nucleic acids, each of which can code for identical or different proteins or peptides, All forms of nucleic acids which encode the desired protein or peptide and can express it at the desired site are suitable. The person skilled in the art knows the respectively suitable forms of nucleic acids and can select the most suitable ones The nucleic acid can originate from any source, eg from a biological or synthetic source, from a gene library or collection, it can be genomic or subgenomic DNA The nucleic acid may contain the elements necessary for its amplification and expression, such as promoters, enhancers, signal sequences, ribosome binding sites, tails, etc.

 The nucleic acid may be a DNA or RNA and may have one or more genes or fragments. The nucleic acid may be an autonomously replicating or integrating sequence, it may be as a plasmid, vector or other form well known to those skilled in the art. It may be linear or circular and single or double stranded. Any nucleic acid active in a cell is suitable here. Since "naked" nucleic acids are not very stable and are rapidly inactivated and degraded in the cell, it is preferred to coat the nucleic acid with a layer, so-called polyplexes being a particularly preferred embodiment.

 In order to protect the nucleic acid, it can be used in the form of so-called polyplexes. Polyplexes are nucleic acid molecules surrounded by a polymer shell. Preferably, a cationic polymer is used as the shell material. It has been found that cationically charged particles can be more easily taken up by the cell than neutral or anionically charged particles, but they also tend to promote nonspecific deposits. For enveloping nucleic acids as active constituents, cationic shell materials are preferred, since nucleic acids can be enveloped and protected very easily with cationic substances. Corresponding methods are well known to the person skilled in the art.

The shell material may be a naturally occurring, synthetic or cationically derivatized natural substance, eg a lipid or a polymer or oligomer. An example a natural oligomer is spermine. Examples of synthetic polymers are nitrogen-containing biodegradable polymers, in particular those with protonatable nitrogen atoms. Particularly suitable are polyethyleneimines, in particular branched polyethyleneimines, which are commercially available. For example, a branched polyethyleneimine having an average molecular weight of 25 kDa, which is commercially available, is suitable. It has been found that this polymer is very compatible with the other constituents of the spray system according to the invention. It is also possible to use lipids, in particular cationic or neutral lipids, as natural, optionally derivatized, layer-forming shell material. Lipids are available in many variants and can be used for example for the formation of liposomes.

 When polyplexes are used as the active ingredient, the ratio of shell material to nucleic acid should be adjusted in a manner known per se so that the nucleic acid is sufficiently protected but can be expressed upon release. If too little shell material is present, the nucleic acid will not be sufficiently protected. If the proportion of shell material is too high, on the one hand problems with the compatibility can occur and on the other hand an excessive amount of shell material can lead to the fact that the nucleic acid can no longer be released and / or can no longer be expressed. In both cases, the efficiency of the transfer suffers. The expert can find the most suitable ratio in a few routine tests. Particularly suitable is a ratio of shell material to nucleic acid in the range of 10: 1 to 1: 4, by weight, proved. Particularly preferred is a ratio of shell material to nucleic acid of 4: 1 to 1: 4. When the polyplexes contain polyethyleneimine as the polymer, the proportion of the polymer can also be given by the molar ratio of polymer nitrogen to the proportion of DNA phosphate (N / P); Preferably, the N / P ratio is in a range of 1 to 10, more preferably 4 to 8.

 The polyplex molecules are designed so that the nucleic acid is protected during storage, transport and application, and then the nucleic acid is released and expressed at the target site. Suitable polymers have been widely described in the literature, and those skilled in the art can choose from a variety of materials the most appropriate one.

Experience with nucleic acids as drugs has been collected in more than 1,400 clinical trials since the first clinical trial in 1989. In addition to retroviral adenoviral gene transfer systems were used, with a great advantage in the efficiency of these systems. Thus, it could be shown that even the binding of a single virus particle is sufficient to infect the target cell. Non-viral gene transfer systems represent a safe alternative to viral systems in terms of immunogenicity and mutagenesis potential. Non-viral gene therapy approaches have been described. The application of naked nucleic acid in combination with physical methods such as electroporation, as well as the use of nanoscale complexes with synthetic carrier systems such as cationic polymers, which are also referred to as polyplexes. Information on the preparation and use of polyplexes can be found, for example, in the article by Godbey WT, Mikos AG, "Recent progress in gene delivery using non-viral transfer complexes" (J Control Release, 2001; 72: 1 15-125) and cationic Liposomes (Lipoplexes) in articles by Lee RJ, Huang L. "Lipidic Vector Systems for Gene Transfer". (Crit Rev Ther Drug Carrier Syst. 1997; 14: 173-206) and Simoes S, Filipe A, Faneca H, Mano M, Penacho N, Duzgunes N, et al., "Cationic liposomes for gene delivery" (Expert Opin Drug Deliv. 2005; 2: 237-254). The complex systems are based on the fact that under physiological conditions the positively charged nucleic acid and the negatively charged carrier material spontaneously assemble into nanoscale particles ('self-assembly ^* ').

 The spray system of the present invention provides a new promising approach for achieving long-lasting gene expression. By forming a film in the form of a gene-activated depot system whose local application can lead to a constant nucleic acid level over a defined period of time in the area of application, advantageous properties are achieved. Dosage frequency and dosage amount can thereby be reduced, unwanted side effects such as transfection of other tissues, so-called off-target effects, prevented, unphysiological protein levels and the burden of the patient with nucleic acid and support material can be avoided and patient acceptance can be improved.

 As stated above, it is believed that the ratio of PA to PAI-1 inhibitor has an influence on the formation of surgical adhesions and scarring. In a further embodiment, therefore, a spray system is provided, which contains as active ingredients a combination of PA and PAI-1 inhibitor or nucleic acids encoding them, wherein the ratio of PA and PAI-1 inhibitor in the range of 5: 1 to 1: 5, using the appropriate nucleic acids, the ratio can be adjusted so that at the target site after expression, a ratio of PA: PAI-1 inhibitor from 5: 1 to 1: 5 results. It has been found that when such a combination is applied, the formation of surgical adhesions can be suppressed particularly effectively.

The spray system according to the invention is characterized in that, when the two components are mixed, the polymer is precipitated very rapidly to form a film, with any active ingredients present in one or both of the components simultaneously being integrated into the film. For this, the two components, which are stored in separate containers before use, are sprayed so that they are sprayed or mixed directly before or sprayed during mixing. The two components of the spray system according to the invention are thus mixed for application. This is preferably done by the two separate components are performed for spraying in a mixing chamber and sprayed directly from it. Preferably, a device is used for spraying, as is known per se, in which, when the spray valve is actuated from two storage chambers, one dose in each case is conducted into a mixing chamber and sprayed together therefrom. In this way, the mixing takes place directly in the spray applicator during spraying, whereby a premature precipitation, which could clog the spray nozzle, is avoided. Successive spraying of the two components from separate spray applicators can not produce high quality film. It is essential for the invention that the two components held separately until use come into direct contact with each other during spraying, so that the film formed by precipitation of the polymer can then deposit at the target location upon impact of the spray. Spray applicators suitable for mixing / spraying two components previously separated are known in the art. A known and suitable for the application according to the present invention device is shown in Figure 8 and is available as a spray kit from Baxter.

 The dose to be dispensed between the two components can be adjusted. It depends on the type of use, the type of components, if necessary, the active ingredient. The two components should be applied at a ratio (based on the volume of solutions / liquids) of 10:90 to 90:10, preferably 25:75 to 75:25 and more preferably in a ratio of 40:60 to 60:40 be mixed. The amount of components dispensed for the film to be formed depends on the desired size and thickness of the film. It can be adjusted in a conventional manner. For administration in the abdomen an amount of 0.5 to 5 ml, preferably 0.7 to 3 ml of each component has been found suitable.

A particularly advantageous formulation, the following combinations proved: lipophilic component: 10% (m / v) PLGA solution (Resomer ^® RG H series) in glycerol formal, tetraethylene glycol or DMI,

 hydrophilic component: water for injections

 Active ingredient component: pDNA / l-PEI polyplexes, dissolved as lyophilisate in the hydrophilic phase (embedded variant A) or dispersed in the lipophilic PLGA solution by means of a homogenizer (embedded variant B).

optionally sucrose with a concentration of 10% (m / v) as cryoprotector for lyophilization The spray system according to the invention is provided for therapeutic use. Areas of application for matrix systems produced thereby are the prevention of postoperative adhesions, in which permanent adhesions can occur after abdominal surgery, triggered by an imbalance between the tissue-specific plasminogen activator and its inhibitor [56, 64, 65]. Critical is a time window of 2 weeks, which includes an acute phase of 2-5 days after surgery. Depot systems which contain a plasmid coding for tPA as the active component are particularly suitable. In addition to the pharmacologically active component of the polymer film additionally provides an anti-adhesive barrier against adhesions. It is also possible, for example, the spraying of the spray system according to the invention via endoscope, z. B. in an endoscopic procedure in the abdominal cavity.

 The invention is further explained in the attached figures.

 The figures show embodiments of the invention and results obtained with them.

 FIG. 1 shows a schematic representation of the pathogenesis of surgical adhesions

2 shows diagrams showing the film quality of selected Resomer ^® RG polymers compared: A) PLGA 50:50, H-series, B) PLGA 75:25, S series. Shown is the percentage of polymer used, which forms the spray film, whereas the remainder is lost as a soluble fraction in the supernatant. Data are presented as means ± standard deviation (n = 4). Statistically significant differences are marked with asterisks (P <0.05 (*), P <0.01 (**)).

3 shows diagrams illustrating the results of examinations of the viscoelastic films: A) storage modulus (G ^'), B) loss modulus (G ") of Resomer ^® RG H- series with different solvents in comparison at a frequency of 1Hz.

Figure 4 shows the film quality which is obtained with the tested solvents: A) In situ formed films using Resomer ^® RG 503 H, B) plotted against the film quality distribution coefficient P. The data are presented as mean ± standard deviation from n = 4 approaches.

Figure 5 shows LD ₅ o values of the solvents tested compared: LD ₅₀ of the solvents tested as a function of incubation time on mesothelial cells. In each case, the metabolic cell viability was determined by ATPlite assay

6 shows diagrams for release kinetics of different formulations in situ formed films: (A) Resomer ^® RG 502 H, polyplexes in a hydrophilic phase, (B) Resomer ^® RG 502 H, polyplexes in a lipophilic phase (C) Resomer ^® RG 504 H, Polyplexes in hydrophilic ler phase, (D) Resomer RG 504 H, polyplexes in the lipophilic phase. Lyophilized polyplexes were incorporated into the films using different polymer solutions (DMI (·), tetraglycol (o) and glycerol formal (T), and the pDNA release was analyzed over 30 days.) Data are presented as means ± standard deviation of the mean (n = 3). specified.

Figure 7 shows the transfection efficiency of lyophilized 1-PEI / pDNA polyplexes on lung cell lines using different cryoprotectants. DNA topology - Lyophilized pDNA / l-PEI polyplexes were separated by agarose gel electrophoresis with addition of heparan sulfate (HS). The polyplexes were resuspended in water for injections (Wfl). The data are presented as means ± standard deviation ((a) n = 7, (b) n = 4)). Statistically significant differences are marked with asterisks (P <0.05 (*), P <0.01 (**)). pCMV-Luc control (C), size markers (L), water for injections (Wfl), Ultra-Turrax ^® (UT), a homogenizer (H).

 FIG. 8 shows a test setup for the production of in situ formed films

Figure 9 is a diagram showing results for the in vitro application of erfmdungs- modern films on mesothelial cells: luciferase gene expression after application Resomer ^® RG 504 H based films on mesothelial cells. Plasmid DNA / 1-ΡΕΙ polyplexes were incorporated into the hydrophilic phase and expression was examined by luciferase assay over a period of 29 days. The emitted photons (RLU) were measured for 10 s after background correction. The results are given as mean ± SEM (n = 3).

FIG. 10 shows results for in vitro application of in situ formed films on mesothelial cells. (A) matrix release 504 H films based and (B) Fluorescence image of the embedded plasmid DNA after staining with Propidium- iodide from Resomer ^® RG. pCMV-tPA-IRES-Luc / I-PEI polyplexes were dissolved in the hydrophilic phase, the spray film was sprayed onto mesothelial cells and the tPA level determined over a period of 29 days by ELISA. The results are given as mean ± SEM (n = 3).

FIG. 11 shows cotransfection of plasmid DNA / siRNA on mesothelial cells: a) PAI-1 and tPA detection in the Western Blot after 48 h, b) tPA / PAI-1 ratio as a function of time. The polyplexes consisting of pCMV-tPA-IRES-Luc (ptPA) or a wild-type plasmid (pUC) and various siRNAs (PAI-1, EGFP) were treated with 1-PEI at an N / P ratio of 10 (relative to the pDNA amount) in HBS. For comparison, the expression of untreated cells (UN) is shown. The determination of the tPA and PAI-1 levels was carried out by means of Western Blot in the supernatant at different times. Figure 12 shows a schematic representation of the pCMV-tPA-IRES-Luc plasmid

 Figure 13 shows a dilution series of 1-PEI / pDNA polyplexes in PBS

 Figure 14 shows a standard curve of the human tPA antigen assay

 Figure 15 shows the transfection efficiency of powdered polyplexes using different cryoprotectants: transfection efficiency of lyophilized 1-PEI / pDNA polyplexes on lung cell lines under (A) using different cryoprotectants (10% (m / v) sucrose or mannose, 4% (m / v) dextran 5000, B) after homogenization of lyophilized polyplexes using 10% (m / v) sucrose as cryoprotectant.

The invention is illustrated by the following examples, without thereby limiting them in any way:

 example 1

 MATERIAL AND METHODS

 nucleic acids

 The plasmid pCMVLuc, which is available as described in [19], contains the luciferase gene (Luc) of the fire fly Photinus pyralis under the control of the CMV promoter, a promoter from cytomegalovirus. The pMetLuc construct also encodes the luciferase gene of the rower crawler Metridia longa, a secreted luciferase enzyme, under the control of the CMV promoter [20].

 The construct pCMV-tPA-IRES-Luc was cloned and is shown schematically in FIG. It contains, in addition to the sequences for the luciferase enzyme (Luc) and the tissue-specific plasminogen activator (tPA), a CMV promoter (CMV-IE, cytomegalovirus immediate-early).

 The cloning of the pCMV-tPA-IRES-Luc plasmid was carried out using the pl-RES-Luc vector [21]. Into this vector, a tissue-specific plasminogen activator (tPA) -coding sequence was cloned into under the control of the CMV promoter using the restriction endonucleases Mlul and Fsel (New England Biolabs Inc., USA). For this purpose, the sequence (insert) from the plasmid pCMV-tPA was amplified by means of polymerase chain reaction (PCR) [22]. In addition to the luciferase gene, the pIRES-Luc vector contained an internal ribosomal entry site (IRES), which allowed translation of both transcripts independently of one another.

 The control plasmid used was pUC21 vector (Invitrogen, Germany), which contains no expression cassette but only the bacterial backbone.

The following oligonucleotides were synthesized: An siRNA was screened against plasminogen activator inhibitor 1 (PAI-1, 5 '-GGAACAAGGAUGAGAUCAG [4, 23] -3') and as control a siRNA against EGFP

(5 ^' -GCAAGCUGACCCUGAAGUUCAU [dT] [dT] -3'). The lyophilized samples were dissolved in resuspension buffer (Quiagen) to 20 μΜ or for the release studies to ΙΟΟμΜ stock solutions, incubated for 1.5 min at 90 ° C, shaken gently at 37 C for 1 h and stored at - 20 ° C in aliquots.

 polyethyleneimine

 Linear polyethylenimine with a molecular weight of 22 kDa was synthesized according to a protocol by Plank and colleagues [24]. Linear PEI was obtained analogously to the procedure by acid hydrolysis of the propionic acid poly (2-ethyl-2-oxazoline) 50 Da, wherein the liberated propionic acid as an azeotrope mixture was continuously withdrawn from the synthesis approach, so that the reaction could proceed almost completely. The free base was then precipitated by means of sodium hydroxide solution at pH 12, washed and lyophilized. The lyophilized 1-PEI was stored at 4 ° C and dissolved in distilled water as needed, adjusted to pH 7.4, dialyzed (ZelluTrans dialysis membranes T2, MWCO 8-10 kDa) and sterile filtered.

 Quantification of the PEI solution was carried out photometrically by means of the copper sulfate method at 285 nm on a spectrophotometer (Ultrospec 3100 Pro) [25]. As a reference, a 1-PEI charge of known concentration was used. The purity of the synthesis product was checked by 'H-NMR spectroscopy (Bruker 250 MHz, Karlsruhe). The measurement of the molecular weight was carried out by gel permeation chromatography with a multi-angle laser scattering detector (GPC-MALLS) and gave a molecular weight of 20-22 kDa.

PLGA polymers

 The following polymers from Boehringer Ingelheim, Germany were used for the production of the films:

• Resomer ^® RG 502, 503 and 504 H

• Resomer ^® RG 752, 755 S

 cell line

Pleural mesothelial cells (human), short Met5A, from ATCC, Germany (CRL-9444) were used. The cell line was cultured in a 1: 2 mixture of Ml 99 (Gibco-BRL, UK) and MCDB 105 (Sigma-Aldrich, Germany) at 37 ° C, 5% CO ₂ and 100% humidity. In addition to 10% fetal calf serum (PPA Laboratories, Austria), an epidermal growth factor (5 ng / ml, Sigma-Aldrich, Germany) and hydrocortisone (400 ng / ml, Sigma-Aldrich, Germany) were added to the medium [26]. Furthermore, the cells were passaged at a confluency of approximately 80% and used until a passage of 20 for experiments. Production of Polvplexes

 The complexes were formed spontaneously by electrostatic bonding forces, the properties of the polyplexes formed thereby being essentially dependent on the ionic strength of the medium, the polymers used and the N / P ratio. This gives the molar molar ratio of protonated nitrogen atom (N) of the polymer structure to the negatively charged phosphate atom (P) in the nucleic acid. To obtain small monodisperse particles, equal volumes of the lower charge density solution, the nucleic acid solution, were pipetted to the higher charge density solution, the polymer solution, and mixed by pipetting up and down (5-8 times). The solution was then incubated at RT for 20 min before further experiments were performed. The medium used was water for injection (siRNA, plasmid DNA lyophilisate) and HBS pH 7.4 (plasmid DNA liquid).

 Production and Characterization of Powdered Polvplexes

 The polyplexes were prepared using pCMVLuc and 1-PEI at a N / P ratio of 10 as described above in water for injections. To test different cryoprotective substances, the polyplexes were incubated after incubation with a 20% (m / v) sucrose solution, a 20% (m / v) mannose solution or a 4% (m / v) dextran 5,000 solution Diluted 1: 2, mixed and aliquoted. These could then be flash frozen in nitrogen and lyophilized for about 24 hours at maximum power in the lyophilizer. The lyophilizates were resuspended to a final concentration of 0.02 μg / μl (same starting concentration) in the respective medium and transfected onto BEAS-2B cells in 96-well plates analogously as described below.

Sucrose was added in powder form after an incubation time of 10 min and the complexes were incubated for a further 10 min, the particle size being controlled by PCS before and after addition of sucrose. After lyophilization, the powder was in a mortar homogenized and pestle and subsequently by means of homogenizer, a cylindrical glass vessel with glass pestle (debris Labortechnik, Germany), or by means of Ultra-Turrax ^® (stage 3, 14 sec, Ika Labortechnik, Germany) PLGA solution to be suspended. Alternatively, the powder was resuspended directly or previously homogenized by mortar in water for injection. The lyophilizates were resuspended to a final concentration of 0.02 μg μl in the respective medium.

 Determination of particle size and zeta potential

The determination of the hydrodynamic diameter of the polyplexes was carried out by means of photon correlation spectroscopy in a semimicrocuvial cuvette with 600 μΐ Polyplex solution in bidistilled water (0.02 μg / μl pDNA), the zeta potential by means of electrophoretic light scattering in a macro cuvette with 1.6 ml Polyplex solution (0.02 or 0, / μ1 pDNA). The following settings were used: 5 measurements (size measurement), 5 runs of 10 cycles per sample (zeta potential); Viscosity of water (0.89 cP) or HBS (1.14 cP); Ref index 1.33; Dielectric constant 78.5; Temperature 25 ° C. The zeta potential was calculated according to Smoluchowski. The evaluation of the size was based on a standard curve. The device was pulsed with polystyrene latex particles 92 nm in size (Duke Scientific Cooperation, CA, USA) and zeta potential reference Bl-LC-ZRZ with a +50 mV charge (Laboratory chemistry, Vienna, Austria) at regular intervals Checked intervals.

 agarose gel electrophoresis

 Agarose gel electrophoresis can be used to determine the degree of complexation of the nucleic acid (plasmid DNA, mRNA) in polyplexes. For this purpose, polyplexes were prepared as described above, mixed with 6-fold concentrated loading buffer and 100 ng each pDNA on a 0.8% agarose gel spiked with ethidium bromide (10 ug / 100 μΐ) applied. For reference, a corresponding size marker was applied. Electrophoresis was carried out at 125 V for approximately 1.5 h in 1x TAE buffer. Subsequently, the bands of the nucleic acid were detected under UV light (360 nm) and recorded by gel camera.

 In the agarose gel, incomplete complexation by free nucleic acid in the gel can be detected, with polyplexes remaining in the gel pocket. Furthermore, depending on the intensity of the signal in the gel pocket, a conclusion can be drawn about the degree of condensation. The weaker the band, the stronger the nucleic acid is condensed by the cationic polymer. The addition of a polyanion (0.1 μg heparan sulfate (HS) / g g pDNA, incubation time 45 min) displaced the nucleic acid from the complex and allowed an integrity check (topology, degradation) of the nucleic acid.

 Example 2

Characterization of in situ formed films

Determination of film quality as a function of biomaterial and solvent

In order to be able to characterize the film formation more closely depending on the solvent used and the polymer type, spraying tests were carried out in petri dishes. For this purpose, a 10% (m / v) polymer solution was prepared in each solvent and sprayed in each case 1 ml of polymer solution (syringe 1) with 1 ml of water for injection (syringe 2) at 1.5 bar. A waiting time of 5 minutes should allow complete formation of the matrix. For the visual inspection of the matrix, the aqueous phase with Brilliant blue G stained and set the distance to the spray area with 11 cm. The further experiments were carried out without fixation, since a more homogeneous film could be achieved thereby. The results are shown in FIG.

 In order to better assess the quality of the matrix, the supernatant was removed and dried in a vacuum system (Speed Vac, Dieter Piatkowski, Germany) to constant weight. Similarly, the matrix in a freeze dryer (Lyovac GT 2, LH Leybold, Germany) was also dried to constant weight, which then determines the percentage of the polymer in the supernatant (loss) and in the precipitate (matrix grade) based on the total amount of polymer used could be.

Determination of tissue compatibility of the solvent

The cytotoxicity of the solvents was determined by means of an ATP-based test assay (ATPlite, Perkin Elmer). For this purpose, cells were seeded in a 96-well plate 24 hours before the experiment, the medium was removed immediately before the experiment, the cells were washed once with PBS and 50 .mu.l serum-containing medium with antibiotic addition (Penicillin / Streptomycin 0.1% (v / v); gentamycin 0.5% (v / v), Gibco-BRL, UK). Then, each 50 μΐ different concentrations of solvent (16-500 ug / ul) diluted in water for injection, added and incubated for different lengths of time at 37 ° C, 5% C0 ₂ and 100% humidity (15, 30, 60, 221 , 360 and 640 min). After the incubation period, the medium was aspirated, the cells were washed once with PBS, 50 .mu.l PBS per well presented and determined cell viability according to the manufacturer. The measurement of luminescence was carried out in a plate reader (Wallac Victor2 / 1420 Mulitlabel Counter, PerkinElmer Inc., USA), using the luminescence of untreated cells (50 μM water for injection) as a reference value with a viability of 100%. For each time point, the concentration of the solvent against the measured cell viability (mean ± standard deviation from n = 4 batches) was plotted and a non-linear standard function was adjusted:

y = min + [(max -min) / (l + (x / EC50) ^auxiliary )]

This function provided a good fit for all solvents: tetraglycol (R2 = 0.9181-0.9900), glycerol formal (R2 = 0.9268-0.9945), dimethyl isosorbide (R2 = 0.9647-0.9894). The LD ₅₀ values were taken from the estimates of the plotted regression curve. It is the concentration at which 50% cell viability could be measured. Viscoelastic properties of in situ formed films

To get an idea of the viscoelastic properties of the matrix, rheological investigations were carried out. For this purpose, the films on the plate of a rotary viscometer (Physica MCR 301) was sprayed and the biomaterials (re somer ^® RG 504 H and 502 H) in a dynamic shear experiment tested depending on the solvent used. Here, a harmonic oscillating shear stress of defined amplitude and frequency is applied to a sample and the resulting shear strain determined by two response quantities, the response amplitude and the response frequency, also called phase shift, is characterized. Both response variables can be mathematically transformed into the memory module G "and loss modulus G ^" ', the memory module characterizing the stored and thus recyclable portion of the introduced kinetic or deformation energy (elastic component) and the loss modulus, a measure of the heat released per oscillation Energy and thus lost share represents (frictional share).

Experiments to determine the release kinetics

 The experiments to determine the release kinetics were carried out in sealable Petri dishes (Petri dishes without absorbent 50x9mm, PA11) at 37 ° C with constant shaking in an incubator. For this, the samples were sprayed as above with water for injection. Lyophilized 1-PEI / pCMVLuc complexes (N / P ratio 10, 10% sucrose, 25 μg pDNA / batch) were previously dispersed in homogenized form (mortar and pestle) in the PLGA solution or resuspended in the water phase. As a control water for injection purposes was used. After spraying, the mixture was waited for 5 minutes, the supernatant was removed (0 h value) and 1 ml of PBS was added. The supernatant was then completely replaced at regular intervals, with the samples stored at -20 ° C until analysis.

The quantification of the released plasmid DNA from the matrix formed in situ was carried out photometrically. For this purpose, the samples were shaken out before the measurement with chloroform (1 ml, 400 g, RT, 10 min) in order to separate PLGA degradation products which would disturb a photometric quantification [27]. The samples were then measured photometrically at 260 nm (Nanodrop-1000, PEQLAB Biotech, Germany). Beforehand, 1-PEI / pDNA polyplexes (pDNA concentration 100 μg / ml) were prepared in water for injection and a standard series was determined by serial dilution with PBS on 5 individual days at 260 nm, on the basis of which the concentration of released complexed plasmid DNA could be calculated. The results are shown in FIG. As a control, unloaded films were examined (background basic correction), small deviations in the volumes were taken into account by weighing the samples over the density of water.

Example 3

 In vitro investigations transfections

To perform in vitro transfection studies, 90,000 to 120,000 cells (24-well plate) per well were seeded 24 hours prior to transfection using only cells to passages 20 for transfection. This resulted in a confluence of approximately 70% on the day of transfection. Immediately prior to transfection, the medium was removed, the cells were washed once with PBS and 200 μΐ (24-well plates) medium without serum submitted. Subsequently, 50 .mu.l of the polyplexes, corresponding to a plasmid DNA amount of 1 .mu.g (24-well plate) were added to the medium introduced. After an incubation period of 4 h at 37 ° C., 5% CO ₂ and 100% atmospheric humidity, the polyplexes were removed, the cells were washed once again with PBS and replaced by serum-containing medium with addition of antibiotics (penicillin / streptomycin 0.1%). v / v), gentamycin 0.5% (v / v), Gibco-BRL, Great Britain).

Carrying out the spraying experiments

 L-PEI / plasmid DNA polyplexes (N / P ratio 10, 100 μg pDNA / batch) were formulated as described above, lyophilized with 10% sucrose, and homogenized using a mortar and pestle, which was dosed by weight and applied either in a sterile filtered PLGA solution or resuspended in the water phase (water for injection) could be resuspended. Water for injections without additives was used as a negative control. As plasmid DNA, pMetLuc and pCMV-tPA-IRES-Luc were used in equal proportion.

Three days before the start of the experiments, Met5A cells were seeded on hanging inserts (1 μM PET Millicell) with a polyethylene terephthalate (PET) membrane, which allowed light microscopic control of the cells. Each 1.5 ml cell culture medium was presented, the inserts 2 minutes in equilibrated therein and then seeded 250,000 cells per well in 1, 5 ml of medium on the membrane. Prior to the experiment, the medium was removed, washed once with PBS, and the samples were sprayed on the cells as described above. Samples were taken daily at the beginning, and every two to three days later, with the medium being changed completely, the samples immediately placed on ice and stored at -80 ° C. until the analytical determination. Determination of transfection efficiency by luciferase activity measurement

To investigate the gene transfer efficiency when using the pCMVLuc plasmid, which codes for the reporter gene luciferase, the luciferase activity 24 h after transfection was measured by washing the cells once with PBS, per well with 100 μΐ lx cell lysis buffer (25 mM Tris / HCl pH 7.8, 0.01% Triton-X 100) and after an incubation time of 10 min at RT were shaken for 60 sec. 100 μΐ luciferin substrate (470 μΜ D-luciferin, 270 μΜ coenzyme, 33.3 mM DTT, 530 μΜ ATP, 1.7 mg (MgC0 ₃ ) ₄ Mg ₂ × 5 H ₂ O) were then automatically added to an aliquot of 50 μΐ, measured 2.67 mM MgS0 _4, 0.1 mM EDTA 0.1 mM, 20 mM tricine) was added and the light emission over a period of 5 sec in a plate reader (Wallac Victor ^2/1420 Mulitlabel Counter, PerkinElmer Inc., USA) become. Before addition of the substrate, the background was also determined over the period of 5 sec. The luciferase activity, measured as emitted photons (Relative Light Units, RLU), was integrated after background correction over a period of 10 seconds and related to the total protein amount of the cell mass. The total protein was previously determined by means of a standard protein assay (Biorad method).

Determination of transfection efficiency via the expression of the metridialuziferase

Initial in vitro spraying experiments were performed with a mixture of 1-PEI / pMetLuc and 1-PEI / pIRES-Luc-tPA polyplexes. The pMetLuc plasmid encodes a cell-lysed luciferase enzyme, the metridialuciferase, and thus allowed the measurement of gene transfer efficiency via enzyme expression in the supernatant of the samples. This luciferase catalyzes the oxidative decarboxylation of luciferin, in this case coelenterazine, with simultaneous light emission at a wavelength of 482 nm. Samples were analyzed at selected sample times using the Ready-To-Glow Automation Kit (Clontech, A Takara Bio Company, France). by this were thawed on ice and the light emission over a period of 5 sec without prior dilution in accordance with the manufacturer's instructions in a plate reader (Wallac Victor ^2/1420 Mulitlabel counter, PerkinElmer Inc., USA) measured. Before addition of the substrate, the background was also determined over a period of 5 sec, so that the luciferase activity (RLU values) after background correction could be integrated over a period of 10 sec and the respective negative controls could be subtracted from the values. Untreated cells served as a negative control for bolus administration (single application of the complete amount of pDNA in water for injection) and unloaded films were used as a negative control for the matrix systems. Determination of total tissue plasminogen concentration by ELISA

As an adjunct to the determination of luciferase activity, the total tissue plasminogen concentration in selected samples was determined by ELISA (Human tPA Total Antigen Assays, Innovative Research, Dunn Laboratory GmbH, Germany) in the supernatant of the cells, with the assay used in addition to free and thus active tPA also the latent and bound to the inhibitor form was detected. Since these were supernatants from the cell culture, the standard was diluted analogously to the samples in cell culture medium of the cells used without FCS. The positive control (bolus dose) was diluted as follows: 1:50 (48 h, 9 d), 1:10 (16, 23 and 29 d). The samples from the inner compartment were filled in (30 μΐ sample ad 100 μΐ), whereas the samples from the outer compartment were analyzed undiluted. The assay was performed according to the manufacturer's instructions and the absorbance at 450 nm over a period of 0.1 sec in a plate reader (Wallac Victor ^2/1420 Mulitlabel Counter, PerkinElmer Inc., USA). The standard curve is shown in FIG. The negative controls were used as described above.

Fluoroscopic micrographs

 After completion of the spray test, the medium was removed and the plasmid DNA remaining in the matrix was stained with propidium iodide. For this purpose, the matrix was incubated with propidium iodide in a 1:10 dilution in PBS for 10 min at RT, washed again with PBS before taking up and taken pictures with an epifluorescence microscope (Axiovert 135, Carl Zeiss, Jena, 10x objective). The excitation of propidium iodide was at 470 ± 20 nm, whereas the emission was detected at 540 ± 25 nm. For the evaluation, the software Axiovision LE 4.5 was used and analyzed with an Alexa 560nm filter at Brightfield.

 Example 4

 Co-transfection of siRNA and plasmid DNA and determination of tPA PAI-1 ratio by Western Blot

The transfection was carried out as above in 24-well plates with some special features. In each case 750 ng of plasmid DNA and 30 pmol siRNA complexed with 1-PEI at an N / P ratio of 10 (based on the amount of plasmid DNA) were used. The change of the medium took place after 6 h. The proteins, the tissue-specific plasminogen activator and plasminogen activator inhibitor type 1 (PAI-1), were analyzed after transfection by Western blot. Since these were secreted proteins, they could be detected in the supernatant of the cells, so that 20 μΐ of the supernatant of the cells were taken at different time points and the samples were pooled per batch (n = 3) and centrifuged at 14,000 rcf and 4 ° C for 10 min to separate dead cells. The samples were always stored on ice and frozen at -20 ° C until final analysis.

 The separation of the proteins was carried out according to their molecular weight by means of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To destroy secondary and tertiary structures of the proteins, the batches were assayed (3.75 μΐ sample, 15 μΐ 4x application buffer (130 mM Tris / HCl pH 7.4, 20% glycine, 10% SDS, 0.06% bromophenol blue, 4% DTT). ad 60 μΐ water for injection) previously denatured for 5 min at 95 ° C. Each 20 μΐ of which were separated by electrophoresis on a 7.5% Tris-HCl gel (Bio-Rad Laboratories GmbH, Germany) and transferred by means of electrotransfer (1 h, 200 mA, transfer buffer) on a polyvinylidene difluoride (PVDF) membrane. After blotting, the membrane was cut at 50 Da (size standard, Precision Plus Protein Standards) and nonspecific protein binding sites in a saturation buffer (5% milk powder in 20 mM Tris / HCl pH 7.4, 137 mM NaCl, 0) for incubation with various primary antibodies. 1% Tween20) for 1 h at RT with gentle shaking. Incubation with the primary antibodies was carried out overnight at 4 ° C. with gentle shaking (mouse anti-alpha-actin 1: 15,000 Chemicon, Germany; mouse anti-huPAI-1 monoclonal 1: 200 American Diagnostics, Germany; hatPA monoclonal 1: 400 Calbiochem, Germany in 1:10 diluted saturation buffer). For detection, the secondary antibody (goat anti-mouse HRP-conjugated, Bio-Rad Laboratories, Germany) was used in a 1: 10,000 dilution (tPA, PAI-1) or in a 1: 20,000 dilution (actin) and the membrane incubated for 1.5 h at RT with gentle shaking. Subsequently, the labeled proteins were detected by ECL chemiluminescence (Amersham Bioscience, USA) on a film (Amersham Hyperfilm ECL, GE Healthcare, German) and analyzed for quantification using Image J Basics Version 1.38. Normalization was via the actin band of the untreated cells.

 statistical evaluation

 The results, unless otherwise indicated, are presented as means ± standard deviations. Statistically significant differences were calculated using an unpaired t-test. Statistical significance was assumed at ot = 0.05 (*) or at a = 0.01 (**).

Crucial for the selection of a suitable solvent were i) the pharmaceutical applicability, ii) a good tissue compatibility, iii) the water miscibility and iv) the solubility of the polymer in the solvent. Based on these parameters, some solvents were selected for further screening. A widely used solvent for use in injectable depot systems is tetraglycol (tetrahydrofurfuryl alcohol-polyethyleneglycol), also referred to as glycofurol [28, 29]. Tetraglycol has been used since the 1960s as a solvent for parenteral (iV, im) in concentrations up to 50% (v / v) and shows low toxicity at this dilution [30].

 Glycerolformal is an odorless, also low toxicity, solvent consisting of a mixture of 1,3-dioxan-5-ol and 1,3-dioxolane-4-methanol [30] and is an excellent solvent for many pharmaceuticals and cosmetics. Nowadays, it is mainly used in veterinary medicine as a solvent for injections. For example, Ivomec ™ 0.27% is approved for subcutaneous administration in pigs and is used at 0.1 ml kg [31].

 For dimetylisosorbide (DMI) and ethyl lactate, there are few studies on the compatibility of the solvents. Ethyl lactate is used as a parenterally administrable vehicle for steroid formulations and, despite the GRAS number, is considered to be relatively toxic with narcotic and slightly hemolytic activity. DMI is used topically as a penetration enhancer with low haemolytic activity [30, 34]. Furthermore, a study by Matschke and colleagues showed that glycerol esters with good compatibility are suitable solvents for PLGA / PLA polymers [33]. In this context, triacetin, a low-toxicity short-chain triglyceride, has been tested [35, 36] and previously described as an alternative to NMP and DMSO for in situ formed depot drug forms [29, 37-39].

Determination of the film quality as a function of the solvent used

 The decisive prerequisite for the formation of an in situ formed depot system is the solubility of the polymer in the solvent. In the literature, solubilities of at least 10% (m / v) are assumed for PLGA-based systems formed in situ [40]. Solubility data in classical solvents such as NMP and DMSO, but also in ethyl lactate, have already been reported for different PLGA polymers [41]. These studies showed that the solubility of the polymers decreased with increasing molecular weight. Further, the amount of water necessary for in situ precipitation correlated with the solubility of the polymer in the solvent used. The better the solubility of the polymer in the solvent used, the more water was needed to form the depot matrix. In contrast, the amount of water required decreased with increasing polymer content.

First formulation and solubility tests were carried out using the model 503 H polymer Resomer ^® RG. For better visualization, the water phase stained with the blue dye brilliant blue. For all solvents tested, a 10% (m / v) polymer solution could be prepared but the films already visually showed significant differences in strength, homogeneity and incorporation of the aqueous phase into the matrix. The results are shown in FIG. As can be seen, using triacetin as the solvent, no homogeneous film could be obtained, but a W / O emulsion was formed which is clearly recognizable. All other solvents resulted in the formation of a film, with the incorporation of the aqueous phase varying significantly. Based on the staining of the matrix, incorporation of the aqueous phase decreased in the following order: triacetin <ethyl-lactate <<tetraglycol <glycerol formal-DMI.

 4 shows the incorporation of the aqueous phase into the polymer matrix with the LogP values of the individual solvents, a dependence of the film quality, which is characterized by a small loss of polymer in the supernatant, on the water solubility of the solvent used becomes clear. It can be assumed that triacetin and ethyl lactate as lipophilic solvents (XlogP> 0) have lower water miscibility than the other solvents, which explains the poor film quality of the matrix. These solvents are therefore considered only for the solution of very populist PLGA polymers. Possibly. is a mixture of solvents more suitable. While no spray film could be produced with the experimental setup using triacetin, ethyl lactate resulted in a very inhomogeneous, holey film structure in which very little dye could be incorporated. In contrast, when using glycerol formal, DMI or tetraglycol in situ films were formed. For this reason, if possible, such solvents with an XlogP value of less than 0 are preferred.

 In order to better evaluate the film quality, the amount of polymer in the supernatant (loss) and in the precipitate (implant) was quantified in spray tests by weighing back the dried matrix. If one now plots the distribution coefficient P of the solvents against the matrix quality, as shown in FIG. 4B, the film quality is linearly dependent on the water-miscibility of the solvent. The graph clearly shows that the matrix formation could be improved with increasing water-miscibility of the solvents, with about 80% of the amount of polymer used being incorporated into the matrix with a P value <0.25.

For the further experiments, different polymers were tested in combination with the solvents glycerol formal, DMI and tetraglycol. Triacetin has not been further investigated due to poor film formation and ethyl lactate due to instabilities and a holey, inhomogeneous film structure and because of its strong hemolytic activity. Tissue compatibility of the solvents

 In addition, the influence of the solvent on metabolic cell bi-lability was investigated over a period of 1 1 hours. This is determined by the ATP value of the cells, a measure of viability. In acute toxicity of substances, the value drops rapidly and thus allows an estimate of the tissue compatibility of the solvent. FIG. 5 shows the LD 50 values calculated from the experiments for all solvents tested as a function of the incubation time.

The compatibility of the solvents decreased in the following order; Glycerol formal>>DMI> tetraglycol. Glycerol formal showed the lowest toxicity of the tested solvents with an LD ₅ o value of approximately 1 g / ml with an incubation time of less than 6 h. In comparison to DMI and tetraglycol, this meant a 220-fold or 400-fold better compatibility after the end of the experiment.

Example 5

Examination of biomaterials

As biomaterials biodegradable copolymers of lactic acid and glycolic acid, poly (D, L-lactic-co-glycolic acid) (PLGA) polymers, the company Boehringer Ingelheim were investigated (trade name Resomer RG ^® ), which are already approved by the FDA for parenteral administration ,

Matrix quality depending on the selected polymer

 As described above, the percentage of polymer that can be incorporated into the film varied depending on the water solubility of the solvent used. Analogously, the matrix quality using different polymers should be considered in more detail. Since tetraglycol was not allowed to evaporate for the polymers tested, there is no data available for this solvent. Figure 2 shows the results for polymers having a composition of (a) PLA / PGA 50:50 with free acid groups (H series) and (b) PLA / PGA 75:25 with esterified end groups (S series). The latter are more lipophilic than the H series due to the higher proportion of lactic acid and the esterified end groups.

The graphs illustrate that with higher molecular weights of the polymers in both series, a larger amount of polymer could be incorporated into the matrix. This effect was significant for DMI in the H-series, and for both solvents in the S-series, 755 S almost 100% of the amount of polymer used in the matrix trained using glycerol formal and Resomer ^® RG (97.6 ± 0.6% ). Comparing the polymer series in combination with the solvents tested, glycerol formal with the S-Series showed a 20% lower loss of polymer used compared to the H series and compared to DMI in both series of polymers. These results are again explained by the different water miscibility of the two solvents, which led to a different solubility of the polymers in the solvent and significantly influenced the kinetics of film formation. While both series can be solved very well in DMI, the S-Series was difficult to dissolve in glycerol formal compared to the H-Series. The latter showed strong swelling behavior. Therefore, the S series in glycerol formal was expected to be near the solubility limit. Combined with the good water miscibility of glycerol formal, rapid and complete film formation resulted.

Viscoelastic properties of in situ formed films

 In a dynamic shear experiment, the viscoelastic properties of the films were investigated. For this purpose, an oscillating shear stress with a defined amplitude and frequency was applied to the sample and the resulting shear strain determined, which is also characterized by amplitude and frequency, also called phase shift. These two response variables can then be mathematically transformed into the memory module G "and the loss modulus G", the memory module characterizing the stored and thus recyclable portion of the introduced kinetic or deformation energy (elastic component) and the loss modulus a measure of the per-vibration into heat emitted energy and thus represents the lost portion (viscous portion). In Figure 3, both modules are shown at an excitation frequency of 1Hz for different H series films.

Comparing the elastic and the viscous portion of both films with each other, one recognizes a different sensitivity of viscoelastic properties to the various solvents. While in the case of 502 H films the viscoelastic properties by the choice of suitable solvent set quite wide left (maximum factor: 29 (G ") and 22 (G ^{^)),} showed 504 H films is independent of the solvent used rheological The latter showed a mechanical strength comparable to muscle fibers (8 -17 kPa) at 2-4 kPa [46] and was predominantly elastically dominated by a loss factor (ΰ70 ^Λ, )> 1, so that these films were similar in the experiment behaved a solid. the only exception was DMI is, were the dominant in situ films with a dissipation factor of 0.85 viscous. with a thickness of> 500 were μηι these films compared to the Resomer ^® RG 502 H films relatively thick (DMI : 500 μηι, Glycerolformal: 600 μπι, Tetraglycol: 800 μηι). The latter spread on the plate of the rheometer and showed a thickness of only 300 μηι regardless of the solvent used.

All films based on Resomer ^® RG 502 H behaved with a loss factor <1 gel-like, with significant differences in solvent strength. Only with the use of tetraglycol could a Resomer RG 504 H comparable strength of the films are achieved at a loss factor of 0.79. DMI and glycerol formal were not nearly comparable with a strength of 130 and 900 Pa, respectively, and showed strongly viscous dominated behavior (loss factor> 0.5).

Example 6

Formulation of in situ formed films

 Based on the experience gained from the spray tests already carried out, a formulation consisting of i) PLGA polymer dissolved in one of the three solvents already used, ii) aqueous phase and iii) plasmid DNA was developed as a model active substance. Theoretically, the plasmid DNA could be incorporated into the matrix both in "naked" and complexed form. However, since "naked" plasmid DNA transfected cells only very inefficiently, the plasmid DNA was complexed prior to embedding in the film with 1-PEI (N / P ratio 10) and incorporated as nanoscale polyplexes in the matrix. As a result, it was additionally able to be protected against a drop in pH within the matrix, which occurs during the degradation of the polymer structure due to the release of polymer monomers within the matrix and is generally a problem for sensitive macromolecules [47].

 The incorporation of the polyplexes into the matrix could be carried out dissolved in the aqueous phase [44] or dispersed in the PLGA solution [28, 45]. However, in preliminary experiments using the example of PLGA / tetraglycol systems, it has already been shown that a direct addition of small quantities of water (about 5%) could cause precipitation of the polymer. At high loadings of the spray film, therefore, the use of highly concentrated plasmid DNA solutions was required, which, however, have a low stability and tend to aggregate formation. It was therefore advantageous to disperse the polyplexes as lyophilisate analogously to protein formulations [28, 45] in the PLGA solution or to resuspend before use in the aqueous phase. Basically, the formulations were structured as follows:

1) The lipophilic phase (1 ml): 10% (m / v) PLGA polymer in glycerol, tetraethylene glycol, or DMI (Resomer ^® RG 502 H, 504 H)

2) Hydrophilic phase (1 ml): water for injections 3) Active Ingredient Component: lyophilized polyplexes resuspended in the lipophilic or hydrophilic phase

Production of powdery polyplexes

 Freeze-drying is one of the standard methods in the pharmaceutical industry for stabilizing formulations during storage. By including the molecules in an adjuvant matrix, formulations can be stabilized during the drying process, it being possible to use different protectors depending on the drying phase. Thus, cryoprotectants prevent crystallization of the solution during freezing. The system solidifies as a supercooled melt without complete phase separation (solidified liquid, glass). In contrast, lyoprotectants provide protection in the further course of drying by replacing the bonds of the active ingredient to the water by forming hydrogen bonds.

 Polyplexes can also be lyophilized with the addition of cryoprotectants and lyoprotectors, so that aggregation can be prevented after resuspension [48, 49]. As a lyophilisate, polyplexes can be stored better [48] and the solution can be concentrated to a plasmid DNA concentration of 1 mg / ml [50]. Sugars such as sucrose or trehalose act as lyo- and cryoprotectants and have been shown to stabilize polyplexes [48, 49]. Water-soluble substances such as these can also accelerate the release of macromolecular drugs from in situ formed PLGA based films. During the matrix formation, water-filled pores are formed by the dissolution of these substances, through which the active substance can subsequently diffuse out of the matrix. A similar effect was described for high loading of the matrix [27, 33].

Various sugars were tested in standard concentrations [48, 49]. The polyplexes based on 1-PEI were resuspended in water for injection after lyophilization without prior homogenization and their transfection efficiency was tested on human bronchial epithelial cells. Good transfection rates were achieved in the concentrations used with disaccharide sucrose with a 10-fold increase in efficiency over freshly prepared polyplexes. The results are shown in FIG. Comparable values have already been described by Talsma and colleagues [48], although osmotic effects, which could lead to an increased uptake of the particles into the cell, can only be expected from a 2-fold concentration [50]. Particle size measurements, however, showed that there was a slight increase in particle size after freeze-drying. Freshly prepared particles had a particle size of ~ 100 nm, whereas depending on the dispersing medium used, the particles grew after freeze-drying at a PI <0.2 to 200-300 nm. To be able to dose the pulverulent polyplexes and incorporate them into the formulation, these should be homogenized in a mortar after lyophilization and dispersed in the PLGA solution using Ultra-Turrax (UT) or glass homogenizer (H). The stability of the polyplexes after homogenization and subsequent resuspension in water for injection was investigated by transfection and agarose gel electrophoresis. The results of the experiments on lung cell lines showed no changes in the transfection efficiency by homogenization (FIG. 15B). Control of the topology of the plasmid DNA with addition of heparan sulfate also revealed no difference between the lyophilized polyplexes in water for injection (Figure 7).

 Both the samples resuspended in water for injections (untreated) and those polyplexes that were pre-homogenized (resorbed) prior to resuspension showed comparable banding patterns for all three dispersion methods (UT, homogenizer, dissolved). Compared to the control, uncomplexed plasmid DNA, a slight increase in the relaxed form was observed in all cases. Degradation of the plasmid DNA was not evident for water for injection.

 Exemplary of the solvents Glycerolformal is shown. There was no difference in banding pattern between untreated or pre-homogenized samples and water control. Using the homogenizer as the dispersion method, no change was observed either. In the case of Ultra-Turrax, the pDNA remained mostly complexed in the pockets of the gel, with untreated samples showing two very weak bands comparable to the other samples. However, it should be noted that using equal amounts of heparan sulfate generally under the influence of Glycerolformal larger amounts of pDNA complexed remained in the pockets. For the homogenised UT samples, a slight smear was visible in the gel, but destruction of the plasmid DNA in the form of fragments could not be observed here either.

Example 7

Determination of the release kinetics of formulated plasmid DNA polyplexes

The release of active substances from implants can in principle be effected by i) diffusion of the active substance from the polymer matrix (diffusion-controlled) or ii) by erosion of the matrix (erosion-controlled) [51, 52]. For the release of active substances from bulkerodierenden polymer matrices as in the case of PLGA systems depending on the drug loading, the molecular weight of the polymer used, and the polymer concentration one or two-phase release profiles are described [53, 54]. In addition, initial release of the active agent may occur until complete precipitation of the polymer. The release kinetics of films formed in situ was investigated as a function of the molar mass of the polymer, of the solvent used and of the embedded variant. The following combinations were tested:

1. Lipophilic phase (1 ml): Resomer ^® RG 502 H or 504 H to 10% (m / v) in glycerol formal, tetraethylene glycol or DMI

 2. Hydrophilic phase (1 ml): water for injection

 3. Active component: lyophilized polyplexes (1-PEI / pDNA) in homogenized form

After the lyophilization, the polyplexes were homogenized in a mortar and either resuspended in the hydrophilic phase (embedding variant A) or dispersed in the lipophilic PLGA solution by means of a homogenizer (embedding variant B). The Freisetzungspro- füe are shown for Resomer ^® RG 502 H and 504 H using different solvent for both variants embedding in FIG. 6

Shown diagram (A): Resomer ^® RG 502 H, polyplexes in hydrophilic phase diagram (B): Resomer ^® RG 502 H, polyplexes in lipophilic phase diagram (C): Resomer ^® RG 504 H, polyplexes in a hydrophilic phase, chart (D): Resomer ^® RG 504 H, polyplexes in lipophilic phase.

If one first considers the incorporation of the polyplexes into the film by dissolution in the hydrophilic phase, a clear influence of the molecular weights of the polymers used and of the solvents used on the release kinetics is evident (FIGS. 6A, C). Incorporation of the active component was using DMI as a solvent in combination with the short chain polymer Resomer ^® RG 502 H not possible (Fig. 6A). During the precipitation of the polymer already 100% of the polyplexes were released. Using the longer chain polymer, the Resomer ^® RG 504 H, was achieved, however, a typical two-phase release pattern. This was composed of an initially diffusion-controlled release (phase 1, concave release profile) followed by an erosion-controlled release (phase 2, linear kinetics) (FIG. 6C). It was noticeable in this context that using DMI in all cases, the highest initial release was achieved, which varied from 10% to 100% depending on the embedding variant and the chain length of the polymer.

In contrast, glycerol formal showed a low initial, followed by a slow diffusion-controlled release. It was only with the beginning of erosion of the matrix erosion that an accelerated release of the polyplexes was observed, which began 15 or 26 days, depending on the chain length of the polymers used. By contrast, films based on tetraglycol showed a moderate initial release of 32% (Resomer RG 502 H) and 50% (Resomer RG 504 H) a moderate to no release in the observed time frame. Only in the case of the longer-chain polymer was it due to the erosion of the matrix to a low release after 26 days (Fig. 6C).

 On the other hand, when the polyplexes were dispersed in the lipophilic PLGA solution (embedment variant B), the initial release rates of the polyplexes as well as the diffusion from the matrix were reduced for all polymer / solvent combinations and the release was mainly erosion-controlled (FIGS. 6B, D). , The tested solvents showed similar release curves with varying degrees of retardation, the release rates increasing in the following order: tetraglycol <<glycerol formal <DMI. While significantly shorter erosion rates for the short-chain 502 H polymer compared to the long-chain 504 H polymer could be seen in the release profile for DMI and glycerol formal (day 17 versus day 29), in the case of tetraglycol no difference between the polymers was possible due to the strong retardation of the matrix (5.4% versus 8.8% cumulative release after 30 days).

In summary, DMI showed the fastest release for all combinations of long or short chain polymer and the different embedding variants. Continuous release up to 100% drug release was achieved with Resomer ^® RG 504 H by incorporation of the drug component into the hydrophilic phase. Polyplexes incorporated in tetraglycol-based films showed no diffusion-controlled release. Over the observed period, an additional 14% of the pDNA amount could be released after initial release, whereby the initial release varied between 0 and 48%. This was relatively high in the case of embedding variant A, whereas no initial release was observed when the polyplexes were dispersed in the lipophilic phase. Films based on glycerol formal showed a low initial release irrespective of the embedding method, although the polyplexes could not be released until after 23 days by erosion of the matrix even when these films were used.

 Example 8

 Investigation of transfection efficiency in vitro

First in vitro release profiles were determined as described above using the reporter gene luciferase. Based on these data and the requirements of the dosage form the polymer Resomer ^® RG 504 H was selected in combination with DMI as a solvent for the film. Both active components should be incorporated in lyophilized form in the hydrophilic phase of the matrix, which was expected from the preliminary experiments with an initial release of active ingredient of 55%. This appears for use in the Range of postoperative adhesions makes sense to cover the acute phase after surgery (day 2-5) [79-81], while avoiding an unphysiological increase in tPA levels due to increased bleeding tendency in the peritoneum. Alternatively, a film with no initial release, composed of Resomer ^® 504 H and glycerol were tested. Again, the polyplexes were dissolved in the hydrophilic phase. The formulations were first tested in vitro using the active component 1, which was complexed with 1-PEI and lyophilized with the addition of 10% sucrose. As a control, the pMetLuc plasmid, which codes for a luciferase enzyme secreted by the cell, was additionally used in a 1: 1 mixture.

 For in vitro testing in the cell assay, mesothelial cells (Met5A) were cultured on inserts and the polymer film was subsequently sprayed onto the cell layer. The use of the inserts made it possible to divide the wells into a two-chamber system with outer and inner compartments comparable to the on-site anatomy in the peritoneum, between which a constant mass transfer was possible, so that the cells could be supplied with medium from the apical and basolateral side. An optical control of the cell morphology was carried out by light microscopy, but was complicated by the sprayed on film. The expression of the reporter gene luciferase could be examined by using the inserts in both compartments over a period of 30 days.

Shown in FIG. 10 is the upper compartment. Compared to the bolus dose in which the entire plasmid DNA dose was administered without delivery system in water for injection purposes, the films formed in situ showed a lesser amount of luciferase by a factor of 10 ³ to 10 ⁴ . The course of gene expression was as described above. In these release kinetics studies, films based on DMI had shown an initial release of 56% of the amount of pDNA used and subsequently a further 38% to day 26 release. Following this release profile, an increased gene expression was observed for the DMI-based film after only 2 days, which dropped to basal levels after a further 7 days and increased again to moderate levels by day 23. In contrast, when glycerol formal was used as the solvent, gene expression was biphasic without initial release of the polyplexes. After a first moderate increase in the expression rate within the first 10 days, another peak with a 2-fold increase in gene expression was detected after 23 days. Low molecular weight fragments in the cell culture medium indicated an incipient erosion.

The tPA expression from the matrix consisting of glycerol and Resomer ^® RG 504 H is shown in comparison to the single dose and at a film without active ingredient component (inactive film) in Figure 10A. For the single administration of the active substance component Without the depot system, very high protein levels were obtained over a period of 29 days, analogous to luciferase expression, whereby a 100 to 40-fold increase in the tPA concentration compared to the basal values could be achieved. Similar concentrations were achieved after intraperitoneal administration of recombinant tPA (alteplase) in plasma [82]. The values that could be achieved using the matrix formulation therefore appear much closer to the physiological conditions. For plasma and peritoneal fluid, free tPA antigen concentrations of 4 to 6 ng / ml are described [82- 84], with an additional 1.3 ng / ml tPA bound to PAI-1 [84]. It is astonishing that even by applying the inactive film the values increased slightly, whereby at the beginning of the release a 4-fold increase could be achieved by the active film (with embedded active ingredient component) in comparison to the inactive one. The effect leveled out by day 16 and tPA levels dropped to basal levels at 2 ng / ml. This is probably due to the fact that barely more polyplexes could be released from the matrix by day 15 (see chapter 7.3). At the end of the experiment this was not completely eroded, so that embedded polyplexes could be detected in the spray film (FIG. 10 B).

 Example 9

 Increase of tissue plasminogen concentration by co-application of siRNA and pDNA

 The increase in the extracellular tPA concentration by application of a tPA-encoding plasmid was successfully demonstrated on mesothelial cells. The extent to which an increase in the tPA / PAI-1 ratio can be achieved by simultaneous application of an siRNA to PAI-1 will be examined below.

Previous studies had shown that 1-PEI is particularly well suited for the in vivo application of siRNA and plasmid DNA [23]. Therefore, pDNA / siRNA / l-PEI polyplexes were prepared at an N / P ratio of 10 (based on pDNA concentration) in HBS and different siRNA sequences were tested against PAI-1. The tPA PAI-1 ratio for application of different pDNA / siRNA combinations is shown in FIG. The siRNA sequence was used, which inhibited the PAI-1 expression in preliminary experiments most efficiently (PAI-1 A), with an optimized concentration of 0.12 μΜ. 48 h after transfection, the tPA / PAI-1 ratio when co-applied to the untreated cells increased to 8-fold, with the application of pDNA alone or in combination with a non-functional siRNA (EGFP siRNA) only an increase of the 4 - or 5 times could be achieved. Using a placebo plasmid (pUC), the siRNA was found to increase the tPA / PAI-1 ratio by a factor of 2. Example 10

 The development of an in situ formed film for the controlled release of poly- plexes was based on an application system from Baxter, as shown in FIG. The two-syringe system was loaded with a lipophilic component (syringe 1) consisting of a biodegradable polymer dissolved in an organic solvent and an aqueous component (syringe 2), water for injections. As the active ingredient lyophilized pDNA / l-PEI polyplexes were used. These could then be dispersed in the lipophilic phase or dissolved in the hydrophilic phase. Both embedment variants were examined for release kinetics as above.

 Example 11

Glycerol formal showed the best tolerability on mesothelial cells in the cell viability test, with LD ₅₀ values being 200 and 400 times higher, respectively, than DMI and tetraglycol. These were below 6 g incubation time at about 1 g / ml, ie with an application of about 780 μΐ pure Glycerolformal die off 50% of the mesothelial cells. Literature data indicate similar LD50 values after iv administration in rodents for DMI and glycerol formal, whereas tetraglycol is more toxic by a factor of 2-3. According to the manufacturer to Ivomec ^®, a veterinary drug against parasites, an LD50 of 4 to 4.8 g / kg body weight (mouse) with iv administration of a 50% strength by weight refer Glycerolformallö- solution. The EMA describes this in a final report by the Committee for Veterinary Products [85]. The acute toxicity after intravenous administration of DMI hardly differs from glycerol formal. With an LD ₅₀ of 5.4 g / kg body weight (rat) with application of 40% DMI in isotonic saline solution (v / v) and an LD ₅₀ of 6.9 g / kg body weight (mouse) with application of a 20% Solution seems even better tolerated DMI after intravenous administration. Tetraglycol has been used as a solvent for parenterals (iv, im) since the 1960's in concentrations up to 50% (v / v) and is considered to be non-irritating at this dilution. The LD50 after intravenous administration without dilution is 3.8 g kg body weight (mouse) lower than described for the other two solvents [86].

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Claims

 claims

 Spray system for generating an in situ formed matrix, the

a) at least one lipophilic component containing at least one based on glycolic acid and lactic acid polymer and at least one biocompatible solvent having an XlogP3 value of less than 0.2, and

b) has at least one hydrophilic component,

wherein the at least two components are separate from each other prior to use and are first mixed for or during spraying, wherein the components form a film when sprayed onto human tissue.

 Spray system according to claim 1, characterized in that it additionally contains an active ingredient which is dissolved in one of the two components or in both components and / or dispersed.

 Spray system according to one of the preceding claims, characterized in that the lipophilic component contains a PLGA polymer and a biocompatible solvent for the PLGA polymer.

 Spray system according to one of the preceding claims, characterized in that the biocompatible solvent has an XlogP3 value of 0.2 to -1.0.

 Spray system according to one of the preceding claims, characterized in that the biocompatible solvent is selected from tetraglycol, dimethylisorbide and / or glycerol formal.

Spray system according to one of the preceding claims, characterized in that the solvent has an LD ₅₀ of at least 1 mg / ml.

 A spray system according to any one of the preceding claims, characterized in that the polymer is a poly (D, L-lactide-co-glycolide) polymer in which the ratio of lactide to glycolide is between 25:75 and 75:25; and / or that the polymer is a PLGA having an intrinsic viscosity number of 0.16 to 0.70 dl / g; and / or that the PLGA polymer has a molecular weight, as measured by gel permeation chromatography, of 10 to 63 kDa.

 Spray system according to one of the preceding claims, characterized in that PLGA and biocompatible solvent are selected so that 5 to 60 parts of PLGA per 100 parts of solvent are dissolved.

Spray system according to one of the preceding claims, characterized in that the hydrophilic component is water, optionally with dissolved or dispersed active ingredient therein.

10. Spray system according to one of the preceding claims, characterized in that the film formed after spraying has a degradation time at the applied location of 2 to 6 weeks.

 11. Spray system according to one of the preceding claims, characterized in that the active substance contains at least one nucleic acid, wherein the nucleic acid preferably RNA, DNA, mRNA, siRNA, miRNA, piRNA, shRNA, antisense nucleic acid, aptamer, ribozyme, catalytic DNA and / or a mixture of the preceding ones.

 12. Spray system according to the preceding claim, characterized in that it contains a nucleic acid which codes for a fibrinolytic factor, preferably tPA.

 13. Spray system according to one of the preceding claims, characterized in that the active substance contains a plasminogen activator inhibitor inhibiting agent.

14. Spray system according to the preceding claim, characterized in that the PAI inhibiting agent is an siRNA.

 15. Spray system according to one of the preceding claims, characterized in that the nucleic acid is complexed with a carrier; wherein the carrier is preferably a carrier with positively charged groups, more preferably polyethyleneimine.

 16. Spray system according to one of the preceding claims, characterized in that the film formed has a two-phase release kinetics.

 17. Spray system according to one of the preceding claims, characterized in that polyplexes are contained with an N P ratio of 1 to 10.

 18. Spray system according to one of the preceding claims, characterized in that tPA-encoding DNA and PAI inhibitor in a ratio of 5: 1 to 1: 5 are included.

 19. Spray system according to one of the preceding claims for use for application to peritoneum, in particular for use for the prevention of surgical adhesions and scarring.