ITMI20081056A1 - NANOSPUGNE BASED ON CYCLODESTRINE AS SUPPORT FOR BIOLOGICAL CATALYSTS AND IN THE VEHICLE AND RELEASE OF ENZYMES, PROTEINS, VACCINES AND ANTIBODIES - Google Patents

Description

Description of the patent for industrial invention entitled:

"CYCLODEXTRIN-BASED NANOSPONGES AS SUPPORT FOR BIOLOGICAL CATALYSTS AND IN THE VEHICULATION AND RELEASE OF ENZYMES, PROTEINS, VACCINES AND ANTIBODIES"

The present invention relates to the use of cyclodextrin nanosponges as supports for enzymes, proteins, vaccines or antibodies, a process for the preparation of enzymes supported by such nanosponges and catalysts obtainable from this process.

State of the art

Proteins perform numerous functions, such as the transport of oxygen, the maintenance of the structure of some tissues, the reception and transduction of signals in the cell, the immune response and catalysis in metabolic processes. This last function is performed by enzymes, which are classified into six classes on the basis of the generic reaction they catalyze.

Many industrial chemical transformation processes have operational disadvantages. Non-specific reactions lead to low yields, the need to often operate at high temperatures and pressures requires the consumption of high amounts of energy and in the "downstream" process a very large amount of water for cooling. The reactors themselves must often be built in materials resistant to the drastic reaction conditions of temperature, pressure, acidity or alkalinity with a consequent increase in plant costs.

All these disadvantages can be eliminated or greatly reduced by using enzymes as biological catalysts. In fact, they operate under mild reaction conditions, have a high reaction rate and are highly specific. Given their efficiency, only small amounts of the enzyme are required to transform large volumes of reagents. They have a beneficial effect on the environment as they reduce energy consumption and reduce the production of pollutants.

Developments in genetic engineering have made it possible to increase the stability, economy and specificity of enzymes and the number of their industrial applications is constantly increasing.

Examples of industrially useful enzymes include alpha amylase, trypsin, cellulase and pectinase for the clarification processes of fruit juices, ligninase to degrade lignin, lipase in the detergency industry and in the production of biodiesel, etc.

The enzymes, like all the catalysts, can be used either in the homogeneous phase (homogeneous catalysis) or in the heterogeneous phase (heterogeneous catalysis).

In the first case, the catalyst is in the same (liquid) phase as the reactants and reaction products. Homogeneous catalysis is characterized by a high selectivity, a great activity, a constant reproducibility. The main disadvantages of this technique consist in a short life of the catalyst, in its possible poisoning and also in the difficulty of separating the reaction products from the catalyst and in the recycling of the latter, which is always problematic.

In heterogeneous catalysis, on the other hand, the catalyst is in a different phase than the one hosting reactants and reaction products. In this case the separation of the reaction products is easy and it is possible to work in a continuous flow. However, less selectivity and activity of the enzyme used is often observed.

The catalytic performance of the heterogenized enzyme depends on both the substrate used and the immobilization techniques. Since the catalytic activity significantly depends on the correct orientation of the active site, the immobilization of the enzyme must be performed in such a way as to minimize changes to the catalytic site. The surface on which the enzyme is supported is responsible for the retention of the enzyme structure and the micro reaction environment that is created can also modify the fields of use of the enzyme, for example, to widen the operating range of pH.

Three immobilization techniques are known:

1) interactions with supports (physical adsorption, ionic bond, covalent bond);

2) crosslinking;

3) entrapment.

In particular, physical adsorption is usually the simplest method to immobilize enzymes. The enzyme is linked to the support through hydrogen, saline and Van der Waals bonds. However, since these interactions are weak, desorption of the enzyme is often observed in relation to the change in temperature, pH, ionic strength or even due to the substrate used.

It is also well known that proteins, peptides, enzymes and their derivatives can be used in the biomedical and therapeutic fields. Proteolytic enzymes can be used to treat cancer or to treat type I mucopolysaccharidosis, while DNA and oligonucleotides are used for example in gene therapy. The administration of these molecules presents various problems and limitations. Most of the protein drugs are in fact poorly absorbed through biological membranes due to the combination of various factors such as high molecular size, hydrophilic nature, ionicity, high surface charge, both chemical and enzymatic instability and low permeability through the mucous membranes. The molecules of a protein nature, following intravenous administration, show a rapid blood "clearance", a significant ability to bind to plasma proteins and a remarkable sensitivity towards proteolytic enzymes. The oral route has the drawback of poor bioavailability. For use in therapy there are different approaches, such as an increase in the dose or the use of absorption promoters, which can cause toxicity problems, or alternative routes of administration. Various systems have been developed for the delivery of enzymes and proteins, such as for example nano- and micro-particles, liposomes, hydrogels. The delivery in a particulate system can protect proteins from degradation, modify their pharmacokinetics and improve their stability in vivo.

Cyclodextrins (CDs) are non-reducing cyclic oligosaccharides made up of 6-8 glucose molecules linked with 1,4-α-glucosidic bond, having a characteristic truncated cone structure. The arrangement of the functional groups of the glucose molecules is such that the surface of the molecule is polar while the internal cavity is relatively lipophilic.

The lipophilic cavity gives the CD the ability to form stable inclusion complexes even in solution with organic molecules of suitable polarity and size.

For this reason, CDs have been studied and have numerous applications in various fields (pharmaceuticals, analytics, catalysis, cosmetics, etc.) in which the characteristics of inclusion compounds are exploited.

In these applications, however, native or chemically modified cyclodextrins are generally used, but never insoluble cross-linked polymers.

Nanosponges are cross-linked cyclodextrin polymers with different bonds that have proved very useful in various applications ranging from environmental decontamination to the delivery and controlled release of drugs.

Description of the invention

It has now been found that cyclodextrin nanosponges are a particularly suitable support for the adsorption of proteins, enzymes, antibodies and macromolecules in general. Using enzymes in particular, it was possible to preserve their activity and efficiency, prolong their operation over time, extend the pH and temperature range of activity as well as allow the conduct of processes in continuous flow. Furthermore, it is possible to convey proteins and other macromolecules by adsorbing or encapsulating them in cyclodextrin nanosponges. The interaction between proteins and nanosponges allows the formulation of prolonged and controlled release systems over time.

The nanosponges usable according to the invention are described for example in WO 2006/002814, prepared by reacting natural cyclodextrins with organic carbonates in the absence of solvents and irradiating or not with ultrasound. Surprisingly it was possible to obtain nanosponges both structurally amorphous and with a significant degree of crystallinity (Figure 1a and 1b). Other types of nanosponges can also be used, obtainable by reacting a cyclodextrin with a polyisocyanate-type polyfunctional crosslinker, as described in WO 98/22197.

A new class of nanosponges containing polyamidoamine bonds have also been used as supports, obtainable for example by the reaction of cyclodextrins with bis acrylamides, the synthesis of which is described in more detail in example 1. The synthesis is preferably carried out in the presence of ultrasounds.

The immobilization of enzymes on said nanosponges includes putting an aqueous solution of enzyme in contact with the cross-linked nanosponges for a period of time ranging from 10 to 720 minutes at temperatures between 4 and 40 ° C. The aqueous solution is suitably buffered at pH between 5 and 9.

The invention concerns both the immobilization process and the enzymes immobilized on cross-linked cyclodextrin nanosponges.

Examples of enzymes that can be advantageously immobilized according to the invention include oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase, in particular amylase, trypsin, lipase, catalase, cellulase, ligninase, pectinase, protease and other enzymes of industrial interest.

The quantity of enzyme immobilized on the nanosponges essentially depends on the incubation time of the polymer solution with the nanosponges, as well as on the nature of the same and on the temperature. In principle, the immobilization yields can be adjusted within various limits, for example from 1 mg to 50 mg per gram of nanosponges. The enzymatic activity is maintained over time for several days and is also more resistant to temperature and pH conditions, thus allowing the carrying out of more efficient enzymatic reactions.

The invention is described in greater detail in the following Examples.

Example 1 - Synthesis of polyamidoamine nanosponges (Figure 2)

0.66 ml of distilled water, 339 mg of 2,2'-bis (acrylamide) acetic (BAC) and 84.7 mg of LiOH.H2O were introduced into a 10 ml flask, supplied with nitrogen flow. agitation and in a nitrogen atmosphere. After obtaining a clear solution, 573 mg of β-cyclodextrin (βCD) and another 63.5 mg of LiOH.H2O were added. The reaction was kept under nitrogen flow for 10 minutes, after which the nitrogen inlet was closed, leaving the reaction to continue under an inert atmosphere, in darkness, at room temperature for 94 hours. Once the reaction is complete, the crude product appears as a cross-linked polymer that has been triturated and swollen with an excess of water for 10 minutes under stirring, then it has been centrifuged, to remove traces of base from the resin. The resin was swollen again in excess of water and subsequently acidified with 1M HCl up to pH 2.5. The acidified resin was purified by washing three times in distilled water with vigorous stirring, followed by centrifugation. Traces of water were extracted with ethanol and leaving the product under vacuum for two days. The final product was obtained as an odorless, fine, white powder. The swelling degrees of the resin in three buffer solutions of pH 2, 4.5 and 7 were evaluated and reported in table 1. The resin was characterized by FT-IR spectrophotometry in KBr tablets and the main functional groups were identified as follows : 707 cm <-1> (secondary dwamide NH), 947 cm <-1> (d C-O-H outside the plane), 1030 cm <-1> (n C-N aliphatic), 1087-1301 cm <-1> (n C-O-C ), 1520 cm <-1> (d secondary amide NH, n C-N), 1650 cm <-1> (n secondary amide C = O, n C = C) of d, 2921 cm <-1> (n asymmetric CH2 ), 3385 cm <-1> (n OH).

The same reaction can be carried out with other stoichiometric ratios and with other types of crosslinkers such as methylene bisacrylamide or methylpiperazine or their mixtures, also using other natural cyclodextrins (alpha and gamma) or their derivatives (e.g. monotosylcyclodextrin, monoazido cyclodextrin, etc.). quickly operating in the presence of ultrasound. Nanosponges with magnetic properties can also be obtained by carrying out the synthesis in the presence of magnetic particles, for example magnetite.

Example 2 - Loading of enzymes on nanosponges

The best immobilization yield was obtained from the cross-linked support described in WO 2006/002814 with molar ratio between β-cyclodextrins and cross-linking agent 1: 2.

To establish the best immobilization conditions, 0.3 mg of 1,2-dioxygenase enzyme was immobilized on 1.0 g of both types of cross-linked media (1: 2 and 1: 4) at two different temperatures (4 ° C and 22 ° C). The support was previously washed with the same solution where the immobilization took place (50 mM Hepes pH 8.0).

The amount of immobilized enzyme was evaluated by the difference between the total one loaded on the nanosponges and that not bound to the resin and present in the washing solutions. The quantities of enzyme were calculated by measuring the activity on the catechol and obtaining the amount in mg of enzyme from the specific activity (activity / mg of protein).

The experiment was conducted by incubating the enzyme and the nanosponges for different times and calculating the immobilization yield.

Figure 3 shows the yield of immobilization obtained after incubation of enzyme and 1: 2 cross-linked support at different times at 22 ° C.

Once the enzymatic loading conditions were established, it was possible to immobilize the enzyme by absorption up to a load capacity of 28.9 mg of protein per gram of support.

Example 3 - Production of muconic acid

The 1,2-dioxygenase enzyme catalyzes the transformation reaction of catechol into muconic acid. This reaction was used to evaluate the activity of the immobilized enzyme. The reaction was followed for 60 seconds through the increase in absorption at 260 nm, due to the formation of the reaction product, muconic acid. The reaction mixture contained 20 µl of wet resin according to WO 2006/002814 with the immobilized enzyme, 1 mM of catechol in 50 mM Hepes pH 8.0 buffer. The activity assays were carried out at 30 ° C in a cuvette with an optical path equal to 1 cm. The absorption spectrum of catechol and muconic acid are shown in Figure 4.

The formation of the reaction product was followed through separation in HPLC, as shown in Figure 5. Reverse phase separation with LiChrospher 100 RP-18 5 µm 250x4 (Merck) column was used. The mobile phase consisted of water and acetonitrile in a 60:40 ratio.

For verification purposes, the two standard solutions of catechol and muconic acid were injected from which it results that the retention time for muconic acid is approximately 2.7 minutes, while for catechol it is approximately 6.7 minutes. The solution leaving the bioreactor was then injected. The presence of a peak at the same retention time confirms the presence of the reaction product. The UV spectrum of the product was compared with that of standard muconic acid and reveals the presence of the characteristic absorption peak at 260 nm.

From the chromatogram (Figure 5) it is evident that the conversion of catechol into muconic cis-cis acid is almost total. Table 2 reports the kinetic constants of the catalyzed reaction.

Example 4 - Enzymatic activity over time

A column (10 x 2.5 mm) was packed with 1 g of hyper crosslinked support described in WO 2006/002814 on which 0.3 mg of catechol 1,2-dioxygenase were immobilized.

At regular intervals (1 day), a 1 mM catechol solution was injected into the bioreactor and, after 1 hour, the column was washed with 50 mM Hepes buffer pH 8.0. The presence of the reaction product leaving the bioreactor was monitored spectrophotometrically and by HPLC analysis. The quantification of the reaction product was carried out using a molar extinction coefficient at 260 nm equal to 17600 M <-1> cm <-1>.

Through the adsorption immobilization tests, a greater stability over time of the protein immobilized on the column was obtained. The enzyme was able to reconvert the catechol into muconic cis-cis acid for about 72 hours as can be seen from the graph in Figure 6.

Example 5 - Influence of temperature on enzymatic activity To investigate the temperature stability of the enzyme immobilized on the hyper-crosslinked support of WO 2006/002814 and of the enzyme in solution, activity analyzes were carried out by incubating the enzyme (20 µl of wet resin with the immobilized enzyme) for 3 minutes in 50 mM Hepes pH 8.0 buffer in a temperature range from 10 ° C to 70 ° C. After this incubation, the residual activity of the enzyme was verified by adding the substrate and monitoring the formation of the product.

As shown in Figure 7, the immobilized enzyme has the greatest activity around 50 ° C and still maintains 70% of activity at 60 ° C.

The optimal temperature range for the enzyme in solution is instead between 30 ° C and 40 ° C. After incubation at 60 ° C, the almost total loss of activity of the enzyme in solution is observed. To monitor the stability of the enzyme catechol 1,2 dioxygenase at two different temperatures, 40 ° C and 60 ° C, both the activity of the immobilized protein and that of the protein in solution were measured at different incubation times at the three selected temperatures. (Figures 8 a and 8 b).

The incubation of the immobilized enzyme in solution was carried out in 50 mM Hepes pH 8.0 buffer at 2 different temperatures. At regular intervals 20 µl of wet resin with the immobilized enzyme were taken and the residual activity measured by adding the substrate and monitoring the formation of the product.

As shown in Figures 7, 8a and 8b, the immobilization process gives the enzyme greater heat stability. For the enzyme in solution there is complete loss of activity already after incubation of the enzyme for 90 minutes at 40 ° C, while the immobilized enzyme after 90 minutes still has 50% of its initial activity. Only after 3 hours there is a complete loss of the enzymatic activity of the immobilized protein. The profile of the loss of the immobilized enzyme activity at 40 ° C shows a biphasic behavior which is probably due to the presence of two catalytic sites stabilized in a different way by the immobilization process. Since so far no cases of allosterism between the two monomers of the enzyme catechol 1,2-dioxygenase have been reported in the literature, this behavior may probably be due to the immobilization process that can create two non-equivalent Iron centers. Figure 8b shows the enzyme activity profile after incubation at 60 ° C. The complete loss of activity is obtained after 15 minutes for the enzyme in solution, while at the same time the immobilized enzyme still has 70% of its initial activity.

Example 6 - Influence of pH on enzymatic activity

To monitor the behavior of the enzyme catechol 1,2 dioxygenase at different pH values, both the activity of the immobilized protein and that of the protein in solution in the pH range of 5.5-10 were measured. The enzyme (20 µl of wet resin referred to in example 1 with the immobilized enzyme) was incubated for 3 minutes in the various buffer solutions at a determined pH value at 30 ° C and subsequently the residual activity was evaluated .

The buffers used are: Mes (for pH 5.5 to 7), Hepes (for pH 7 to 8.5) and Ches (for pH 8.5 to 10). The buffer concentration was appropriately chosen to keep the ionic strength constant.

As shown in Figure 9, the activity profiles are different, which shows that the immobilization process affects the catalytic site of the enzyme. The optimal pH for the enzymatic activity of the immobilized protein is 9.5, while the highest activity for the enzyme in solution is recorded at a pH of 8.5. Furthermore, as evidenced by the first part of the curve, the immobilized enzyme already has an activity of 40% at pH 6.5, while the protein in solution appears to have only 8% of activity with respect to the point of maximum activity.

Example 7 - HRP activities

To confirm the ability of the support to retain other enzymes by adsorption, the commercially available horseradish peroxidase (Sigma Aldrich) was tested.

0.3 mg of enzyme were incubated with the nanosponges of WO 2006/002814 and for 62 hours at 4 ° C in a potassium phosphate buffer solution pH 7.4.

The concentration of the protein was estimated from the absorption at 402 nm using a molar extinction coefficient of 93500 M-1 cm <-1> (Keilin and Hartree, 1951) and the immobilization yield was calculated from the difference between the amount of protein incubated and amount of protein not bound to the support.

The enzymatic activity was measured on the substrate 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) following the formation of the cationic radical ABTS. + In the presence of H2O2e peroxidase at 414 nm (figure 10) . The reaction mixture was composed of 20 µl of wet resin with the immobilized enzyme, 1 mM ABTS, 15 µM H2O2 in phosphate buffer 50 mM pH 7.4.

Table 1. HRP load on different NS

Table 2. Kinetic constants of 1,2-dioxygenase

Example 8 - Immobilization of lysozyme

5 ml of a solution of 200 ppm of lysozyme were incubated with increasing and known quantities of nanosponge referred to in example 1 at room temperature for 24 hours. At the end of this time the suspension was centrifuged at 3000 rpm and the supernatant analyzed by UV-Vis spectrophotometer at 281 nm. From the observed absorbance, knowing the initial one, the amount of lysozyme adsorbed by the nanosponges was calculated by difference. The results are shown in Figure 12 from which it is evident that NS have a very high affinity for lysozyme and are able to bind over 80% of the protein, a percentage that tends to increase as the concentration of protein in solution increases.

Example 9 - Delivery and release of albumin

50 mg of albumin are incubated with 50 mg of nanosponge overnight under stirring in 5 ml of water. Then the mixture is lyophilized. A solid product is obtained which contains 50% albumin. A dissolution test was used to evaluate the release kinetics of albumin from nanosponges. 25 mg of nanosponges of example 1 containing 12.5 mg of albumin are suspended in 25 ml of phosphate buffer at pH 7.4. The sample is left under stirring for 24 hours at 37 ° C. At predetermined times, 0.5 ml of sample are withdrawn and replaced with 0.5 ml of buffer. The sample taken is centrifuged and the supernatant analyzed with a spectrophotometer at a wavelength of 278 nm. The results are shown in Figure 11.

Claims (8)

CLAIMS 1. Cross-linked cyclodextrin nanosponges in crystalline or amorphous form as supports for enzymes, antibodies, proteins, vaccines and macromolecules in general. 2. Nanosponges according to claim 1 as an enzyme carrier. 3. Nanosponges according to claim 1 or 2 wherein the cyclodextrins are crosslinked with polyamidoamines in the presence or absence of ultrasounds. 4. Process for the immobilization of enzymes which includes putting an aqueous solution of enzyme in contact with the cross-linked nanosponges for a period of time ranging from 10 to 720 minutes at temperatures between 4 and 40 ° C. 5. Process according to claim 4 wherein the aqueous solution is buffered at pH between 5 and 9. 6. Enzymes supported on cross-linked cyclodextrin nanosponges. 7. Enzymes according to claim 6 selected from oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase. 8. Enzymes according to claim 7 selected from amylase, trypsin, lipase, catalase, cellulase, pectinase, ligninase, protease.