Crosslinked polysaccharide beads and their biomedical uses

Abstract

The present inventions relates to beads as biocompatible material adapted for use within the human or animal body. Said beads are highly useful for tissue engineering, in situ tissue regeneration, as well as for drug and/or cells delivery. In addition, said beads may support biotechnological applications such as cell carriers.

Claims

1. A crosslinked polysaccharide bead having a spherical or ovoid shape obtained by a) preparing an alkaline aqueous solution comprising at least two polysaccharides and a cross linking agent; b) in the absence of surfactant, dispersing said alkaline aqueous solution into an hydrophobic phase consisting of a vegetal oil, in order to obtain a water-in-oil (w/o) emulsion; and c) transforming the w/o emulsion into beads by placing said w/o emulsion at a temperature from about 4 C. to about 80 C. for a sufficient time to allow the cross-linking of said at least two polysaccharides; wherein said polysaccharides are selected from mixtures of dextran and pullulan, or mixtures of dextran, pullulan and fucoidan, wherein said crosslinked polysaccharide bead is free of any organic solvent and surfactant, and wherein said crosslinked polysaccharide bead has a mean diameter of more than 10 m.

2. The bead according to claim 1, wherein the vegetal oil is selected from canola oil, corn oil, cottonseed oil, safflower oil, soybean oil, extra virgin olive oil, sunflower oil, palm oil, MCT oil, and trioleic oil.

3. The bead according to claim 2, wherein the vegetal oil is canola oil.

4. The bead according to claim 1, wherein the polysaccharide is a mixture of pullulan/dextran.

5. The bead according to claim 4, wherein the mixture of pullulan/dextran is in a ratio of 75:25 w/w.

6. The bead according to claim 1, wherein the cross-linking agent is selected from the group consisting of trisodium trimetaphosphate (STMP), phosphorus oxychloride (POCl.sub.3), epichlorohydrin, formaldehydes, carbodiimides, and glutaraldehydes.

7. The bead according to claim 1, wherein the alkaline aqueous solution further comprises a porogen agent.

8. The bead according to claim 7, wherein the porogen agent is selected from the group consisting of sodium chloride, calcium chloride, ammonium carbonate, ammonium bicarbonate, calcium carbonate, sodium carbonate, sodium bicarbonate and mixtures thereof.

9. The bead according to claim 1, wherein the alkaline aqueous solution further comprises hydroxyapatite.

10. The bead according to claim 1, wherein the alkaline aqueous solution further comprises a drug.

Description

FIGURE LEGEND

(1) FIG. 1: Macroscopic view of beads as macro/microcarriers. The top panel evidences the beads after crosslinking (left), after freeze-drying (middle), and after hydratation (right). The bottom panel is an example of beads observed on confocal microscopy incubated with vascular cells that attached to the bead surfaces for further use in vitro in a bioreactor, or in vivo for cell delivery. FITC-dextran was used in the composition of the beads and cells were previously labeled with TRITC-phalloidin for identification on the bead surface.

(2) FIG. 2: The morphology of freeze-dried porous heads was analyzed by scanning electron microscopy (left, scale bar: 50 m). After rehydradation in PBS, hydrated beads were observed with Environmental Scanning Electron Microscopy (ESEM Philips XL 30, Netherlands) (right, scale bar: 200 m).

(3) FIG. 3: Fluorescent porous beads prepared with FITC-dextran were observed with a fluorescent microscope before size calibration (left, size ranging from 20 m to 1 mm) and after calibration (middle, diameter: 620 m). Cell infiltration inside porous beads was assessed using confocal microscopy (right). Cells were identified by labeling with TRITC-phalloidin. Cells infiltrated the beads and were observed within the pores (mean diameter of beads: 660 m)

(4) FIG. 4: Macroscopic views and confocal images of injectable porous microbeads. On the top left panel, beads were labeled with alcian blue to evidence the beads coming out of the needle. Different beads were prepared that varied in their size: 222 m, 977 m and 16423 m.

(5) FIG. 5: Fluorescent beads were injected subcutaneously without leakage into a female C57 black mouse using a G25 gauge needle (left). Subcutaneous tissue was excised 24 hours later for histopathology analysis (right). Beads remained at the site of implantation and were visually assessed in the subcutaneous sample.

(6) FIG. 6: Fluorescent beads (arrows) were observed on 8 m sections of subcutaneous tissue 24 hours after injection (left, fluorescent microscopy). Beads (arrows) were also observed on alcian blue/nuclear red stained sections (right, light microscopy). Scale bars: 100 m.

(7) FIG. 7: Polysaccharide bead according to the invention covered with Fibroblast 3T3 after 2 days of culture.

EXAMPLES

(8) Preparation of Macro/Micro Beads

(9) A water-in-oil (w/o) emulsification process was performed to obtain polysaccharide micro/macro beads.

(10) Beads were prepared using a blend of pullulan/dextran 75:25 (pullulan, MW 200,000, Hayashibara Inc; Dextran MW 500,000, Pharmacia), prepared by dissolving 9 g of pullulan and 3 g of dextran into 40 mL of distilled water.

(11) Chemical cross-linking was carried out using trisodium trimetaphosphate STMP (Sigma) under alkaline condition. 100 L of 10M sodium hydroxide was added to 1 g of the polysaccharide blend, followed by the addition of 100 L of water containing 30 mg of STMP. This polysaccharide/NaOH/STMP mixture was then dispersed into 100 mL of canola oil under mechanical stirring for 10 min.

(12) The w/o emulsion was then cross-linked at 50 C. for 20 min. Resulting heads were collected by centrifugation (2000 rpm, for 3 min), washed extensively with PBS (10) then 0.025% NaCl solution, calibrated according to their size using nylon filters and freeze-dried for 24 h until complete removal of water (FIG. 1, top).

(13) Human endothelial cells cultured with beads can be observed on their surface (FIG. 1, bottom). For confocal analysis, FITC-dextran was used and cells were labeled with TRITC-phalloidin.

(14) In another experiment, (w/o) emulsification process was conducted using a high performance disperser (Polytron Homogenizer) in order to obtain smaller beads (<50 m).

(15) Preparation of Porous Macro/Micro Beads

(16) In another experiment, porous microbeads could be prepared. In this process, porogen agent such as NaCl (14 g) was added into the polysaccharide solution before cross-linking.

(17) The resulting macro/micro beads were further characterized by confocal analysis and electronic microscopies. Beads prepared with NaCl as a porogen agent were porous (FIG. 2). The morphology of freeze-dried porous beads was evidenced by scanning electron microscopy (FIG. 2, left). After rehydradation in PBS, hydrated porous beads were observed with Environmental Scanning Electron Microscopy (FIG. 2, right)

(18) Interestingly, using a process of crosslinkinging, porous micro or macrobeads were obtained (FIG. 3, left). By changing the experimental conditions, micro/macro beads of different sizes could be obtained from 1 m to a size higher than 1 mm. Polytron equipment provided beads with a size comprised between 1 and 30 m, while using mechanical stirring provides beads with a size comprised between 10 m and 1 mm (FIG. 4).

(19) Through calibration, the inventors have met the burden to obtain a large scope of beads with different size. More precisely, the inventors have obtained polysaccharides heads according to the invention of a size of: 100 to 200 m; 200 to 300 m; 300 to 500 m; 500 to 700 m; and 700 to 1000 m.
Cell Infiltration

(20) The Inventors assessed cell infiltration inside porous microbeads using confocoal microscopy. After incubation of beads with human endothelial cells, the cell infiltration inside the FITC-labeled porous beads was thus observed (FIG. 3, middle and right panels).

(21) Injection of the Beads

(22) The microbeads of the invention have a size highly appropriate for injection through a needle (FIG. 4), and allow a depot of a bead suspension. Fluorescent beads (100-200 m) were injected subcutaneously into female C57 black mice using a G25 gauge needle, with no immediate nor late leakage observed (FIG. 5). No reaction was observed at the injection site 24 hours later. After sacrifice, subcutaneous tissue was excised and analyzed with histopathology techniques.

(23) Beads were observed on 8 m sections, either using a fluorescence microscope (FITC-labeled green beads and red autofluorescence background) or a light microscope following alcian blue/nuclear red staining protocols (FIG. 6).

(24) Incorporation of Nano-Hydroxyapatite

(25) Nano-hydroxyapatite (n-HA) was prepared by wet chemical precipitation using a 0.6M solution of Phosphoric acid (H3PO4 Rectapur, Prolabo, France) and a 1M solution of calcium hydroxide (CaOH2 Alfa Aesar, Germany). The suspension of n-HA was included in the alkaline solution of polysaccharides in the starting solution at a 6% w/w. The resulting polysaccharide macrobeads contained n-HA dispersed in the 3D structure of the beads. The inventors have then discovered bone formation after implantation of the said beads in a condyle defect in rats.

(26) Preparation of Polysaccharide Beads According to the Invention with a Thrombolytic Agent:

(27) Beads according to the invention were prepared in the range of 1 to 10 microns containing tPA (American Diagnostica, tPA single-chain recombinant tissue plasminogen activator (tPA)).

(28) 9 g of pullulan, dextran 3 g, 1.2 g of fucoidan and 14 g NaCl in were mixed in 40 ml of water. 300 mg of the solution were isolated and 30 L, of 10 M NaOH were added before mixing. 30 L, of STMP (300 mg dissolved in 1 mL of STMP water) were then added. The mixture is injected into the oil (30 ml of canola oil, 0.35 mg of Span 80+Tween 80 0.12 mg) and the mixture was stirred with polytron (small propeller) at full speed for 2 minutes. The breaker is then put in an oven at 50 C. for 20 minutes. After removing the beaker from the oven, the mixture is balanced in 10 PBS under magnetic stirring for 30 min. The supernatant was removed and two rinses in 0.01% SDS, 3 rinses (1 h) in saline 0.025% NaCl were performed. Finally, a lyophilisation step is performed.

(29) The inventors obtained different types of beads with the following composition of polysaccharide: 75% pullulan+25% dextran 25% DEAE pullulan, 75% neutral pullulan 50% pullulan+(25% dextran, 25% neutral dextran) 25% DEAE pullulan, 75% neutral pullulan, 50% DEAE dextran, 50% neutral dextran.

(30) The activity was then assessed with 5 mg of beads in Eppendorf tubes. For this purpose, 50 L of t-PA (200 UI/mL) or beads according to the invention containing t-Pa were incubated 1 hour, and rinsed 3 times with 500 L of buffer solution (PBS-0.1% HSA-0.01% Tween 20).

(31) Colorimetric measurement after adding 50 L of 2 mM substrate in S444 demonstrated that more than 30% of native t-PA activity was maintained in the beads.

(32) Infiltration of Cells on Polysaccharide Heads According to the Invention

(33) The seeding of the cells is performed in sterile 1.5 ml eppendorf tubes. In each of those tubes, 4 mg of lyophilized beads are placed within each tube. A cell suspension is prepared, containing from 2 to 310.sup.6 cells in 15 to 20 L of a culture medium for each of the eppendorf tubes. The cell suspension is placed within each eppendorf tube, which are then incubated for 30 min. 500 L, of culture medium is then added in each tubes. The tubes are then incubated for 2 h at 37 C. in order to optimize the adhesion of the cells on the beads. The beads are then transferred in culture wells. 500 L of culture medium is added to each wells. The cells are thus infiltrated in the beads of the invention and cultivated the appropriate time.

(34) The inventors performed said techniques for having heads infiltrated with: fibroblasts 3T3 (see FIG. 7), human umbilical vein endothelial cells, human mesenchymal stem cells, or lewis rat mesenchymal stem cells.

(35) The inventors thus obtained polysaccharide bead comprising cultivated cells.