FIGURE 4
Fractional release of drugs from hydrogels in PBS buffer
Slow controlled release of theophylline in PBS at room temperature from hydrogels prepared with BSA and PEG of various molecular masses
Operational stability of acid phosphatase immobilized in BSA-PEG, Mr 10,000, in presence of 10 mM of p-nitrophenylphosphate (flow rate of 1,25 ml/min).
-i—i—i—|—i—i—i—i—r—r~r—i—i i—r—i—r
I I [ I I I I I I I I I I I I I I,....J, I I L_J I I I I 1.. ,1 , I.
100 200 300 400 500 600 700
ALBUMIN BASED HYDROGEL
This is a continuation of application Ser. No. 08/159,559, filed on Dec. 1, 1993, which was abandoned on the filing hereof of application Ser. No. 08/591,941, filed on Jan. 23, 1996.
FIELD OF THE INVENTION
This invention relates to the preparation of high water content bioartificial hydrogels obtained from the crosslinking of albumin type proteins, such as bovine serum albumin (hereinafter referred to as BSA) with bifunctionalyzed polyethylene glycol, or other suitable bifunctionalyzed polyethylene oxide, of various molecular masses, in different proportions, in an aqueous solution.
BACKGROUND OF THE INVENTION
The term "hydrogel" refers to a class of polymeric materials which are extensively swollen in a aqueous medium, but which do not dissolve in water. In general terms, hydrogels are prepared by polymerization of a hydrophylic monomer under conditions where the polymer becomes cross-linked in a three dimensional matrix sufficient to gel the solution. Bioartificial or semi-synthetic hydrogels may also be prepared by the covalent addition of the hydrophylic polymer to the surface of a protein so that the polymer and protein form a further covalently cross-linked three dimensional matrix. This class of hydrogels made from a synthetic polymer and a biopolymer has been recently reviewed and it has been proposed to name this new class of biomaterials as bioartificial polymeric material or hydrogel (Giusti, P et al, Trends in Polymeric Science, (:261-267, 1993).
The usefulness and multiplicity of desirable applications of hydrogels in medicine and other areas as well as their composition and methods of their preparation have been well documented. For example, in a two volume book series, Hydrogels in medicine and pharmacy, vol. 1 & 2, N. A. Peppas ed. CRC Press publ., USA, 1986; or other books such as Polymer in Medicine, R. M. Ottenbrite and E. Chiellini, Technomic publ., USA, 1992, as well as several scientific publications such as: The design of hydrogel for biomedical applications, by P. H. Corkhill, C. J. Hamilton and B. J. Tighe in Crit. Rev. Biocompatibility, 5: 363^135, 1990; Bioartificial Polymeric Materials: a new method to design biomaterials by using both biological and synthetic polymers, P. Giustic, L. Lasseri and L. Lelli, Trends in Polymeric Science, 9: 261-267, 1993.
Some of the more notable biomedical applications of hydrogels include contact lenses, non-toxic in-vivo controlled drug release implants, wound dressings, cell growth matrices. Novel or improved hydrogels having improved properties, applications or ease of preparation are of course required.
The present invention concerns a novel bioartificial hydrogel prepared from the cross-linking of a polyethylene oxide, or derivative thereof, preferably polyethylene glycol (hereinafter arbitrarily referred to in its abbreviated form as PEG), with albumin type protein.
With the benefit of the above described background of hydrogels, the known prior art will now be addressed.
PRIOR ART
With regards to bioartificial hydrogels, methods for the covalent addition of hydrophylic polymers such as monomethoxy-polyethylene glycol (hereinafter referred to
2
as mPEG) to the surface of a protein have been successfully developed using a variety of reagents (review in Biotechnol. Appl. Biochem. 17: 115, 1993; TIBECH, 4: 190, 1986 & 6: 131, 1988). mPEG has generated a great deal of interest
5 when it was first used to modify the surface of proteins and enzymes in the aim of reducing their immunogenicity and to increase their blood clearance time for potential in vivo applications (J. Biol. Chem, 252:3578,1977 and 252:3582, 1977). Such soluble modified proteins or enzymes have
10 shown an increase in their structural resistance against proteolytic action of proteases (J. Ferment. Bioeng., 71: 137-139,1991), an increase of their rate of adsorption by the gastro-intestinal tractus (Biol. Chem. Hoppe Seyler 101: 3715, 1990) and an increase of their cellular influx (Proc.
15 Natl. Acad. Sci. USA, 84: 1487, 1987). Such modified enzymes have also shown a good activity as microparticulate suspensions in organic solvents (Enzyme Microb. Technol., 14: 96-100, 1992; TIBECH, 4: 190, 1986 & 6: 131, 1988). mPEG can even be used as a blood substitute
20 when covalently bound to the protein heamoglobin (J. Prot. Chem. 10: 61-67, 1991). When the surface of a material is covered by mPEG, the protein adsorption on this surface is reduced (2nd Congress on Biomaterials, USA 1984) as well as the complement activation and the platelet adhesion
25 (Biomaterials, 13: 113-118, 1992).
Recently, the preparation of a gel using a mixture of copolymerized acrylic derivatives of chymotrypsin and polyethylene oxide acrylate derivatives has been described in the literature (Int. J. Peptide Protein Res. 38: 273-277,
30 1991). The gel was utilized successfully in organic solvents for peptide synthesis. However, the authors do not offer any mention regarding the characterization of the protein aggregates obtained using this procedure. The protaneous gel cannot be considered as an hydrogel since the reactions were
35 performed in an organic medium as opposed to an aqueous medium.
A detailed review on the chemistry of polyethylene glycol and its biotechnological and biomedical applications is found in (Polyethylene glycol chemistry, Biotechnological
40 and biomedical applications, Milton Harris ed. Plenum Press, 1992) as well as the book entitled Hydrogels in medicine and pharmacy, ed. by N. A. Peppas, especially the chapter 4 of Volume 2 which focuses on poly(ethylene oxide) and related hydrogels. Many PEG based hydrogels
45 are therein described without any mention of cross-linking of PEG with a protein. If is demonstrated that PEG can be considered as an useful polymer and hydrogel by itself for many biomedical uses because of its specific and advantageous properties: it is a non thrombogenic material, it
50 presents a low toxicity, it is approved by FDA (Food & Drug Agency) as polymer in foodstuffs and pharmaceutical products used via injection, topical or oral routes. When PEG was converted into a cross-linked network (an hydrogel), it passed regulatory requirements. One of the most popular
55 polymer for hydrogel synthesis was for some time the polyHEMA which unfortunately suffered biocompatibility problems which ultimately restricted its usage.
Presently the majority of hydrogels are prepared from a mixture of polymer or block copolymer cross-linked by
60 radiation or chemical reagent or simply prepared by polymerization and reticulation of a monomeric solution. One important drawback arising from these methods are the inherent difficulties in achieving reproducibility in specific porosity and degree of cross-linking which ultimately affect
65 many characteristics of the resulting hydrogel. Control of the chain length of the polymer used, the hydrophylicity, and the molecular masses of the copolymer (cross-linker) along
3
with controlled mild aqueous reaction conditions, are the key to success in achieving reproducibility, size pore control, water swelling control, and geometry control of the hydrogel.
U.S. Pat. No. 4.101380, the specification of which is incorporated herein by reference, discloses a very wide variety of reagents useful to activate polyethylene oxide in the aim of obtaining a bifunctionalyzed polyethylene oxide or polyalkene oxide. When those reagents are used to cross-link PEG with a gelatin preformed membrane, a cross-linked gelatin-PEG membrane was obtained and was characterized by an high liquid swelling capacity. However, other embodiments described in the patent provided very low yield of protein cross-linking (in the order of of about 2%). In said patent, there is no mention or suggest that advantageous hydrogels could be obtained by cross-linking of an albumin or an other native protein in an aqueous solution with activated polyethylene oxide. Moreover, said patent states that the use of a carbonate derivative of polyethylene oxide is not recommended and not useful should attempts be made to obtain cross-linking of the polymer with a protein or enzyme. This is explained as being due to the high pH required for the subsequent cross-linking reaction which could induce denaturation of enzymes or proteins.
The present invention demonstrates in sharp contrast that the use of a carbonate polyethylene oxide derivative should be very useful for the preparation of cast hydrogels having reproducible specific geometries as required, for example, when making contact lenses or devices for controlled drug release. Although the pH of reaction is higher than 9 during cross-linking with a protein or enzyme, the present invention confirms that PEG carbonate derivatives are useful reagents for enzyme or protein modifications which prevent denaturation of many enzymes (catalase, peroxidase, superoxide dismutase, acid phosphatase, glucose oxidase, lysozyme, and asparaginase).
U.S. Pat. No. 4,161,948 discloses synthetic membranes (artificial skin) for wound dressings. Said patent suggests that when preparing the membranes, it is preferable that the 5-amino acid polymers employed be cross-linked with a diol such as polyoxyethylene glycol, in order to achieve properties resembling those of natural human collagen. Polypeptides as a source of polyamino functional groups were not mentioned in said patent. According to said patent, 8-amino acid polymers are prepared by a polymerisation process which results in a statistically distributed polymer in a wide range of molecular weights. The fabrication of the artificial skin described in said patent is obtained via a complex multi step process. It is obvious, from the disclosure of said patent, that the inventors, are experiencing great difficulty in controlling the porosity and the cross-linking of the synthetic membrane and are resorting to a complex method.
U.S. Pat. No. 4,752,627 discloses a contact lens which contains polymerized ethylene glycol moieties as side chains to the major polymer (HEMA, polyvinyl pyrrolidone) components. Said patent explains that the use of PEG as a side chain component contributes to the ability of the lens to resist to the absorption of proteins present in human tears. Depending on the amount of the hydrophobic cross-linker used to prepare the hydrogel, an equilibrium water content varying from 18 to 60% was obtained. Such a degree of water swelling is unfortunately not sufficient to allow free diffusion of oxygen similar to the corresponding rate in a solution.
U.S. Pat. No. 4,814,182 discloses the preparation of a controlled release device based on a hydrogel coated with an
4
impermeable layer on at least one side of the device. The hydrogel was obtained from the polymerization of block copolymers comprising both hydrophylic and hydrophobic domains, preferably polyethylene oxide methacrylate which
5 reacted with a substituted or unsubstituted cyclic mono or polyether having from 3 to 6 ring atoms or cyclic imine. The use of a protein as a polyamino groups source is not mentioned in said patent.
U.S. Pat. No. Reissue 33,997 discloses a contact lens
10 based on the cross-linking of collagen with an ethylenicaUy unsaturated compound of varying length from one to six carbons. The hydrogel described therein displayed good biological stability to protease digestion and protein fouling (adsorption).
15
SUMMARY OF THE INVENTION
Unexpectedly, a family of novel hydrogels has been discovered. The novel hydrogels are polymerized hydro
20 phylic water-swellable gels essentially consisting of a crosslinked mixture of: (a) a bifunctionalized polyethylene oxide, activated with a suitable activating agent, dissolved in an aqueous solution; and (b) albumin type protein albumin. The novel hydrogels are based on the cross-linking of a protein,
25 namely albumin of various sources including, for example, BSA, with a bifunctionalyzed polyalkene oxide preferentially polyethylene oxide, and most preferably polyethylene glycol, or a mixture of bifunctionalyzed polyalkene oxides of various molecular masses (Mr 2,000 to 35,000) dissolved
30 in aqueous .solution, in adequate proportions.
The present invention also provides a method and conditions for preparing the novel hydrogels.
A variety of biomedical applications for the novel hydrogels are also envisaged and method for preparing the prod
35 ucts related to those applications are claimed. Additionally, it has been found that the mechanical properties of the novel hydrogels can be improved by adding to the casting solution unreactive PEG or other inert polymers of high molecular masses (Mr>100,000).
40 Accordingly, the novel hydrogels possess many advantageous properties such as shape retention and shape memory, they can reach very high water content (more than 94% (w/w) based on the dry weight of the hydrogel). The novel hydrogels also possess good mechanical and optical prop
45 erties. The hydrogels further possess more characteristics which may render them extremely useful in the pharmaceutic and medical areas due to their advantageous properties such as biocompatibiliry, resistance to proteases action, slow release of various drugs, hydrophylic surface, good oxygen
50 permeability, controlled porosity, and other desirable properties.
These and other objectives can be achieved by practising the teachings disclosed herein. It is to be noted that specific 55 examples relating to the possible applications of the novel hydrogels will be described and do not represent an exhaustive enumeration and consequently do not limit all other possible applications of the novel hydrogels which are to be contemplated and understood.
60 BRIEF DESCRIPTION OF THE DRAWINGS
These and other advantages of this invention will be readily apparent when considered in light of the following description when considered in light of the drawing set forth 65 with comprises the following figures:
FIG. 1 is a graphical representation of the effect of the molecular masses of PEG (horizontal scale) on the gelling
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