Patent Application
20130029397
The present disclosure relates generally to biomineralization of a membrane at the cell surface of living cells. More particularly, the present disclosure relates to producing cell-surface directed and associated non-naturally occurring bioinorganic membranes with living cells.
During evolution, some classes of living cellular organisms developed the ability to manipulate inorganic materials. Diatoms, a large class of eukaryotic unicellular algae believed to have originated prior to the Jurassic period, are one such organism. Diatoms have cell walls comprised of silica and are capable of forming diverse inorganic and hybrid materials with unique functionality and complex nano and micro-scale architectural features. In general, organisms capable of forming bioinorganic membranes (such as diatoms) form these membranes by producing a matrix (generally a protein matrix) which serves as a template for the deposition of the bioinorganic membrane and by manipulating the chemical composition of cellular microenvironments.
To date, artificial deposition strategies for classes of organisms which are evolutionary distinct from organisms such as diatoms have yet to produce bioinorganic membranes which rival the functionality and structural features possessed by the cell walls of diatoms. Traditionally, cell immobilization methods result in cell entrapment within bulk materials, creating significant diffusion barriers hindering survival of the cell. Also, current technology (generally based on passive silica deposition) creates thin coatings with poor mechanical strength around the cells which are brittle amorphous structures that degrade and are poorly resistant to various physiological fluids over time.
Further, current artificial deposition strategies result in the formation of membranes having indiscriminate pore morphology which tends to cause slower molecular diffusion into and out of the cell. Pore morphology, however, is an important feature for the viability of cells having associated cell surface bioinorganic membranes. For cells to remain viable, the associated bioinorganic membrane must allow the free diffusion of small molecules while excluding the passage of other large molecules and cells.
Therefore, it would be desirable to have a method for producing and associating a non-naturally occurring bioinorganic membrane with a cell surface of a living cell which allows for the design and control of pore morphology.
The present disclosure provides methods for forming a bioinorganic membrane by attaching at least one polypeptide to a surface of at least one cell, wherein the cell does not form a bioinorganic membrane in nature, and wherein said at least one polypeptide associates with a non-naturally occurring bioinorganic material, wherein said non-naturally occurring bioinorganic material is rich in silica.
In certain embodiments, the cell is a eukaryotic cell, a pancreatic beta cell, or a prokaryotic cell, such as a prokaryotic cell from one species of the genus Pseudomonas.
In certain embodiments, the method further includes exposing the cells to a biomineralization solution, wherein the solution is mildly acidic and rich in silica. The biomineralization solution may be low in methanol and formed by hydrolyzing tetramethyl orthosilicate in an acid.
The present disclosure also provides methods for forming a bioinorganic membrane by attaching a polypeptide to a surface of a bio-film, wherein the bio-film does not form a bioinorganic membrane in nature, and wherein said at least one polypeptide associates with a non-naturally occurring bioinorganic material, wherein said non-naturally occurring bioinorganic material is rich in silica.
In certain embodiments, the bio-film is a surface of a pancreatic islet.
In certain embodiments of these methods, said polypeptide is attached directly to the surface of the cell or the bio-film. For example, the polypeptide may be bound, link, or associated with at least one group, wherein said at least one group is part of the cell or the bio-film.
In other embodiments of these methods, said polypeptide is attached indirectly to the surface of the cell or the bio-film. For example, wherein the surface of the cell or the bio-film is attached to a ligand, wherein the ligand includes a reactive group, wherein the reactive group of the ligand binds to an intermediate group, and wherein the intermediate group includes a first portion and a second portion, the first portion of the intermediate group may be attached to the reactive group of the ligand and the second portion of the intermediate group may be attached to said polypeptide.
The present disclosure further provides a bio-structure including at least one cell, at least one polypeptide, and a non-naturally occurring bioinorganic membrane. A first portion of said polypeptide may be attached either directly or indirectly to a surface of the cell. A second portion of said polypeptide is associated with a form of silica, wherein said polypeptide and the form of silica form part of the non-naturally occurring bioinorganic membrane.
The present disclosure still further provides a bio-structure including at least one bio-film, at least one polypeptide, and a non-naturally occurring bioinorganic membrane. A first portion of said polypeptide may be attached attached either directly or indirectly to a surface of the bio-film. A second portion of said polypeptide is associated with a form of silica, wherein said polypeptide and the form of silica form part of the non-naturally occurring bioinorganic membrane.
In certain embodiments, said polypeptide is selected from the group consisting of: a silicatein protein, a naturally occurring polyamine rich peptide, a non-naturally occurring polyamine rich peptide, a derivate of a silicatein, a derivative of a silaffin, a thiolated peptide, and a polypeptide that includes at least one free hydroxyl group. The polypeptide that includes at least one free hydroxyl group may include at least one amino acid selected from the group consisting of: serine, threonine, and hydroxyproline.
In certain embodiments, said polypeptide is a silaffin. The silaffin may be derived from at least one species selected from the genera consisting of: Thalassiosira and Coscinodiscus. The silaffin may be derived from at least one species selected from the group consisting of: Thalassiosira pseudonana, Coscinodiscus wailesii, and Coscinodiscus concinnus.
In certain embodiments, said polypeptide is selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO4, and SEQ ID NO. 5.
These and other features, aspects, and advantages of the present invention will become better understood with reference to the following drawings, descriptions and claims.
pseudonana
pseudonana
pseudonana
The above-mentioned aspects of the present disclosure and the manner of obtaining them will become more apparent, and aspects thereof will be better understood by reference to the following description of the embodiments of the disclosure, taken in conjunction with the accompanying drawings, figures, schemes, formula, and the like, wherein:
a is a scanning electron micrograph of a diatom illustrating silification of the cell wall.
b is a greater magnified image of a region of
a is a mammalian cell unexposed to a non-naturally occurring bioinorganic material-rich environment.
b is a scanning electron micrograph of a mammalian cell in suspension after association of a non-naturally occurring bioinorganic membrane to the cell surface.
a is a scanning electron micrograph of Pseudomonas aeruginosa cells prior to exposure to a non-naturally occurring bioinorganic material-rich environment.
b is a scanning electron micrograph of Pseudomonas aeruginosa cells after after association of a non-naturally occurring bioinorganic membrane to the cell surface.
a is a scanning electron micrograph of Nitrosomonas europaea cells prior to exposure to a non-naturally occurring bioinorganic material-rich environment.
b is a scanning electron micrograph of Nitrosomonas europaea cells after after association of a non-naturally occurring bioinorganic membrane to the cell surface.
a is a graph presenting oxygen flux measurements of Pseudomonas aeruginosa cells during biomineralization.
b is a graph presenting oxygen flux measurements of Nitrosomonas europaea cells during biomineralization.
a is a transmission electron micrograph illustrating biomineralization of Max8 peptide associated INS-1 cells.
b is a magnified and localized transmission electron micrograph of a region of the cellular membrane of the INS-1 cell of
a is scanning electron micrograph illustrating biomineralization of a silaffin associated INS-1 cell.
b is lower magnification scanning electron micrograph of the INS-1 cells of
The embodiments of the disclosure presented and/or described below are not intended to be exhaustive or to limit the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art may appreciate and understand the principles and practices of various aspects and embodiments discussed herein.
Unless specifically stated otherwise, as used herein, the term “about” refers to a range of plus or minus (+/−) 10% (e.g., 1.0 encompasses the range of values from 0.9 to 1.1).
With reference to
The astonishing patterns found on the silica rich cell walls of many diatoms are clues to the utility of these structures. These patterns are channels through the silica rich protective naturally occurring cell walls that enable these organisms to freely exchange nutrients and waste material with their environments. The naturally occurring cell wall of the diatoms provide functionalities far superior to cells that are merely encased, entrapped or coated with materials such as silica rich layers. The patterns are the result of the deposition of silica facilitated by the association of specific moieties on the diatoms cell membrane that are evolved to interact with silica and to direct the formation of silica surface.
Many of these moieties in diatoms are polypeptides that include stretches that are lysine rich and that interact with dissolved silicic acid. These polypeptides include a class of proteins referred to as silaffins. For a further discussion of the purification and characterization of such proteins, please see Poulsen and Kroger, JBC, Vol. 279. No. 41, October 8, pp. 42993-42999. Amino acid sequences for 3 of the proteins disclosed in Poulsen and Kroger can be found listed herein as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. Some of the embodiments of the instant invention include associating silaffins with the surfaces of either prokaryotic cells or eukaryotic cells, other than diatoms, and under suitable conditions, produce viable cells that include a patterned, non-naturally occurring bio-membrane having a structure that is directed by the association of the silaffins with various moieties such as proteins, carbohydrates or lipids that are present on the cellular membranes of the cells.
Still other embodiments of the invention include associating synthetic polypeptides, such as the MAX8 peptide (SEQ ID NO. 4) disclosed in Altunbas, et al, AcsNANO, Vol. 4, No. 1, pp. 181-188 (2010), with the surface of a cell (that does not naturally form a biomineral rich cell membrane) in order to facilitate the formation of a biomineral rich membrane having a pattern directed by moieties on the cell surface that interact with the peptide. These cells further remain viable.
Still other embodiments include polypeptides such as the one disclosed herein as SEQ ID NO. 5, which has physio-chemical properties similar to MAX8. The peptide of SEQ ID NO. 5 is designed to be less cytotoxic than MAX8 but still able to augment the cell surface directed formation of a biomineral rich membrane around, at least, a portion of cell surface that does not form biomineral rich cell walls in nature.
Broadly, the present disclosure provides materials and methods for cell-surface directed association of non-naturally occurring bioinorganic membranes with the cell surface of living cells which do not form biomineral rich cell walls in nature. Referring to
With reference to step 204, association of the non-naturally occurring bioinorganic membrane with the cell-surface is induced. As will be explained in further detail below, induction of this association may occur in various manners, but in general accordance with the disclosure, involves introduction of the living cells to a bioinorganic material-rich (or even saturated) environment (such as a silica-rich buffer).
Further, as used herein, non-naturally occurring biomineral membranes are mineral rich structures, generally having a pattern that includes pores and are associated with cells that are not associated with such biomineral rich structures in nature. In some embodiments, the biomineral membrane may exist in nature as, for example, a silica rich cell wall in a diatom, but, as used herein, the same biomineral composition is defined as non-naturally occurring because in its inventive embodiment it is associated with a cell type, such as a prokaryotic cell, or an animal cell, or a higher plant cell, that it is not associated with in nature.
Still another wholly unexpected embodiment is that biomineral rich cell membranes (pseudo cell walls) can be formed on surfaces of cells such as Pseudomonas, stem cell like P19 murine embryonic carcinomas and mouse pancreatic β-islets cells by maintaining these cells in contact with a biomineral rich buffer for a length of time, even in the absence of the addition of exogenous polypeptides, such as silaffins. As illustrated in more detail herein, these cells remain viable and are able to exchange nutrients and products produced by the living cells with their environments. Without wishing to be bound by any theory, it appears as if naturally occurring moieties on the surface of cells, such as pseudomonas P19s, and β-islets, can direct the formation of a non-naturally occurring biomineral rich membrane (pseudo cell wall) by their ability to accumulate a biomineral such as silica from a biomineral rich buffer.
Referring next to steps 206 and 206′ of
With reference to step 206′ specifically, cell survival and physiological functionalities of the living cells having the associated bioinorganic membrane may also be assessed. For example, proton (
Next, and with reference to step 208 of
According to one embodiment of the present disclosure, a non-naturally occurring bioinorganic membrane may be associated with a cell surface of a living cell (evolutionary distinct from diatoms) by exposing the cell surface to a biomineralization buffer (rich or saturated in the biomineral). In some embodiments the silica solution is acidic before it is introduced into the physiological buffer. The resultant neutralization of the silica increases the rate of polycondensation of the silicate into an amorphous state that is well-suited for biomineral deposition. For example,
Another embodiment of the present disclosure, represented in the schematics of
With reference to
Similar to
In accord with the instant disclosure, peptides 404, 508 (including polyamine groups 406, 510 attached thereto) have an overall net positive charge under the buffer conditions utilized herein. In general, peptides 404, 508 may comprise any one (or combination thereof) of a silaffin protein, silicatein protein, a polyamine rich naturally occurring cell surface peptide, a synthetic polyamine rich peptide, a silaffin derivative, a silicatein derivative, thiolayted peptides, peptides that have free hydroxyl groups including amino acids such as serine, threonine, hydroxyproline, and SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and the like.
Silaffin peptides within the scope of the present disclosure include, but are not limited to, silaffin proteins derived from Thalassiosira pseudonana, Coscinodiscus wailesii, Coscinodiscus concinnus or any combination thereof. Additionally, silaffin peptides may be isolated from any diatom, produced recombinantly, or produced synthetically.
It should be understood that embodiments of the present disclosure depicted in
Further, in some embodiments, association of the silaffins to the cell surface can be accomplished by taking advantage of integrin/ligand binding interactions. Peptides with affinities for specific cell surface integrins can be readily produced synthetically or in transgenic bacteria. Simple chemical modification can be employed to attach a thiol (—SH) group to the terminus of the peptide chain. When introduced into solution, the peptides will bind to surface integrin receptors, studding the cell with gold binding thiol groups. Gold nanoparticles can then be added to the media and allowed to attach to the thiol groups studding the cell. Silaffins, produced by transgenic diatoms and chemically modified to express a thiol group on the peptide chain terminus, can then be introduced. The gold affinity of the thiol modified silaffins will induce aggregation onto the nanoparticles. Once the cell has been decorated with silaffins, immersion into silica rich solution will result in the silaffin governed nanopatterning of a silica shell. Alternative binding strategies can be applied to the same experimental motif Biotinylated peptide termini could be chemically produced to couple with avidin coated microbeads. The more direct approach of creating a ligand/silaffin fusion protein in the recombinant diatom could also be used to associate the silaffin to the cell membrane; however, the relative ease of cellular adaptability would be compromised. Nanoparticle junctions prevent the need to create new recombinant silaffin proteins for every ligand explored. Self assembly of silaffins onto a nanoparticle would also allow for peptide concentration dependant morphological structure control. Direct ligand/silaffin binding would place an upper limit on silaffin concentration to the number of integrins expressed on the membrane. Precise control of silaffin concentration will be necessary in order to manipulate the pore morphology and diffusional characteristics of the coating.
Referring next to
Thus the present disclosure also provides materials and methods for the association of a non-naturally occurring bioinorganic membrane 608 to the cell surface of living cells which are associated with a structure. Such embodiments employ the additional steps relating to associating (or culturing) cells onto a structure. Embodiments of the present disclosure as depicted in
Unlike the artificial deposition strategies (which create bioinorganic membranes around cells) of current technologies, embodiments of presently disclosed non-naturally occurring bioinorganic membranes, formed by the cell surface directed and associated materials and methods disclosed herein, are biocompatible, strong, and chemically resistant. Further, the bioinorganic membranes generated by the present disclosure possess relatively rapid (compared to current technologies) rates of molecular diffusion critical for maintenance of cell viability.
Embodiments of the materials and methods described above allow for uses in the association of non-naturally occurring bioinorganic membranes with the surface of living cells, both prokaryotic and eukaryotic. Additionally, embodiments of the present disclosure allows for uses in sensors and adaptive drug delivery devices as well as for the implantation of foreign cellular material into a host without the need for global suppression of the immune system of the host. Further, the bioinorganic membrane disclosed herein can be used for regulation of the release of a wide range of molecules in products such as pharmaceutical agents, nutrients, gasses, and biological products. Even further, methods of the present disclosure may also be employed in applications with structures other than living cells. For example, the present disclosure may be used with drug carrying structures, such as hydrogels, polymer particles, liposomes, and micelles in order to create controlled release drug delivery devices.
Materials and Methods: A biomineralization solution was prepared by hydrolysis of tetramethyl orthosilicate (TMOS) in a weakly acidic aqueous solution. The methanol byproduct of the hydrolysis reaction was removed by rotary evaporation. Suspended mouse (P19) cells were then exposed to media containing the mildly acidic silica-rich solution, resulting in the polycondensation of a biomineral membrane. The solution was then diluted prior to bulk gelation. Tetramethyl orthosilicate (TMOS, Sigma-Aldrich) was hydrolyzed in a 1:16 mol ratio (TMOS:H2O) deionized water solution using 1 μl of 0.04 molar hydrochloric acid initiator per 1 g of solution. The mixture was stirred vigorously for 10 minutes until clear. The methanol produced by the hydrolysis reaction was removed from the solution by rotary evaporation under vacuum at 45° C. (30% reduction in solution volume). The resulting saturated silica solution was refrigerated prior to use or used immediately. Biomineral layer formation was induced by exposing cells to a α-MEM media solution supplemented with 30 μl per ml of the previously prepared saturated silica solution and 50 μl per ml phosphate buffered saline. The cells were incubated in this solution for 10-30 minutes (longer times producing thicker mineral deposits). After mineralization, the solution was removed and fresh silica free media was reintroduced to the cells.
Results: With specific reference to
Further, and with reference to
Experiment 1, described above, demonstrates both that cells which are evolutionary distinct from diatoms (do not form biomineral membranes by extracting anionic biominerals from the environment) surprisingly form such membranes after exposure to the biomineralization buffer disclosed herein. Further, Experiment 1 demonstrates these cells surprisingly retain their cellular activity and functionality, thus demonstrating the associated membrane disclosed herein possess mesoporosity enabling necessary cellular transport and diffusion of cellular material.
Materials and Methods: Mucopolysaccharide-rich P. aeruginosa and N. europaea biofilms were immersed in a mildly acidic silica-rich biomineralizing buffer. P. aeruginosa PA01 (ATCC 97) was obtained from American Type Culture Collection (Manassas, Va.), and biofilms were grown at 37° C. in modified glucose media (10 mM glucose, 50 mM HEPES, 3 mM NH4Cl, 43 mM NaCl, 3.7 mM KH2PO4, 1 mM MgSO4, and 3.5 μM FeSO4). N. europaea (ATCC 19718) was obtained from ATCC, and biofilms were grown in ATCC medium 2265 (25.0 mM-(NH4)2SO4, 43.0 mM-KH2PO4, 1.5 mM-MgSO4, 0.25 mM-CaCl2, 10 μM-FeSO4, 0.83 μM-CuSO4, 3.9 mM-NaH2PO4, and 3.74 mM-Na2CO3). The biofilms were mineralized in freshly filtered growth medium supplemented with 25 μl per ml of the saturated silica solution described previously for ˜20 min prior to media exchange. Scanning electron microscopy images of the biofilims prior to and after membrane formation are presented in
The SEMS were taken after fixing the biofilms on the membrane (
Results: As is observed in
With reference to
Additionally, viability florescent staining (with STYO9 green) was also performed on both the P. aeruginosa and N. europaea biofilms (not depicted) (staining of control cells with and propidium iodide was also performed). The results of the staining analysis found no statistically significant variation between control and biomineralized cell populations. These results indicated that the silica matrix was sufficiently porous to allow for the diffusion of dissolved gasses and nutrients. Biophysical transport of nutrients and electron acceptors regulates synthesis and maintenance of cells within the biofilm and is limited by the concentration boundary layer formed at the biofilm-fluid interface. No significant change in oxygen flux, substrate flux, or stoichiometric metabolic ratio was observed after encapsulation (p<0.02, α=0.05), suggesting that cells survived the encapsulation process intact. No observable differences were noted at 10× magnification in stained samples analyzed using confocal microscopy. There were no large regions of lysed cells within the matrix (2 μm slices), which one would expect if diffusion limitations or nutrient transport was significantly altered by silica encapsulation.
With reference to
Referring now to
Referring now to
This application is a continuation of International Application No. PCT/US2011/021032, filed Jan. 12, 2011, which claims the benefit of U.S. Provisional Patent Application No. 61/294,209, filed Jan. 12, 2010, the disclosures of which are hereby incorporated by reference in their entirety.
This invention was made with government support under grant number RR025761 awarded by the National Institute of Health (NIH), and under grant number W911NF-09-1-0447 awarded by the U.S. Army Research Laboratory's Army Research Office (ARO). The U.S. government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61294209 | Jan 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2011/021032 | Jan 2011 | US |
| Child | 13546834 | US |
