Polymeric composite materials with antimicrobial and biodegradable properties and uses thereof

Abstract

A composite material for the production of a medical device having an antiseptic action includes a matrix of alginate in which the complex of iodopovidone is dispersed. The composite material is used particularly for the production of films, micro-capsules, and suture threads with iodine controlled release.

Claims

1. A medical device with antiseptic action, configured as a self-supporting film comprising a matrix of water soluble alginate in which a complex of povidone-iodine (PVPI) is dispersed as an antiseptic agent, said self-supporting film being a substantially anhydrous composition consisting of said water soluble alginate, said complex of povidone iodine, and a plasticizer; wherein said water soluble alginate is present in an amount of 55% to 80% by weight, and said povidone iodine is present in an amount of 45% to 20% by weight, wherein the amount percentages are relative to a total amount of said alginate and povidone-iodine, and wherein said plasticizer is present in an amount of 20% to 35% by weight relative to the total amount of said alginate and povidone-iodine.

2. The medical device according to claim 1, wherein the matrix is sodium alginate or potassium alginate.

3. The medical device according to claim 1, configured as a film, having antiseptic activity and adapted for the release of PVPI.

4. The medical device according to claim 1, the device consisting of 48% wt-57% wt sodium alginate; 20% wt-25% wt glycerol; and 18% wt-32% wt PVPI; wherein the amounts of sodium alginate, glycerol and PVPI add up to 100%.

5. The medical device according to claim 1, wherein said self-supporting film is obtained by mixing sodium alginate in water, heating to about 100 C., to ensure complete dissolution of the alginate in water, cooling to room temperature, adding said plasticizer, further adding a PVPI aqueous solution, pouring dropwise the obtained solution on a substrate and maintaining at room temperature to dry.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the appended drawings:

(2) FIG. 1 shows photographies of films after 48 incubation with the following compositions: pure NaAlg (a); NaAlg 70%, 30% glycerol (plasticizer) film (b); NaAlg 57%, 25% glycerol, 18% PVPI film (c), 52% NaAlg, 22% glycerol, 26% PVPI (d), 48% NaAlg, 20% glycerol, 32% PVPI; two identical films were incubated in plastic Petri capsules containing Candida albicans fungi for all the experiments.

(3) FIG. 2 shows in the box (A) the growth curves for E. coli in water (line with rhombi), in water containing beads of CaAlg (line with squares), in water containing beads of CaAlg/PVPI (line with triangles); in the box (B), the growth curves for C. albicans in the presence of sodium alginate beads (line with squares), and iodopovidone beads (line with triangles) are shown; and

(4) FIG. 3 is a dynamic release scheme for 1 g PVPI (line with rhombi) and from 8 g (line with squares) of calcium alginate beads in water; PVPI release is quicker (within 15 minutes) in the solution containing 1 g of beads than that containing 8 g.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Example 1Preparation of a Sodium Alginate/PVPI Film

(5) For the preparation of the composites according to the invention, the following materials were prepared: alginic acid sodium salt having a viscosity of 15000-20000 cps, glycerol99.5 (GC) and anhydrous sodium chloride (all purchased from Sigma-Aldrich); 100 ml of PVPI solution in water containing 10% PVPI, 1% of which was active iodine purchased from Pharmatek PMC.

(6) Three different NaAlg-PVPI films were produced to carry out antibacterial and antifungal tests. The films were prepared as follows: 10 ml distilled water with 0.3 g NaAlg were mixed and heated by using a hot plate during 1 hour up to about 100 C., to ensure the complete dissolution of alginate in water.

(7) Next, the viscous solution was let to cool to room temperature, and 0.1 ml glycerol as the plasticizer was added. The amount of added glycerol was kept constant in all the successive preparations of the solution. Different volumes (1.0 ml, 1.5 ml, and 2.0 ml) of a PVPI aqueous solution were slowly mixed to the NaAlg/glycerol solutions.

(8) The final solutions were mixed by using a Vortex mixer and left to rest for a period of time to ensure the absence of bubbles in the vials. By using a pipette, the prepared solutions were poured dropwise on substrates of 1 cm.sup.2 and left at laboratory room temperature to dry.

(9) According to the above-mentioned procedure, the following films were prepared: film 1: 57% NaAlg, 25% glycerol, and 18% PVPI; film 2: 52% NaAlg, 22% glycerol, and 26% PVPI; film 3: 48% NaAlg, 20% glycerol, and 32% PVPI. Percentages are expressed by weight.

Example 2Preparation of Calcium Alginate Microcapsules (Beads) and of Sutures Containing PVPI

(10) By way of example, the above-mentioned solution, which was used for the production of the film 3 (48% NaAlg, 20% glycerol, and 32% PVPI), was used for preparing CaAlg/PVPI beads and suture threads, respectively.

(11) For the production of the beads, the NaAlg/PVPI solution was introduced by dipping dropwise or, respectively, for the production of suture threads, it was continuously supplied by a syringe in a 10% calcium chloride solution in distilled water. The formation of the beads and the suture threads was immediate; however, the so-obtained products were left in the calcium chloride solution during 30 minutes to ensure a high cross-linking.

(12) Next, the thus-formed materials were rinsed with distilled water to ensure the complete removal of calcium chloride traces and let to dry overnight. The cross-linking time may be varied in order to obtain a higher or lower cross-linking, as a function of the desired application.

Example 3Test to Assess the Antibacterial/Antimycotic Activity of Sodium Alginate/PVPI Films

(13) In these tests, the activity of NaAlg/PVPI films coated on slides against two different microorganisms, particularly, Gram-negative bacterium E. Coli and fungus C. albicans known as the agent responsible for oral and genital opportunistic infections in humans was tested. The above-mentioned coated slides, herein below referred to as substrates, were also tested with different concentrations of bacteria and fungi to better determine the antimicrobial characteristics thereof.

(14) In order to verify the antibacterial properties of the sodium alginate/iodopovidone films, an E. Coli culture incubated overnight with a final concentration of 810.sup.8 cells/ml was diluted to final concentration of 10.sup.6 cells/ml, and 500 L of this solution were deposited on a plate with fresh Luria-Bertani (LB) culture medium.

(15) Subsequently, the plates were placed in an incubator at 37 C. for two hours (to allow the evaporation of the residual culture medium) and the different substrates (two for each LB plate) were placed. To assess the antifungal activity of the sodium alginate/iodopovidone films, the initial inoculum of 10.sup.8 cells/ml was diluted to 10.sup.5 cells, and 100 L of this solution were deposited on a fresh Sabouraud dextrose agar (SDA) culture medium. Next, the plates were placed in the incubator at 37 C. for two hours and two different substrates were placed on each of them.

Analysis of the Antibacterial Activity of the Films

(16) The sodium alginate/PVPI films with the following compositions (a) pure sodium alginate (b) 70% NaAlg, 30% glycerol (plasticizer); (c) 57% NaAlg, 25% glycerol, 18% PVPI; (d) 52% NaAlg, 22% glycerol, 26% PVPI; (e) 48% NaAlg, 20% glycerol, 32% PVPI were prepared and tested after 24 hours incubation; films with the following compositions were tested after 24 hours incubation: (f) 57% NaAlg, 25% glycerol, 18% PVPI; (g) 52% NaAlg, 22% glycerol, 26% PVPI and (h) 48% NaAlg, 20% glycerol, 32% PVPI.

(17) At the end of the incubation period of 24 hours, the formation of a bacterial film on both coatings was observed. On the films containing 18% PVPI, the formation of a thin inhibition area free from bacteria without the formation of a bacterial biofilm upon or around the films was observed after 24 hours incubation.

(18) Similarly, for those films containing 26% and 32% of the PVPI complexes, no growth upon or around the films was detected after 24 hours, with the formation of inhibition areas around the films. Samples were incubated during 48 hours to study the longevity of the antibacterial effect obtained after 24 hours. For films containing 18%, 26%, and 32% PVPI complexes, no bacterial growth was observed upon or around the films without significant changes of the inhibition areas around them.

(19) The PVPI complex is very active even when it is incorporated in the NaAlg matrix. Furthermore, the formation of an inhibition area around all the films containing PVPI complexes indicates that PVPI is released from the NaAlg matrix, thus making these compounds not only surface active, but also inhibitory.

Analysis of the Antimycotic Activity of Sodium Alginate/PVPI Films

(20) With reference to the activity of the prepared films against Candida albicans, after 24 hours incubation, it was observed that the control samples comprising pure sodium alginate and 70% NaAlg, 30% glycerol did not show any antifungal activity. In fact, a growth of fungi Candida albicans is noticed upon and about the substrates. However, when the samples containing the PVPI complex were incubated, no growth was detected upon and around the films. Furthermore, it was observed that there was drug release during the incubation process, since Candida albicans was not able to grow not even in those regions far from the films, due to the formation of a large inhibition region.

(21) Similar results were obtained after 48 hours incubation of the NaAlg/PVPI films.

(22) Films of pure NaAlg and glycerol-plastified NaAlg were both invaded by the fungi, as seen in FIGS. 1 (a) and 1 (b), while the films containing the PVPI complex show areas on the surface thereof that are free from fungi, as well as a large inhibition area around them, as shown in FIGS. 1 (c), 1 (d), and 1 (e).

(23) Experimental tests carried out with NaAlg/PVPI films show a higher efficiency against Candida albicans fungi than against E. coli bacteria, creating larger inhibition areas around the coated films.

Example 4Test for Verifying the Antibacterial/Antifungal Activity of Calcium Alginate/PVPI Beads

(24) Microcapsules of calcium alginate/PVPI obtained according to the procedure of the example 2 by depositing droplets of NaAlg/PVPI aqueous solutions in a container containing an excess of CaCl.sub.2 dissolved in water were tested for their antibacterial and antimycotic activity. The obtained beads have an average dimension of about 3 mm. The calcium alginate beads allow the complete encapsulation of the PVPI complex in water, while the same amount of PVPI complex is immediately released in water after the dropwise addition.

(25) The antibacterial/antimycotic properties of the calcium alginate/iodopovidone beads were tested by a turbidity assay. Particularly, a C. albicans culture incubated overnight and which reached confluence was diluted to an OD.sub.600 of 0.1, then 1 g of calcium alginate/PVPI beads was added to the culture, and the optical density at 600 nm was measured every 30 minutes with a spectrophotometer (Thermo Scientific).

(26) The same turbidity assay was performed with E. coli, but, in this case, 12 g of calcium alginate beads were added to the bacterial culture.

(27) As shown by the results in FIG. 2A, the growth curves of fungi C. albicans which contacted the aqueous solution containing 1 g of CaAlg beads are similar to those of the control, consisting in water only. On the contrary, the curve relative to fungi growing with 1 g of calcium alginate/iodopovidone beads shows that the cell population never starts to grow, thus indicating the cell death. The same results were obtained with E. coli cultures as shown in FIG. 2B, but a higher amount of sodium alginate/iodopovidone beads, i.e., 12 g, was necessary to stop the bacterial growth. This is because the minimum bactericidal concentration of 210.sup.3 M iodopovidone was higher than the respective minimum fungicidal concentration of 110.sup.3 M.

Quantification of PVPI Release from Calcium Alginate/PVPI Beads

(28) The dynamic release of PVPI from calcium alginate beads was measured by dipping a different number of freshly prepared beads corresponding to 1 g and 8 g in water, then monitoring absorbance at =360 nm at time intervals of 5 minutes. In this manner, the amount of PVPI encapsulated in the beads is calculated, then the amount thereof that is necessary to reach the minimum bactericidal concentration (MCB) and the minimum fungicidal concentration (MFC) of PVPI within each culture is calculated.

(29) As shown in FIG. 3, the release from the solution containing 1 g of beads reaches more quickly the release plateau (within 25 minutes) compared to a solution containing 8 g of CaAlg/PVPI beads.

(30) Furthermore, by constructing a calibration curve of PVPI in water, it was possible to quantify the amount of drug within the beads. Particularly, 1 g of beads contains about 1.710.sup.4 M PVPI, while 8 g of beads incorporate about 1.410.sup.3 M PVPI. This element, in association with the data for MBC and MFC, allowed calculating the amount of CaAlg/PVPI beads necessary to stop the growth of C. albicans and E. coli corresponding to 1 g and 12 g, respectively.

(31) The controlled release characteristics of the beads, combined with the antiseptic properties, biocompatibility and biodegradability thereof, make such beads particularly suitable in clinical applications as internal and external antiseptic fillers for wounds, and do not require removal interventions.

(32) The threads obtained by the procedure of the example 3 can be used as suture threads for surgical sutures. By virtue of the incorporation of PVPI in the alginate matrix, such sutures have antimicrobial properties suitable to significantly reduce the infection risk of the wound after surgery.

(33) Furthermore, since such sutures are also biodegradable, they are ideal for those patients who do not need to be re-hospitalized for suture removal. Biodegradation times turn out to be sufficient to allow healing, while maintaining the required bonding function.

(34) Furthermore, the described composite materials find application as antimicrobial coatings, or as packaging materials for food and biomedical industries, as well as acting as purification and sterilization materials against bacterial contaminations.

LEGENDA

(35) Time=tempo Release=rilascio