Patent Application
20230263944
This disclosure generally relates to the field of functionalized surfaces for cell capture and expansion.
Medical implants surfaces and ex vivo tissue culture surfaces often lack necessary biological molecules needed to maximize cell numbers and specificity on the surfaces. To improve their performance, the surfaces have been modified by grafting different biomolecules such as extracellular matrix (ECM) proteins, antibodies, growth factors, and peptides. Surfaces with immobilized antibodies, for example, have shown immense potential in selecting cell types and capturing them on the surface based on their surface antigens which have since then been utilized for vascular stents and cell purification systems. Surfaces modified with ECM-derived peptides were shown to promote cell adhesion, proliferation, and differentiation making it ideal for tissue engineering scaffolds and cell expansion platforms. However, each of these types of molecules lack the ability to carry certain integral functions needed to successfully recruit selected cell subtypes and promote their subsequent spreading, proliferation and/or functionality.
Therefore, improvements are desired in the functionalization of surfaces to ameliorate cell capture, survival and function.
In a first aspect, there is provided a surface functionalized with cross linking groups, the surface adapted to receive antibodies and/or fragments thereof that bind a specific subtype of cells capturing the cells onto the functionalized surface and/or that bind cellular products, the surface comprising: an antibody binding biomolecule comprising a linker region which is covalently crosslinked to functional groups on the surface and an antibody binding region that binds to any non-variable region of the antibodies or the fragments thereof and controls the orientation of the antibodies and/or the fragments thereof such that the variable regions are away from the surface, and the antibody binding biomolecule having a molecular weight of less than 10,000 g/mol; and a cell interacting biomolecule comprising a linker region which is covalently crosslinked to functional groups of the surface and a cell interacting region, the cell interacting biomolecule has a three dimensional structure such that the cell interacting region is oriented away from the surface when the linker region has crosslinked with the surface, and the cell interacting biomolecule has a molecular weight of less than 10,000 g/mol; wherein a concentration of the antibody binding biomolecule and a concentration of the cell interacting biomolecule on the surface are each independently controlled within a predetermined range that allows both the antibodies and/or the fragments thereof and the cell binding biomolecule to be functional.
In one embodiment, the molecular weight of the antibody binding biomolecule is between about 500 g/mol to about 2000 g/mol.
In one embodiment, the molecular weight of the cell interacting biomolecule is between about 500 g/mol to about 2000 g/mol.
In one embodiment, the antibody binding biomolecule further comprises one or more spacing regions comprising polyethylene glycol (PEG) and/or glycine.
In one embodiment, the cell interacting biomolecule further comprises one or more spacing regions comprising polyethylene glycol (PEG) and/or glycine.
In one embodiment, the cell interacting region of the cell interacting biomolecule comprises a peptide derived from extracellular matrix proteins.
In one embodiment, the antibody binding region of the antibody binding biomolecule comprises a RRGW peptide.
In one embodiment, the linker region of the antibody binding biomolecule comprises sulfosuccinimidyl 4-(n-maleimidophenyl)butyrate (Sulfo-SMPB).
In one embodiment, the linker region of the cell interacting biomolecule comprises Sulfo-SMPB.
In one embodiment, the cell interacting region of the cell interacting biomolecule comprises a RGD peptide.
In one embodiment, the surface is one of a flat surface, an interior cylindrical surface, an helix/screw-shaped material or an exterior surface of a microbead.
In one embodiment, the surface is a slide or multiwell plate.
In another embodiment, the surface is of polystyrene or of cobalt-chrome (CoCr).
In one embodiment, a ratio of the molecular weight of the antibody binding biomolecule to the molecular weight of the cell interacting biomolecule is between about 1:10 to about 10:1.
In one embodiment, the cells are cancer cells, primary cells or in vivo cells.
In one embodiment, the cells are endothelial progenitor cells such as endothelial colony-forming cells.
In a second aspect, there is provided a vascular stent device comprising the surface defined in the first aspect.
In a third aspect, there is provided a microcarrier comprising the surface defined in the first aspect.
In a fourth aspect, there is provided a method of producing a surface having a dual function of capturing cells and/or cell products and promoting proliferation, adhesion, spreading, differentiation and/or function of the cells, comprising: providing an activated surface having a crosslinking chemical group bound to one or more linker molecules; crosslinking an antibody binding biomolecule to the one or more linking arms at the linker region of the antibody binding biomolecule by adding a predetermined concentration of the antibody binding biomolecule onto the surface, the antibody binding biomolecule having an antibody binding region that binds to the Fc region of antibodies and/or the fragments thereof and controls the orientation of the antibodies and/or the fragments thereof such that the variable regions are away from the surface, and the antibody binding biomolecule having a molecular weight of less than 10,000 g/mol, and crosslinking a cell interacting biomolecule to the one or more linkers at the linker region of the cell interacting biomolecule by adding a predetermined concentration of the cell interacting biomolecule onto the surface, the cell interacting biomolecule having a cell interacting region that interacts with cellular receptors of the cells to promote the adhesion, spreading, proliferation, activation and/or function of the cells on the surface, a three dimensional structure such that the cell interacting region is oriented away from the surface when the linker region has crosslinked with the surface, and a molecular weight of less than 10,000 g/mol, thereby obtaining a conjugated surface; and incubating the conjugated surface with the antibodies and/or the fragments thereof and allowing the antibodies and/or the fragments thereof to bind the antibody binding biomolecule.
In one embodiment, prior to step a) an optimization step is performed to determine the predetermine concentration of the antibody binding biomolecule and the predetermined concentration of the cell interacting biomolecule such that the surface has an optimized dual function of both capturing the cells and promoting the proliferation, adhesion, and/or spreading of the cells.
In one embodiment, a first washing step is performed between step b) and step
In one embodiment, prior to step a), activating an inert surface to obtain the activated surface.
It is provided a surface functionalized with cross linking groups, the surface adapted to receive antibodies and/or fragments thereof that bind a specific subtype of cells capturing the cells onto the functionalized surface and/or that bind cellular products, the surface comprising an antibody binding biomolecule comprising a linker region which is covalently crosslinked to functional groups on the surface and an antibody binding region that binds to any non-variable region of the antibodies or the fragments thereof and controls the orientation of the antibodies and/or the fragments thereof such that the variable regions are away from the surface, and the antibody binding biomolecule having a molecular weight of less than 10,000 g/mol; and a cell interacting biomolecule comprising a linker region which is covalently crosslinked to functional groups of the surface and a cell interacting region, the cell interacting biomolecule has a three dimensional structure such that the cell interacting region is oriented away from the surface when the linker region has crosslinked with the surface, and the cell interacting biomolecule has a molecular weight of less than 10,000 g/mol; wherein a concentration of the antibody binding biomolecule and a concentration of the cell interacting biomolecule on the surface are each independently controlled within a predetermined range that allows both the antibodies and/or the fragments thereof and the cell binding biomolecule to be functional.
The surface according to the present disclosure is functionalized to have a dual function by covalently binding two biomolecules on the surface. The first function of the surface is to capture a specific type of cell and/or a specific cellular product. This is achieved with an antibody-binding molecule which is conjugated on the surface and then further incubated with antibodies to allow their immobilization. By changing the immobilized antibodies on the surface, different cell types and/or different cellular products could be specifically targeted. Therefore, the surfaces as encompassed herein can recruit any cell type or product as long as there is a corresponding antibody or combination of antibodies to capture the desired group of cells or products. The second function is the adhesion, spreading, proliferation, differentiation, activation and/or other functional attributes of the cells on the surface which is achieved with a cell-interacting biomolecule that is covalently conjugated on the surface. The term “biomolecule” as used herein refers to molecules that are found in biological systems, that are derived from biological systems or molecules that are engineered/synthesized to be biocompatible and interact with biological systems. The biomolecule may comprise a peptide, a protein, a nucleic acid, a nucleic acid sequence, a lipid, a glycoprotein, a fluorophore, other biopolymers, or any combination thereof. The biomolecule may be a bioactive molecule such as synthetic polymers or synthetic molecules which interact with the cells.
Antibodies do not provide cells with the necessary signals to fully adhere, proliferate, or differentiate/function. Cell-interacting molecules such as ECM-derived peptides on the other hand lack the ability to capture cells under dynamic conditions due to slow adhesion kinetics and significantly smaller structure. Therefore, the combination of the two biomolecules in the present disclosure allows for an improved surface functionalization technique having the dual functions conferred by the antibody binding biomolecule and the antibody as well as the cell-interacting biomolecule. It is encompass that the antibodies described herein also can be seen as cell-interacting. For example antibodies can be used to block or activate cell receptors. Accordingly, antibodies and the smaller biomolecules could be used together to target distinct cell-activating pathways or mechanisms. Thus, the antibodies described herein can be used for cell capture and/or to activate or block a cell receptor, wherein the peptides can be used to trigger a secondary signal.
The surface according to the present disclosure can have any desired geometry such as a flat, concave, convex or complex geometry. For example, the surface may be a cell culture surface, the surface of a cardiovascular stent, the surface of microbeads or microcarriers. The surface can be an inert surface that is modified to have functional groups or a commercially available surface that has functional groups, which are covalently bound to the biomolecules. The functional groups may be amine, carboxylic acid, thiol, carbonyl or any other groups suitable for the formation of the covalent bond with the biomolecules. The functional groups may be present in the underlying surface or introduced through surface modification (e.g. chemical or plasma treatment of the surfaces; adsorption of polymers or other molecules containing these functional groups). In some embodiments, the surface modifications are applied to polymers or metals (e.g. CoCr). In some embodiments, prior introduction of functional groups on the substrate for example by coating (e.g. with polymers that contain nitrogen or other functional groups such as polylysine or polydopamine), chemical modification (e.g. silanization) or plasma treatments (as we have done on CoCr) are needed.
In some embodiments, the surface is used for cellular assays. For example, the antibodies immobilized on the surface can be specific to cellular products that are desired to be quantified. Cellular products include secreted molecules such as hormones and cytokines or any signaling molecule. Other examples include yeast or bacterial cells that have been modified to secrete a specific product. In certain embodiments, antibodies can be used for cell capture and other antibodies can be further immobilized for quantification of their cellular products. Cellular products are not necessarily secreted by the cells as they can be released in the medium after cell lysis or permeabilization of cell membranes. The quantification analysis of the cellular products binding to the antibodies can be done by enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), western blot or any other suitable means depending on the assay. The cellular assays include cell culture assays, and in one example, the antibodies target cytokines to reduce the amount of harmful cytokines in the medium, or to “track” what the cells are secreting. In this case, the molecules secreted could be quantified at the end of the culture by adding a secondary antibody and detection using conventional sandwich ELISA assays—similar to ELISPOT assays. Making reference to
The antibody binding biomolecule has an antibody binding region 113 comprising an amino acid sequence or a nucleic acid sequence that binds to any non-variable region of antibodies or antibody fragments. In the embodiment exemplified in
The antibody binding biomolecule optionally has one or more spacing regions 112 to improve the performance of the antibody binding biomolecule. The spacing regions can be used to reduce the steric hindrance induced by the surface and/or to minimize non-desirable and non-specific protein adsorption on the surface when in contacts with fluids containing other proteins such as blood, plasma or cell culture medium. The spacing regions can comprises polyethylene glycol, amino acids (such as G and C), propylene sulfoxide, and/or other compounds with similar polar functional groups, net charge, hydrogen bond acceptor groups, and hydrogen bond donor groups.
Most of the antibody surface immobilization techniques available for in vitro studies or for in vivo cell capture applications according to the prior art rely on direct adsorption, surface conjugation via primary amines, or interactions with bio-affinity bacterial proteins. The simplest method to immobilize antibodies on surfaces is adsorption, but this method can lead to a reduction in antigen binding due to desorption and conformations with reduced availability of antigen-binding sites. Although antibody conjugation via primary amines, carboxylic acids or other functional groups on antibodies is less susceptive to desorption, this technique suffers from a lack of control over antibody orientation on the surface due to the prevalence of primary amine throughout the antibody structure. Directional antibody immobilization can be achieved via binding of the Fc region to surfaces grafted with bacterial products such as protein A or G. Disadvantages of this strategy include the immunogenicity of these bacterial proteins and their high affinity for albumin, which may increase fouling in the presence of biological fluids. Furthermore, protein A and G have a large molecular weight of more than 30,000 g/mol and more than 20,000 g/mol respectively which could explain why these two proteins are not suitable for the present dual function surface.
Due to the disadvantages and limitations of such immobilization methods it is desirable to use easily synthesized peptides or nucleic acids, such as for example aptamers, that are able to both immobilize antibodies and control their orientation. For instance, the short peptide sequence RRGW has a strong association toward the Fc region of mouse immunoglobulin G (lgG). With this and other short synthesized peptides, it becomes convenient to screen a large number of antibodies in a single experiment through an easy switch between different antibodies. The additional advantage of using these peptides to immobilize antibodies rather than proteins, such as protein A or G, is their small size that provides better control in grafting the molecule on the surface and minimizing steric hindrance.
The cell interacting biomolecule 120 has a linker region 121 that is bonded to the surface by a covalent bond or crosslink 102. The linker region 121 comprises a crosslinker that can be a maleimide crosslinker, a carbodiimide crosslinker, an imidoester crosslinker or a N-Hydroxysuccinimide Ester (NHS Esters) crosslinker. For example, the crosslinker can be selected from the group consisting of disuccinimidyl substrate (DSS), sulfosuccinimidyl 4-(n-maleimidophenyl)butyrate (Sulfo-SMPB), sulfosuccinimidyl 4-(n-maleimidophenyl)cyclohexane-1-carboxylate (Sulfo-SMCC), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Sulfo-NHS and N-κ-maleimidoundecanoyl-oxysulfosuccinimide ester (Sulfo-KMUS). Depending on the available functional groups on the surface, the linker region might consist of one or more of these crosslinking arm. Thus, for example the linker region has a bilinker or a trilinker arm.
The cell-interacting biomolecule 120 has a cell-interacting region 123 that interacts with the cellular surface molecules such as cell surface receptors. The interaction may be direct, such as integrin binding to ligands on the surface, and/or indirect such as receptor binding with ligands that trigger receptor activation and downstream signaling within the cell. Examples of such cell surface receptors are integrins, growth factor receptors, other signaling proteins, as well as other components present in the cell membrane or on its surface which can relay signals to the cells. In one embodiment, the cell-interacting region comprises a ligand that can be a peptide or small protein derived from extracellular matrix (ECM) proteins, growth factors, hormones, other proteins which bind cell-surface receptors or molecules engineered to bind to these receptors such as an engineered aptamer. Surfaces modified with ECM-derived peptides promote cell adhesion, proliferation, and differentiation making it ideal for tissue engineering scaffolds and cell expansion platforms. The ligand can be derived from common motifs that contribute to the cell adhesive properties of various ECM proteins including fibronectin, vitronectin, fibrinogen, osteopontin, and some collagens. These motifs may be between 3 to 100 or even between 3 to 20 amino acids. For example, RGD is a common motif that can be used as ligand. Other examples include but are not limited to REDV, DEGA, YIGSR, RNIPPFEGCIWN, PHSRN, KRSR GFOGER, GPEILDVPST and IKVAV. The ligand can be derived from common motifs that promote cell survival and proliferation present in ECM proteins or growth factors such as vascular endothelial growth factor, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, insulin-like growth factor or others. The ligand can be derived from motifs that contribute to cell differentiation or function such as cell signaling proteins or hormones. Examples of these ligands include hormone peptides such as adrenocorticotropic peptide (ACTH-(4-10), MEHFRWG-OH), growth factor-derived peptides such as FREG peptide (DPHIKLQLQAE) and cell migration-mediated peptides such as SRSRY peptide. When cells are captured on the surface, the cell interacting region enters into contact with molecules at the cell surface which promotes the adhesion, proliferation, spreading, differentiation and/or functional response of the cells on the surface. The cell interacting biomolecule has a three dimensional structure such that the cell interacting region is oriented away from the surface when the linker region has crosslinked with the surface. This reduces the steric hindrance exerted by the surface. “Oriented away from the surface” can be defined as being at a distance from the surface of at least 8, 9, or 10 angstrom. This is achieved by reacting the biomolecules with the linker that was introduced on the surface through their terminal crosslinking group, thus controlling the biomolecule's orientation. In some embodiments, the cell interacting region comprises a peptide at the end tail of the biomolecule.
The cell interacting biomolecule has a total molecular weight smaller than about 10,000 g/mol, about 9,000 g/mol, about 8,000 g/mol, about 7,000 g/mol, about 6,000 g/mol, about 5,000 g/mol, about 4,000 g/mol, about 3,000 g/mol, about 2,000 g/mol or about 1500 g/mol. In some embodiments, the molecular weight of the antibody binding protein is between about 500 g/mol to about 2000 g/mol, between about 650 g/mol to about 1750 g/mol, or between about 800 g/mol and about 1500 g/mol.
The cell interacting biomolecule optionally has one or more spacing regions 122 to improve the performance of the antibody binding biomolecule. The spacing regions can be used to reduce the steric hindrance induced by the surface and/or to minimize non-desirable and non-specific protein adsorption on the surface when in contacts with protein-containing fluids. The spacing regions can comprises polyethylene glycol, amino acids (such as G and C), propylene sulfoxide, and/or other compounds with similar polar functional groups, net charge, hydrogen bond acceptor groups, and hydrogen bond donor groups. The spacing may be a capping end 124 consisting of one or more amino acids.
Surface modification strategies that can combine a small biomolecule (less than 10,000 g/mol) and antibodies are scarce and non-controllable due to the vast difference in their molecular weight. For example, a peptide can be over 150 times smaller than antibodies, therefore it can easily bind to the structure of the antibody itself and not the surface if they are simply mixed in solution. Reported methods mainly consist of conjugations of different growth factors or morphogenesis proteins independently or using a simple mixture of those molecules with no control over overall resulting surface density or the orientation of the biomolecules and antibodies. The techniques known today have been limited to the immobilizing of either antibodies alone or ECM-derived molecules alone as each of these biomolecules has a unique function. Some of these known technologies also rely on adsorption forces to modify the surface with the biomolecules of interest which is easily washed away upon exposure to aqueous conditions such as cell culture media and bodily fluids. Thus previously known techniques lack control over the resulting surface concentration of the grafted molecules which hinders the success of the technology due to regulatory constraints. They also mostly lack the ability to control the orientation of both biomolecules which reduces the biological effect of the surface.
The above mentioned problems are overcome by the surface as proposed herein and according to the present disclosure. The cell interacting biomolecule and the antibody binding biomolecule crosslinked to the surface have similar sizes. Furthermore, as previously described, the biomolecules are covalently bound to the surface with a controlled orientation and the antibodies are also immobilized with a controlled orientation due to the presence of the conjugated antibody binding biomolecule. During surface functionalization, applying molecules similar in size avoids interactions in solution which may impact the final surface density of the antibodies and cell-interacting biomolecules. The molecular weight ratio between the cell interacting biomolecule and the antibody binding biomolecule is between about 2:1 to about 1:2, about 1:3 to about 3:1, about 1:4 to about 4:1, about 5:1 to about 1:5, or between about 1:10 to about 10:1. Furthermore, because the biomolecules are conjugated to the surface, the resulting surface concentration of the biomolecules and antibodies can be tightly controlled by changing the concentration applied to the surface. The combination of the small size of the biomolecules, the control of the orientation of the molecules, the similarity in size between the biomolecules, and their respective function yields an advantageous surface that can be used for cell capture and retention across most of the surface. Applications for such a surface include but are not limited to dynamic cell culture, a mimic of biological cell interfaces or walls, vascular stents, engineered grafts and microcarrier applications.
The cell-interacting biomolecules and the antibodies can be applied as mixtures. For example, mixtures of cell-interacting biomolecules can be applied to confer different functional attributes such as cell adhesion and cell differentiation. To achieve this, cell-interacting biomolecules can be mixed in different ratios with antibody-binding peptides prior to antibody immobilization. Similarly, mixtures of antibodies can be applied to optimally capture one cell type or cellular product which expresses corresponding antigens, or to capture more than one desired cell type. This can be achieved by mixing antibodies in different ratios during the antibody immobilization step. As an example, after grafting the linking arm to surfaces, a mixture of RRGW peptide, RGD-TAMRA peptide and a growth factor-derived peptide motif can be mixed and conjugated via free thiol groups. Due to the similar size of these peptides, the surface density can be adjusted by changing the concentration of these peptides in solution during covalent grafting. Next, a mixture of antibodies such as CD31 and CD309 can be applied in solution to immobilize both antibodies in the same step. By conjugating the cell-interacting biomolecules and the antibody binding biomolecule prior to immobilizing the antibodies, several cell-interacting biomolecules and several antibodies can be applied while retaining control over the final surface density of each component. No additional reaction steps are required to add more than one cell-interacting biomolecule or more than one antibody on the surface aside from adjusting concentrations in solution during surface modification steps.
The surfaces of the present disclosure are prepared with the following method. Making reference to
Second, the linker 203 is reacted with the functional groups to form a covalent bond as shown in
Then, an antibody binding biomolecule 204 is crosslinked to the linker 203 at the linker region of the antibody binding biomolecule by adding a predetermined concentration of the antibody binding biomolecule 204 onto the surface 201 thereby forming the complete biomolecule. Due to the antibody binding biomolecule covalently linking with the surface, the concentration of the immobilized antibodies on the surface 201 can be controlled by the concentration of antibody binding biomolecule in the mixture prior to application on the surface. The antibody binding biomolecule 204 has an antibody binding region that binds to the Fc region of antibodies and controls the orientation of the antibodies such that the variable regions are away from the surface, and the antibody binding biomolecule has a molecular weight of less than 10,000 g/mol.
Further, substantially in conjunction or substantially simultaneously, a cell interacting biomolecule or mixture of cell-interacting biomolecules 205 is crosslinked to the linker 203 at the linker region by adding a predetermined concentration of the cell interacting biomolecule 205 onto the surface 201 thereby forming the complete biomolecule. The cell interacting biomolecule 205 has a cell interacting region that interacts with cellular receptors of the cells to promote the adhesion, spreading and/or proliferation of the cells on the surface 201. The cell interacting biomolecule has a three dimensional structure such that the cell interacting region is oriented away from the surface when the linker region has crosslinked with the surface, and a molecular weight of less than 10,000 g/mol. The surface may then be washed to remove the biomolecules that have not reacted to the linker groups.
Finally, antibodies 206 are incubated on the surface and allowed to bind to the antibody binding biomolecule 204. Depending on the application of the surface, a secondary antibody is used to detect the presence of the first antibody. Because the antibody or mixture of antibodies is not immobilized directly onto the surface 201 but is linked to the surface through a previously deposited antibody binding biomolecule, the orientation of the antibodies can be controlled. The concentration of antibodies is tightly controlled by changing the concentration of the antibody binding biomolecule 204. Contrary to applying antibody binding biomolecule, the direct conjugation of antibodies on the surface via amine or carboxylic acid functional groups reduce the control over their orientation. Due to the prevalence of such groups throughout the antibody structure, the Fab regions—necessary for cell capture—might be immobilized on the surface leading to diminished-activity of the antibody.
The dual function obtained for the surface of the present disclosure is shown in
The following example uses a RRGW biomolecule and a RGD biomolecule however the concentrations described are in no means limiting. In fact, the concentrations in the present method can be optimized for different antibody binding biomolecules and cell interacting biomolecules as performed in the example below for the combination of RRGW biomolecule and RGD biomolecule.
Surface Functionalized with RGD Biomolecules and RRGW Biomolecules
To form the surface, the first step was to graft Sulfo-SMPB— a heterobifunctional linker—to activate an aminated surface and to introduce free maleimide groups available for reactions. To have a better control over the dual conjugation strategy, two peptides that are relatively equal in size and have terminal thiol groups were synthesized to have similar chemical reaction kinetics with the surface. The two synthesized peptides, antibody-binding peptide [RRGW(PEG3)C] (
The CCC experimental design was used to build surface responses model with two factors, the first one was the concentration of the RGD-TAMRA peptide in solution, and the second one was the concentration of the RRGW peptide in solution. The outputs of the model were the fluorescence intensity of RGD-TAMRA and the fluorescence intensity of the secondary antibodies. This model contains a 2 by 2 factorial design, a central point and 4-axial points that test the extremes conditions of each factor. The RGD-TAMRA (
Spots of 2 μL (around 0.64 μL/mm2) of each mixture were added to be conjugated on aminated surfaces activated with sulfo-SMPB and incubated statically for two and a half hours in the dark, and then the slides were rinsed twice with PBS. Slides were then blocked by Dako serum-free protein block, followed by covering the entire surface with: first 10 pg/mL mouse anti-CD144 and second by 10 pg/mL F(ab′)2-goat anti-mouse IgG conjugated with Alexa Fluor 488. Slides were lastly washed twice with PBS and RO water, respectively. The spots were then imaged using the same confocal microscope using a 10× objective. Every spot was imaged twice, once at a green wavelength to detect the immobilized antibodies, and second at a red wavelength to detect the RGD-TAMRA. For each condition, 3 spots with 6 images for each spot were analyzed. The central point was triplicated to increase the confidence level and decrease the error in the model.
For the results of the bifunctional surface modification experiment, the effect of two independent variables (the concentrations of both peptides, RGD-TAMRA and RRGW, in solution) on two response variables (the TAMRA intensity and the secondary antibodies intensity) was determined using the fit model option in JMP®, a statistical software. Independently for each response, the lack of fit was verified and considered significant at P<0.05. For each measured output, only parameters or factors that have p<0.05 were considered to have a significant effect.
As described above, to combine both antibodies and ECM-derived peptides, different concentrations of both RRGW and RGD-TAMRA were mixed and spotted on the surface. The CCC design was built to assess the effect of both peptides in the final responses. These surface responses were predicted by measuring the fluorescence intensities of both TAMRA and Alexa Fluor 488, which are conjugated to RGD and the secondary anti-mouse antibodies, respectively (
Interestingly, no significant interaction occurred between the two peptides on the measured surface responses. This model strongly suggests that both peptides could be conjugated on the surface in a very controllable manner.
By applying the present method with commercially available aminated polystyrene surfaces, the behavior of endothelial colony forming cells (ECFCs) towards the modified bifunctional surfaces was tested in static conditions (
To test whether the bifunctional surfaces can impart two significant effects on cells of interests, ECFCs derived from human peripheral blood were either seeded on the modified surfaces under static conditions or flown over the surfaces in a perfusion loop under 1 dyn/cm2 wall shear stress. The RGD-TAMRA peptide and anti-CD309 antibodies immobilized using the RRGW peptide were selected due to each of their demonstrated positive effects on ECFC spreading and ECFC capture respectively. The two molecules were mixed together at the center point concentrations of the previously described surface model. Under static conditions, the number of ECFCs on antibody surfaces and bifunctional surfaces were significantly higher than the controls (unmodified surfaces) due to the presence of the cell capturing effect associated with the antibody. On the other hand, the surface area of the ECFCs on the RGD-TAMRA and the bifunctional surfaces were significantly higher than the controls due to the presence of the cell spreading effects associated with the peptide. Combining these two pieces of data, a maximum cell coverage is seen (
Under dynamic flow conditions this effect was even more evident as shown in
As described herein, surfaces were modified by a combination of two molecules, where each one had a unique function. Combining antibodies with peptides on the surface with control over surface concentrations and orientations has proved to confer desirable advantages to the surface. Without wishing to be bound by theory, it is believed that the antibodies would increase the selectivity of capturing circulating cells, while RGD— the peptide—would promote the adhesion and proliferation of the captured cells. To achieve this, two peptides, RRGW and RGD-TAMRA, were combined in different concentrations and conjugated on the PureCoat′ aminated polystyrene surfaces, followed by the addition of primary antibodies. Combining these relatively similar-sized peptides at the chosen range of concentrations increased the opportunity of having a controlled method as neither of these peptides were expected to interact with each other or block the surface. This was confirmed by the central composite design, where no significant interactions were observed between the two conjugated peptides on the surface. This model was also used to predict the surface responses that result from the combination of both molecules on the surface (
The cell adhesion experiments demonstrated the significant effect of the bifunctional method on improving the cell coverage on the surface (number of cells and their spreading) when compared to antibodies or peptides alone. This technique is useful in blood contacting medical devices for the aim of accelerating reendothelialization by capturing and recruiting EPCs on the surface which reduce biocompatibility issues. It is also very valuable for the modification of cell culture systems as it enables both selection (
Results showed significant trends resulting from varying the concentration of each of two peptides in their respective detected signals and no significant effects of either of the molecules on the other's signal response, suggesting that the method is very controlled with no significant interactions between the two molecules. Therefore, it is possible to choose the desired response by simply choosing the concentrations of each of the molecules in the mixture from an identified range of operation.
As encompassed herein, the modifications can be applied to different aminated substrates—as shown on polystyrene (
Also encompassed are variations to this exemplary method. Indeed, the method offers flexibility for applications on different material surfaces with different functional groups (i.e. not only primary amines) by simply using a different linker. It can also be used to immobilize any peptides with a terminal thiol group and any IgG antibody which makes it versatile for screening purposes and useful for various applications. The same principle can also be used to immobilize biomolecules other than peptides and antibodies. For example, growth factors and full native proteins can be combined with antibodies or peptides using the same strategy. The present technology allows for the creation of surfaces that can better mimic the complex multi-functional processes that are involved in cell recruitment in vivo.
Unsuccessful Surface Functionalization with Protein G
The CD144 or the vascular endothelial cadherin (VE-Cadherin) antigen is a 140 kD glycoprotein. It is a calcium-dependent transmembrane cell-cell adhesion molecule localized at the intercellular boundaries of endothelial cells, hematopoietic stem cells, and perineurial cells. CD144 is thought to play a role in vascular development, permeability, and remodeling.
A 6 well plate was seeded with the following conditions in each well (W1-W6):
The functionalized surface was rinsed with phosphate buffer saline (PBS) and reactive oxide water. HUVEC cells were used and were seeded in each well in serum free medium at a concentration of 19,000 cells/mL. The plate was incubated for 3 hours on an incubation shaker then imaged under the microscope. The plate were left to incubate for another 21 h (total 24 h) at 37° C. 5% CO2 and imaged again.
Results at 3 h are shown in
Results at 20 h are shown in
While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations, including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
The present application is claiming priority from U.S. Provisional Application No. 63/051,608 filed Jul. 14, 2020, the content of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/050968 | 7/14/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63051608 | Jul 2020 | US |
