Patent Application
20230382976
This application contains a sequence listing filed in electronic form as an ST.26 file entitled “222112-1020 Sequence Listing” created on Aug. 212, 2023 and having 18,611 bytes. The content of the sequence listing is incorporated herein in its entirety.
Crosslinking of collagen is an effective method to modify the stability of collagen compositions and materials and to optimize their mechanical and structural properties. Crosslinked collagen materials are used extensively in various medical and industrial applications. For example, crosslinked collagen materials are used to replace or augment hard or soft connective tissue, such as skin, tendons, cartilage, bone, and interstitium. Crosslinked collagen materials have been implanted surgically, and numerous injectable crosslinked collagen formulations are currently available for various cosmetic applications. Toxic chemicals typically are used for crosslinking collagen and additives (growth factors, small molecules, drugs) are incorporated to achieve biological responses. However, these can lead to detrimental off-target effects. However, there remains a need in the art for improved methods of producing crosslinked collagen materials and endowing them with greater functionality at the same time.
Existing systems and devices aimed at modeling tissue barriers utilize plastic membranes to separate types of cells in culture. The use of a barrier material can preclude the use of a truly physiologically relevant and viable system. For example, semi-permeable membranes (such as plastic membranes) can block the tissue interface, and prevent the interaction of cells and other elements introduced into the systems, for example antagonists or agonists (small molecules, peptides, nucleic acids, etc). Accordingly, there is a need to address the aforementioned deficiencies and inadequacies and a need for more physiologically-relevant model systems.
Oligonucleotides are capable of binding protein. A specific type of oligonucleotide structure is DNA aptamer. These are single stranded oligonucleotide sequences, which are capable of forming tertiary structures. This enables them to upon targeting and purification have extremely high specificity for a specific biological structure. Moreover, as disclosed herein, DNA aptamers can activate cell signaling receptors, rather than simply passively attaching to the receptor. Specifically, relevant are extracellular matrix proteins, notably collagen. This protein forms fibers in solution, which are typically crosslinked to form a 3D network by chemical means such as with cytotoxic glutaraldehyde. DNA is capable of forming a complex with collagen, which initiates and promotes collagen fiber formation. These fibers are then DNA-collagen complexes. As disclosed herein, DNA aptamers are capable of forming these complexes and the fiber properties are dependent on both the aptamer sequence, geometry, and relative concentration. These fibers begin to form spontaneously upon combining DNA and collagen solutions. DNA aptamers are also capable of being conjugated together to form 3D assemblies.
Disclosed herein is the use of DNA aptamer assemblies of varying DNA length, structure, and sequence to both bind to collagen and additional extracellular matrix (ECM) components, to then act as a biocompatible, degradable, reversible, or permanent 3D crosslinkers between proteins, and to service as a biologically functional material when using the appropriate aptamer sequence.
Therefore, disclosed herein are compositions comprising collagen fibers and one or more additional ECM components crosslinked with a plurality of one or more DNA aptamers. Also disclosed are devices and implants made from or coated with collagen fibers additional ECM components crosslinked with a plurality of one or more DNA aptamers. Also disclosed are methods of making collagen fibers. Also disclosed are kits for producing collagen fibers. Also disclosed herein are compositions comprising a plurality of one or more DNA aptamers in a collagen fiber matrix that stabilizes the DNA aptamers.
In some embodiments, at least one of the one or more DNA aptamers selectively binds a growth factor or cytokine. In some embodiments, at least one of the one or more DNA aptamers selectively binds a cell receptor, such as a stem cell receptor. In some embodiments, at least one of the one or more DNA aptamers selectively binds an extracellular matrix protein.
In some embodiments, the DNA aptamers comprise from 15 to 100 nucleotides, including 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 nucleotides.
In some embodiments, the DNA aptamers comprise from 1 to 20 stem loops, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 stem loops, such as 1 to 10 stem loops, or 1 to 5 stem loops.
In embodiments, an aptamer comprises one or more of SEQ ID NO:1-5 or SEQ ID NO:9-15. In some embodiments, the plurality of one or more DNA aptamers comprises 2 or more DNA aptamer sequences, including 2 to 4 DNA aptamer sequences, connected by a linker molecule to form an aptamer assembly. In embodiments, aptamers or complexes thereof can be sulfo-SANPAH functionalized prior to or during use.
In some embodiments, the collagen comprises type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type XI collagen, or any combination thereof.
In some embodiments, the one or more additional ECM components comprises an ECM protein, proteoglycan, polysaccharide, or combination thereof. For example, in some embodiments, the ECM protein comprises elastin. In some embodiments, the ECM protein comprises a fibronectin, laminin, or a combination thereof. In some embodiments, the ECM proteoglycan core protein comprises versican, perlecan, aggrecan, decorin, biglycan, testican, betaglycan, agrin, fibromodulin, lumican, neurocan, or a combination thereof. In some embodiments, the ECM glycosaminoglycan comprises heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparin, hyaluronic acid or a combination thereof.
In some embodiments, the selection of cells and ECM components used in the disclosed compositions and methods is based on the tissue type of interest. In some embodiments, the tissue type is a blood vessel, which can comprise an intimal Layer, a medial layer, and/or an adventitial layer. In some embodiments, the intimal Layer contains endothelial cells with type IV collagen, laminin, proteoglycans, and/or von Willebrand factor. In some embodiments, the medial Layer contains vascular smooth muscle cells with type I collagen, type III collagen, type IV collagen, elastin, laminin, fibrillin, fibulin, fibronectin, and/or proteoglycans. In some embodiments, the adventitial Layer contains fibroblasts, myofibroblasts, macrophages, T cells, B cells, mast cells, dendritic cells, microvascular endothelial cells, pericytes, adipocytes, lymphocytes, and vascular progenitor cells with type I collagen, type III collagen, elastin.
In some embodiments, the tissue type is myocardial tissue. Therefore, in some embodiments, this includes cardiac myocytes, fibroblasts, leukocytes, and cardiac vascular cells. In some embodiments, this also includes glycoproteins, such as collagen, elastin, fibronectin, laminin, osteonectin AKA secreted protein acidic and rich in cysteine (SPARC), thrombospondin 1, 2 and 4, tenascin, osteopontin, periostin, cysteine-rich protein-61, and/or connective tissue growth factor. In some embodiments, this also includes the matrix proteoglycans, such as hyalectans (versican, aggrecan, neurocan, and brevican), basement membrane proteoglycans (perlecan, collagen type XVIII, and agrin), cell surface proteoglycans (syndecans and glypicans), and/or small leucine-rich proteoglycans (SLRPs, such as biglycan, decorin, lumican, and osteoglycin). In some embodiments, this also includes glycosaminoglycans, such as hyaluronan and/or heparan sulfate.
In some embodiments, the tissue type is bone tissue. Therefore, in some embodiments, this includes osteoblasts, osteoclasts, osteocytes with type I collagen, osteopontin, osteocalcin, bone sialoproteins, proteoglycans, and/or apatites.
In some embodiments, the tissue type is cartilage tissue. Therefore, in some embodiments, this includes chondrocytes with hyaluronic acid, type II collagen, proteoglycans, fibronectin, type IX collagen, type VI collagen.
In some embodiments, the tissue type is nervous tissue. Therefore, in some embodiments, this includes astrocytes, glial cells, neuron with laminin, fibronectin, type IV collagen, hyaluronic acid, and/or proteoglycans.
In some embodiments, the tissue type is liver tissue. Therefore, in some embodiments, this includes hepatic stellate cells with types I, III, IV, and V collagen, laminin, fibronectin, tenascin, nidogen, and/or proteoglycans.
In some embodiments, the tissue type is kidney tissue. Therefore, in some embodiments, this includes renal cells with type IV collagen, laminin, nidogen, fibronectin, and/or proteoglycans
In some embodiments, the tissue type is muscle tissue. Therefore, in some embodiments, this includes muscle cells with type I, III, V, VI, IV, IX, XI collagens, laminin, fibronectin, and/or proteoglycans.
In some embodiments, the tissue type is gut tissue. Therefore, in some embodiments, this includes enterocytes and other gut related cells with type I, III, IV, and VI collagens, laminin, fibronectin, and/or proteoglycans.
Other aspects of aptamer complexes, methods of use, and kits comprising the same are also disclosed herein.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
In certain aspects, described herein are compositions comprising collagen fibers one or more additional ECM components crosslinked with a plurality of one or more DNA aptamers. In embodiments, at least one of the one or more DNA aptamers can selectively binds a growth factor or cytokine. In embodiments, at least one of the one or more DNA aptamers selectively binds a cell receptor. In embodiments, the cell receptor is a stem cell receptor. In embodiments, at least one of the one or more DNA aptamer selectively binds an extracellular matrix protein. In embodiments, the DNA aptamers can be 15 to 100 nucleotides. In embodiments, DNA aptamers comprise from 1 to 5 stem loops. In embodiments, a plurality of one or more DNA aptamers comprises 2 to 4 DNA aptamer sequences connected by a linker molecule to form an aptamer assembly. In embodiments collagen comprises type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, or any combination thereof.
Described herein are methods of crosslinking collagen one or more additional ECM components. In embodiments, methods for crosslinking collagen and one or more additional ECM components, comprise mixing collagen monomers and the one or more additional ECM components with a plurality of one or more DNA aptamers under conditions suitable for a crosslinking reaction. In embodiments, collagen monomers and DNA aptamers can be mixed at a ratio of from about 8% to about 30%. In embodiments, at least one of the one or more DNA aptamers selectively binds a growth factor or cytokine. In embodiments, at least one of the one or more DNA aptamers selectively binds a cell receptor, which can be cell receptor is a stem cell receptor. In embodiments, at least one of the one or more DNA aptamers selectively binds an extracellular matrix protein. In embodiments, DNA aptamers comprise from 15 to 100 nucleotides. In embodiments, DNA aptamers comprise from 1 to 5 stem loops. In embodiments, the plurality of one or more DNA aptamers comprises 2 to 4 DNA aptamer sequences connected by a linker molecule to form an aptamer assembly. In embodiments, collagen comprises type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, or any combination thereof.
Described herein are kits for crosslinking collagen and one or more additional ECM components. In embodiments, kits comprise collagen monomers, one or more additional ECM components, and one or more DNA aptamers. In embodiments, at least one of the DNA aptamers selectively binds a growth factor or cytokine. In embodiments, at least one of the DNA aptamers selectively binds a cell receptor. In embodiments, the cell receptor is a stem cell receptor. In embodiments, at least one of the DNA aptamers selectively binds an extracellular matrix protein. In embodiments, DNA aptamers comprise from 15 to 100 nucleotides. In embodiments, DNA aptamers comprise from 1 to 5 stem loops. In embodiments, a plurality of one or more DNA aptamers comprises 2 to 4 DNA aptamer sequences connected by a linker molecule to form an aptamer assembly. In embodiments, collagen comprises type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, or any combination thereof.
Described herein are methods of differentiating osteoblasts. In embodiments, methods comprise contacting a composition as described herein with one or more osteoblasts. In embodiments, the osteoblasts are human primary osteoblast. In embodiments, the osteoblasts are mammalian osteoblast. In embodiments, the aptamer selectively binds to or otherwise stimulates a VEGF receptor. In embodiments, the VEGF receptor is VEGF-R2. In embodiments, the aptamer comprises a nucleotide sequence configured to specifically bind to VEGF-R2.
Described herein are methods of transfection. Methods of transfection can comprise contacting a cell in need thereof with a composition as described herein in the presence of one or more nucleotides, wherein the one or more nucleotides are structurally distinct from the DNA aptamer. In embodiments, the contacting is in the further in the presence of calcium phosphate.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere.
Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.
The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
As used herein, the term “DNA aptamer” refers to a single stranded deoxyribonucleic acid (DNA) whose distinct nucleotide sequence determines the folding of the molecule into a unique three dimensional structure. Aptamers comprising 15 to 120 nucleotides can be selected in vitro from a randomized pool of oligonucleotides (1014-1015 molecules). The “DNA aptamer” comprises a degenerate sequence, and can further comprise fixed sequences flanking the degenerate sequence. The term “DNA aptamer” as used herein further contemplates the use of both native and modified DNA bases, e.g. beta-D-Glucosyl-Hydroxymethyluracil.
As used herein, the term “DNA aptamer” refers to an oligonucleotide molecule that binds to a target protein. In some embodiment, the DNA aptamer binds to a specific region or amino acid sequence of the target protein.
As used herein, the term “bind,” the term “binding” or the term “bound” refers to any type of chemical or physical binding, which includes but is not limited to covalent binding, hydrogen binding, electrostatic binding, biological tethers, transmembrane attachment, cell surface attachment and expression.
For purposes of the present invention, the term “oligonucleotide,” the term “polynucleotide,” the term “nucleotide,” and the term “nucleic acid” refer to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, and usually more than ten. The exact size of an oligonucleotide will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded.
For purposes of the disclosed invention, the term “polynucleotide” includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.
For purposes of the disclosed invention, the term “residue,” the term “amino acid residue,” or the term “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
Disclosed herein are DNA aptamers that can be used to crosslink collagen into fibers. The DNA sequence used to produce the aptamer can be selected using routine methods based on desired characteristics, such as protein binding.
DNA aptamers are short, single-stranded DNA oligonucleotides capable of specific binding to defined targets. The advent and success of SELEX technology in 1990s may be attributed to the feasibility to chemically synthesize pools of random oligonucleotides, the availability of the polymerases for nucleic acid amplification, as well as the improvement in sequencing techniques. The molecular recognition between aptamers and their corresponding targets relies on the three-dimensional conformations of the aptamers, hence the specific nucleic acid sequences. By substituting just a few nucleotides, the conformation of an oligonucleotide may change. Consequently, the structural diversity of a DNA or RNA pool containing combinatorial sequences may be infinitely expanded, thereby creating panels of aptamers for a wide variety of binding targets. The evolution process for selecting DNA aptamers typically covers the following steps: 1) chemical synthesis of a combinatorial oligonucleotide library having 1013-1016 single stranded nucleic acid molecules, 2) exposure of the library to the targets to differentiate binding strands from spectators, 3) extraction and amplification of eluted survivors, 4) enrichment of the stronger survivors by iterative binding to targets and by involving counter selection if necessary, and, finally, 5) sequencing to identify individual candidates.
The SELEX process (systematic evolution of ligands by exponential enrichment) for engineering DNA aptamer sequences generates several potential candidates of varying length. As the disclosed data shows, fiber formation is dependent on both ssDNA length and the relative amounts of ssDNA and collagen in solution. Thus, the choice of sequence from the SELEX process is important as the ideal recipe for fiber formation will be different for each candidate sequence. Fibers form above a threshold binding value of 0.05 μg ssDNA/μg collagen, but also required the appropriate amount of ssDNA and collagen in solution (8-30% mass fraction DNA in solution) (
The length of the DNA aptamer comprising the sequence (i) or (ii) or the sequence (I) or (II) (hereafter, simply referred to as the “DNA aptamer according to the present invention”) is, for example, 150 mer or shorter, 140 mer or shorter, 130 mer or shorter, 120 mer or shorter, or 110 mer or shorter, and preferably 100 mer or shorter, 90 mer or shorter, 80 mer or shorter, 70 mer or shorter, 60 mer or shorter, or 50 mer or shorter.
The DNA aptamer according to the present invention may arbitrarily comprise a base analog, another artificial base, another modified base, or the like, in addition to Ds.
The DNA aptamer according to the present invention may be modified with the addition of other substances, such as polyethylene glycol (PEG) (e.g., a PEG polymer of about 20 to 60 kDa), an amino acid, a peptide, inverted dT, a lipid, a dye, a fluorescent substance, an enzyme, a radioactive substance, and biotin. Such substance may be linked via a known linker, if needed. Examples of linkers that can be used herein include a nucleotide linker, a peptide linker, and a linker containing a disulfide bond. It is generally known that a half-life of the DNA aptamer is extended by conjugating PEG to the DNA aptamer. In certain aspects, two or more aptamers as described herein can be linked with a linker, for example a PEG linker or polymer of PEG moieties.
A method for producing the DNA aptamer according to the present invention is not particularly limited. A method known in the art may be employed. For example, the DNA aptamer according to the present invention can be chemically synthesized based on the sequences indicated above in accordance with a known solid-phase synthesis method. Regarding a method of chemical synthesis of nucleic acids, see, for example, Current Protocols in Nucleic Acid Chemistry, Volume 1, Section 3. Many life science manufacturers (e.g., Takara Bio Inc. and Sigma-Aldrich Corporation) provide contract manufacturing services concerning such chemical synthesis, and such services may be used. A DNA aptamer may be prepared by synthesizing several fragments based on the DNA aptamer sequence and then ligating the fragments via, for example, intramolecular annealing or ligation by a ligase.
The DNA aptamer according to the present invention prepared via chemical synthesis is preferably purified by a method known in the art before use. Examples of methods of purification include gel purification, affinity column purification, and HPLC.
In certain aspects, aptamers as described herein can have two ends that are configured to bind separate targets. In an embodiment, an aptamer as described herein has an end configured to bind collagen, and another end configured to bind another target (for example VEGF-R2 or other VEGF-R's).
The disclosed aptamers are in some embodiments able to bind a protein of interest. Examples protein targets include growth factors, cytokines, cell receptors, and extracellular matrix proteins.
Aptamers can be purchased from commercial vendors, such Integrated DNA Technologies, BasePair Technologies, Inc., AptSci., and Aptagen. In addition, DNA aptamers can be generated using the SELEX process or modified SELEX methods.
Examples of pathogen proteins for which DNA aptamers have been developed include Anthrax Protective Antigen, bipd (type iii secretion protein), bope (type iii secretion protein), Botulinum neurotoxin type A, bpsI2748 (putative oxidoreductase), Clostridium difficil toxin a, Clostridium difficil toxin b, ETEC K88 fimbriae protein, Francisella tularensis subspecies (subsp) japonica bacterial antigen, Iron-regulated surface determinant a, Iron-regulated surface determinant b, Iron-regulated surface determinant c, Iron-regulated surface determinant h, Leishmania infantum H2A antigen, Leishmania infantum KMP-11, mannose-capped lipoarabinomannan, microcystin-LA, microcystin-LR, microcystin-YR, and -LA, Mycobacterium avium sp. Paratuberculosis Major Antigens, Mycobacterium tuberculosis cfp10, Mycobacterium tuberculosis esat6, Mycobacterium tuberculosis esxg, Mycobacterium tuberculosis methionyl-tRNA synthetase (MRS), Mycobacterium tuberculosis mpt64 protein, Mycobacterium tuberculosis polyphosphate kinase, Plasmodium falciparum erythrocyte membrane protein 1, Plasmodium lactate dehydrogenase, Protein A, Salmonella typhimurium ompc, Staphylococcus aureus clumping factor a, Staphylococcus aureus clumping factor b, Staphylococcus aureus Enterotoxin B, Staphylococcus aureus enterotoxin c1, Staphylococcus aureus Protein A (SpA), Staphylococcus aureus α Toxin, T. cruzi excreted secreted antigens, Type IVB Pili, and Ustilago maydis RNA-binding protein Rrm4.
In embodiments of the present disclosure, a protein target is a vascular endothelial growth factor receptor (VEGF-R). In an embodiment, a protein target is VEGF-R2.
Embodiments of VEGF-R protein targets according to the present disclosure include (or variants there of having greater than 70% sequence identity to the sequences listed; greater than 80% sequence identity; greater than 90% sequence identity; greater than 95% sequence identity):
In embodiments of the present disclosure, a protein target is a receptor tyrosine kinase, a G protein coupled receptor, or an integrin. In embodiments of the present disclosure, a protein target is a glucagon receptor, a Hepatocyte Growth Factor Receptor, a estrogen receptor, an androgen receptor, an epidermal growth factor receptor, an insulin receptor, an immune receptor, a neurotrophin receptor, a fibroblast growth factor receptor, an adrenergic receptors, an ephrin receptor, an NM DA receptor, a Nerve growth factor receptor, or an olfactory receptor.
DNA Aptamers against glucagon receptor are described in Wang G, et al. Scientific Reports 2007 7(7179), which is incorporated by reference in its entirety for the teaching of this aptamer. DNA Aptamers against Hepatocyte Growth Factor Receptor are described in Boltz A, et al. J Biol Chem. 2011 286: 21896-21905, which is incorporated by reference in its entirety for the teaching of this aptamer. DNA Aptamers against estrogen receptor are described in Sett A, et al. Transl Res. 2017 183:104-120, which is incorporated by reference in its entirety for the teaching of this aptamer. DNA Aptamers against androgen receptor and glucocorticoid receptor are described in Zhang L, et al. Genome Res. 2018 28(1):111-121, which is incorporated by reference in its entirety for the teaching of these aptamers. DNA Aptamers against epidermal growth factor receptor are described in Chen C H, et al. PNAS 2003 100(16):9226-923, which is incorporated by reference in its entirety for the teaching of these aptamers. DNA Aptamers against insulin receptor are described in Nucleic Acids Res. 2015 43(16):7688-7701, which is incorporated by reference in its entirety for the teaching of these aptamers. DNA Aptamers against α4 integrin are described in Kouhpayeh, S, et al. J Cell Biochem 2019 120(9):16264-16272, which is incorporated by reference in its entirety for the teaching of these aptamers. DNA Aptamers against Toll-like receptor 4 (TLR4) are described in Fernández G, et al. Mol. Ther. 2018 26(8):2047-2059, which is incorporated by reference in its entirety for the teaching of these aptamers. DNA Aptamers against fibroblast growth factor receptor are described in Ueki, R, et al. Chem Commun (Camb). 2019 Feb. 26; 55(18):2672-2675, which is incorporated by reference in its entirety for the teaching of these aptamers. DNA Aptamers against ephrin receptors are described in Affinito A, et al. Mol. Ther. Nucleic Acids 2020 20:176-185, which is incorporated by reference in its entirety for the teaching of these aptamers. DNA Aptamers against NM DA receptor are described in Lee G, et al. ACS Chem Neurosci. 2014 5(7):559-567, which is incorporated by reference in its entirety for the teaching of these aptamers. DNA Aptamers against Nerve growth factor receptor are described in Jarvis T C, et al. Structure 2015 23:1293-1304, which is incorporated by reference in its entirety for the teaching of these aptamers.
Examples of viral proteins for which DNA aptamers have been developed include Alfalfa Mosaic virus RNA-coat protein complex, bacteriophage ff gene 5, dengue-2 virus envelope protein domain iii, Ebola Virus VP35 interferon inhibitory domain, Foot-and-mouth disease virus RNA-dependent RNA polymerase, gp130, HBV capsid, HBV core protein, HBV recombinant truncated P protein, HBV surface antigen, HCV core antigen, HCV Envelope Glycoprotein E2, HCV nonstructural protein 3 protease, HCV NS2 protein, HCV NSSA, HCV ns5b replicase, HCV RNA-Dependent RNA Polymerase, HES 1 protein icp27, HIV gp120, HIV integrase, HIV LTR, HIV Nucleocapsid Protein, HIV Tat, HIV-1 gag, HIV-1 Reverse transcriptase, HIV-1 Tar RNA, HPV-16 E7 Oncoprotein, HSV gd protein, HSV US11, HTLV-I tax, Human norovirus (Gii.4) capsid P domain, Human norovirus capsid protein vp1, Influenza A Hemagglutinin, Influenza A NS1 protein, influenza A polymerase PA subunit, influenza protein e, MS2 coat protein, papillomavirus 16 e7 oncoprotein, RABV glycoprotein, rex, Rift Valley fever virus N protein Pool, sars-cov, sars-cov nucleocapsid protein, severe acute respiratory syndrome (sars) coronavirus ntpase/helicase, SP6 RNA polymerase, and Venezuelan equine encephalitis virus capsid protein.
Examples of toxins for which DNA aptamers have been developed include Colicin E3, gliadin peptide, Ricin A-chain, shiga toxin, t-2 toxin, tetanus toxoid, type a botulinum neurotoxin, α-bungarotoxin snake venom, and β-bungarotoxin.
Examples of prions for which DNA aptamers have been developed include bovine prion protein, cellular prion protein, mouse prion, recombinant human (rhu) cellular prion protein (PrPC) 23-231, and Syrian golden hamster prion protein rPrP23-231.
Examples of mammalian proteins for which DNA aptamers have been developed include 4-1BB, Acetohydroxyacid synthase, activated protein c, AGE-human serum albumin, AlkB, Alzheimer's Disease Amyloid Peptide, AM L1 Runt domain, AMPA receptor, amyloid-like fibrils, angiogenin, angiopoietin 1, angiopoietin 2, anti-MPT64 antibodies, anti-NF-kB p65, antivesicular stomatitis virus polyclonal antibodies, ApoE, arginine-rich motif (ARM) model peptide, ATR/TEM8 Von Willebrand factor type A (VWA), B-cell-activating factor-receptor, B52 (SR protein RNA splicing), b7, basic fibroblast growth factor, Bcs1, bovine catalase, bovine factor ix, bovine follicle-stimulating hormone a subunit, bovine serum albumin, bovine thrombin, brain natriuretic peptide (bnp), C-C chemokine receptor type 5, c-Met, C-reactive protein, c-telopeptide (ctx) of human type i bone collagen, c4, c7, calsenilin, cardiac troponin i (ctni), cathepsin E, cd133, CD16, cd18, CD28, CD30, cd31, CD40, cd44, Cdc42, CGRP peptide, CHK2, cholesterol esterase, cJun/cJun, complement factor c5a, Connective tissue growth factor, crdl1, CTAP III/NAP2, ctla-4, CYT-18, cytochrome c, cytochrome P450 51A1, cytoplasmic tail of BACE, DC-SIGN protein, DNA binding domain of TCF-1, DNA polymerase b, E-Selectin, e6 oncogene protein, endostatin, epidermal growth factor receptor, epithelial cell adhesion molecule, Erk2, Estrogen receptor a, Eukaryotic translation initiation factor 4G, factor d, Factor IX, factor ixa, factor viia, factor x, factor xa, fibrinogen, fibronectin, fibronectin binding protein a, fibronectin binding protein b, fractalkine, G-protein-coupled receptor kinase 2, G6-9 anti-DNA autoantibody, ga733-1, ghrelin, glyceraldehyde-derived pyridinium, glycine receptor (glyr), Glycoprotein VI, gonadotropin, gonadotropin-releasing hormone I, gper neurotensin, HA Binding Domain of Human CD44, HbA1c, heat shock factor, hemoglobin, heptaocyte growth factor, histone H4, hmg-1, hnspA2 human nonpancreatic secretory phospholipase A2, hsp90-binding immunophilin, human acetylcholinesterase, Human Cardiac Troponin I, human CD73, human complement 5, human dicer, human epidermal growth factor receptor 2, Human epidermal growth factor receptor-3 (HER3), human erythropoietin-a (rHuEPO-a), human gp73, human growth hormone, human heat shock factors 1, human heat shock factors 2, Human Hepatocyte Growth Factor, human interleukin-8, human matrix metalloprotease 9 (hMMP-human neutrophil elastase, human periostin, human plasma, human Platelet-Derived Growth Factor chain B, Human Pro-Urokinase, human Rad51, human rad51, human RNase H1, human thyroid stimulating hormone, Human tPA, human transferrin receptor, human β2-microglobulin, immunoglobulin e, immunoglobulin g, insulin, insulin receptor, integrin α4, integrin αVβ3, interferon γ, interferon γ induced protein 10, interferon-inducible t-cell α chemoattractant, interleukin-10ra, interleukin-12, interleukin-16, Interleukin-17, interleukin-23, interleukin-6, interleukin-6 receptor, interleukin-8, k ras-derived farnesylated peptide, kallikrein-related peptidase 6 (klk6), keratinocyte growth factor, L-selectin, L7Ae protein, leptin, leptin r, lipocalin-2, Lysozyme, mAb198, macrophage migration inhibitory factor, MAGE-A3111-125, matn2, Mek1, metastatic enzyme heparanase (HPSE1), MetJ (methionine repressor), migraine-associated calcitonin gene-related peptide, mitochondrial GTPase NOA1, mitogen-activated kinase, monoclonal antibody 83-7, mouse CCL1, mouse glycoprotein 2, mouse monoclonal antibody ma20, mouse vcam-1, MRCKα-KD, MUC1, MUC16 ca125 ovarian cancer cell marker, murine c-type receptor dec205, murine Interleukin-10 (IL-10), murine myelin, mutant KRAS (G12V), MutS, myelin basic protein, N-methyl D-aspartate (NMDA) class of ionotropic glutamate receptors, negative elongation factor e, neuropeptide Y, Neurotrophin Receptor, neutrophil elastase, NF-IL6, nogo-66 receptor, nts-1, nuclear factor of activated T cells, nuclear factor κβ, nucleolin, nucleophosmin, oncogene tiam1, Oncogenic Protein Shp2, osteopontin, osteoprotegrin, ovarian cancer biomarker HE4, OX40, p-cadherin, P-selectin, P2X2 receptors, p43, p50, p53R175H, PAI-1, PAK1, pancreatic adenocarcinoma up-regulated factor, PDGF-BB, Pepocin, Peroxisome proliferator-activated receptor δ, phospholamban, Plasminogen Activator Inhibitor-1, plg, prostate specific antigen, prostatic acid phosphatase, protein kinase C-d, PTFase, Pulmonary Surfactant Protein A, quinoprotein glucose dehydrogenase (PQQGDH), Ras, Ras-binding domain of Raf1, recombinant human apc, recombinant human growth hormone (rhgh), rela (p65), Retinol binding protein 4 (RBP4), rev nuclear export signal, Rho, RIG-I, RNA binding domain, rna polymerase σ, RNase, RUNX1, schlerostin, se-selectin, Sec7 domain of cytohesin 1, SelB (elongation factor for selenocystein incorporation), serine protease urokinase-type plasminogen activator, sphingomyelinase, SRP19, substance P, t-cell 4-1bb, tenascin C, TFIIIA, TGF-b Receptor II, thrombin, Thrombospondin 2, thyroid transcription factor 1, toll-like receptor 2, transferrin, transforming growth factor receptor b2, transforming growth factor-b1, transforming growth factor-β type III receptor, TrkB, trypsin, tumor necrosis factor receptor super family member 9, tyrosine kinase RETC634Y, tyrosine phosphatase 1b, Tyrosine phosphatase SHP2, UBLCP1, unglycosylated epidermal growth factor receptor viii ectodomain, urokinase plasminogen activator, vascular endothelial growth factor, vascular endothelial growth factor 165, vasopressin, vimentin, von willebrand factor, von willebrand factor a1-domain, ZAP, Zinc Finger Proteins, α-fetoprotein, α-synuclein, β-arrestin 2, β-catenin, β-Conglutin, and β-site amyloid precursor protein cleaving enzyme1 (bace1).
Additional examples of proteins for which DNA aptamers have been developed include ara h 1 allergen, asp f 1 allergen, bacterial RNA polymerase, caenorhabditis elegans bcl-2 homolog ced-9, CFP, Concanavalin A, cry j 2 allergen, E. coli core RNA polymerase, electric eel acetylcholinesterase, eotaxin, erf1, Escherichia coli methionine repressor, Escherichia coli release factor 1, f(ab′)2 fragments of saxitoxin (stx) antibodies, GFP, heterogenous ribonucleoprotein I (hnrnp I), horse radish peroxidase, i-scei endonuclease, initiation factor 4a, innexin 2, inosine monophosphate dehydrogenase, lup an 1, mitochondrial processing peptidase, okadaic acid monoclonal antibody, peptidoglycan, sA from Thermus aquaticus, streptavidin, subtilisin (protease), systemin, T4 DNA pol, t7 ma polymerase, taq dna pol, tbp (tata box protein), Tet Repressor, tfiib, TIMP1, tobacco protoplast protein, yeast RNA polymerase II (Pol yeast TATA-binding protein, yeast TFIIB, and YFP.
In some embodiments, the DNA aptamer binds a cell target. Examples of non-cancerous mammalian cells for which DNA aptamers have been developed include 3T3-L1 adipocytes, Adult mesenchymal stem cells, BJAB cells expressing c-kit, C666-1, CD81 T-cells, Cell internalization, Differentiated PC12 cells, cho-k1 cells expressing human endothelin type b receptor (etbr), HEK-293, Transformed tonsillar epithelial cells, Human jaw periosteal cells, Human platelets, Inflamed endothelial cells, Malaria-infected RBCs, Mature white adipocytes, MCF-10AT1, MiaPaCa-2 secretome, Mitochondria, NP69, Osteoblasts, PC-3, PC:cholesterol liposomes, Rabies virus-infected live cells, RSV transformed SHE cells, and Transformed tonsillar epithelial cells. A cell target can be a protein receptor or cell-surface receptor, for example a VEGF-R.
Examples of pathogenic microorganisms for which DNA aptamers have been developed include African Trypanosomes, Alicyclobacillus spores, Anthrax spores, Bacillus spores, Bacillus thuringiensis, Campylobacter jejuni, Cryptosporidium parvum, Escherichia coli DH5α, Escherichia coli K12, Escherichia coli NSM59, Escherichia coli O111:B4, Escherichia coli 0157:H7, Francisella tularensis, Lactobacillus acidophilus, Leishmania major promastigotes, Listeria monocytogenes, Mycobacterium tuberculosis, Porphyromonas gingivalis, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella choleraesuis, Salmonella enteritidis, Salmonella O8, Salmonella paratyphi A, Salmonella typhimurium, Shigella dysenteriae, Staphylococcus aureus, Streptococcus mutans, Streptococcus oralis, Streptococcus pyogenes, Streptococcus sanguis, Treponema denticola, Trypanosoma cruzi, Tuberculosis, Vibrio alginolyticus, and Vibrio parahemolyticus.
Examples of cancer cells for which DNA aptamers have been developed include Acute myeloid leukemia (AML) cells, Adenocarcinoma, BG-1 ovarian cancer cells, Brain Tumor-Initiating Cells, Breast cancer, Burkitt lymphoma cells, Cancer stem cells, Colon cancer cell SW620, Colorectal cancer cell line DLD-1, CT26 intrahepatic tumor, Epithelial cancer cells, Gastric cancer cell-line HGC-27, Gefitinib-resistant H1975 lung cancer cells, Glioblastoma multiforme, Hepatocellular carcinoma, HER2 positive cell line, HPV-transformed cervical cancer cells, Human breast cancer MDA-MB-231, Human cholangiocarcinoma QBC-939 cells, Human gastric carcinoma AGS, Human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III, Human hepatocarcinoma, Human pancreatic ductal adenocarcinoma, Human U87MG glioma cells, Leukemia cells, Liver cancer, MCF-10AT1, MDA-MB-231 breast cancer, Metastatic colorectal cancer, Metastatic hepatocellular carcinoma cells, MS03 cancer line, Ovarian cancer cell TOV-21G, Ovarian serous adenocarcinoma cell CAOV-3, Pancreatic cancer cells, Primary Cultured Tumor Endothelial Cells, Primary human chronic lymphocytic leukemia, Ramos cells, Rat brain tumor microvessels, Small-cell lung cancer cells, SMMC-7721 liver carcinoma cells, and Vaccinia virus-infected lung cancer A549 cells.
Examples of nucleic acid targets for which DNA aptamers have been developed include Bacillus subtilis RNase P P5.1 stem-loop element, DNA/RNA motifs, HCV IRES, HIV-1 TAR element, PCA3 RNA, Target A-site 16S rRNA, and Yeast phenylalanine tRNA.
Examples of viral targets for which DNA aptamers have been developed include apple stem pitting virus, Arbovirus, Bovine viral diarrhea virus type 1, Fish Pathogen Viral Hemorrhagic Septicemia Virus, Herpes simplex virus type 2, Hirame rhabdovirus, HIV-1 subtype C envelope pseudovirus, Human cytomegalovirus, Human Norovirus, Influenza A/H1N1, Influenza A/H3N2, Influenza A/H5N1, Influenza B/Tokio/53/99, Influenza B/05/99, Singapore grouper iridovirus, Soft-shelled turtle iridovirus, Tobacco Necrosis Virus, Vaccinia virus, and Vesicular stomatitis virus (VSV).
In some embodiments, the DNA aptamer binds a small molecule target. Examples of fluorophores for which DNA aptamers have been developed include aniline-based quencher, Cibacron Blue 3GA, Cy3, DFHBI, Dihydropyrene photo-switch compound, Dimethylindole Red, DMABI, DMHBI, Fluoroscein, Hoechst derivative 7, Reactive Blue 4, Reactive Brown 10, Reactive Green 19, Reactive Red 120, Reactive Yellow 86, Sulforhodamine, Tetramethylrhodamine, and Thiazole orange.
Examples of pharmaceuticals for which DNA aptamers have been developed include (1-3)-b-D-glucans, 2-anilinophenylacetic acid, Acetamiprid, Aminoglycoside antibiotic, Chloramphenicol, Citrulline, Codeine, Cyclosporin A, Danofloxacin, Daunomycin, Diclofenac, Digoxin, Gentamicin, Globo H, Glutathione, Hematoporphyrin, Heteroaryldihydropyrimidine, Ibuprofen, Kanamycin, Lividomycin, Lysergamine, Metergoline, Moenomycin A, Neomycin, Paromomycin, Poly-γ-D-glutamic acid (g-PDGA), R-Thalidomide, Small Ergot Alkaloids, Spectinomycin, Streptomycin, Sulfadimethoxine, Tetracycline, Theophylline, and Tobramycin.
Examples of toxins and environmental hazard small molecules for which DNA aptamers have been developed include 2,4,6-trichloroaniline (TCA), Abrin toxin, Acetamiprid, Aflatoxin B1, Aflatoxin M1, Bisphenol A, Brevetoxin, Carcinogenic aromatic amines, Chinese Horseshoe Crab endotoxin, cylindrospermopsin, Digoxin, Dinitroaniline, Ethanolamine, Fumonisin B1, Isocarbophos, Lipopolysaccharide, Neurotoxin anatoxin-a, Ochratoxin A, Okadaic acid, Omethoate, P-aminophenylpinacolylmethylphosphonate, Pentachlorophenol, Phorate, Polychlorinated biphenyls, Profenofos, Staphylococcus aureus enterotoxin A, Trinitrotoluene, and zearalenone.
Examples of amino acids and peptides for which DNA aptamers have been developed include Arginine, Citrulline, Glutamic acid, Glutathione, Histidine, His Tag 6x, Isoleucine, L-arginine, L-tryptophan, P-amino phenylalanine, P-amino phenylalanine, Peptide: Asp-Gly-Ile, Peptide: Gly-Glu-Leu, Peptide: His-Phe, Peptide: Leu-Ala-Ser, Peptide: Lys-Ala-Ile, Phenylalanine, S-adenosyl methionine, S-Adenosylhomocysteine, Tachykinin substance P, Tryptophan, Tyrosine, and Valine.
Examples of metals for which DNA aptamers have been developed include Cadmium, Nickel, Palladium ion, Uranyl ion, and Zinc.
Examples of biologics and signaling molecules for which DNA aptamers have been developed include Acetylcholine, Biotin, cAMP, Cellulose, Cholic acid, CoA, Cortisol, Cyanocobalin (vitamin B12), Dehydroisoandro sterone-3-sulfate, Deoxy-corticosterone-21 glucoside, Deoxycholic acid sodium salt, Dopamine, Flavin, Fructose, Galactose, Glucagon, Glucose, Hemin, Hormone Abscisic Acid, N-acetylneuraminic acid, n-glycolylneuraminic acid (neu5gc), Nicotinamide, R-Thalidomide, Sialyl Lewis X, Sialyllactose, Sphingolipid S1P, Sphingosylphosphorylcholine, Steroid, Thiamine pyrophosphate, Thyroxine hormone, thyroxine hormone, Urea, Vasopressin, Vitamin D, Zeatin, and β-estradiol.
Examples of nucleosides and nucleotides for which DNA aptamers have been developed include 8-hydroxy-2′-deoxyguanosine, Adenosine, ADP, AMP, ATP, GMP, GTP, Guanine, and Xanthine.
Examples of synthetic small molecules for which DNA aptamers have been developed include 4-chloroaniline (4-CA), Biotin pyridocarboxamide derivative, Bis-boronic acid receptor, L-tyrosinamide, Methylphosphoic acid, N-methyl mesoporphyrin IX, P-nitrobenzene sulfonyl, and Tartrate.
The crosslinking reaction may be carried out by combining collagen and a DNA aptamer as disclosed herein at relative amounts effective to produce collagen fibers. 8 to 30% mass fraction of ssDNA in solution.
The crosslinking reaction may be carried out at a temperature according to the judgment of those of skill in the art. In certain embodiments, the crosslinking reaction is carried out at about 0-50° C., about 20-50° C., about 20-45° C., about 20-40° C., about 20-35° C., or about 20-30° C. In other embodiments, the crosslinking reaction is carried out at about 0° C., about 5° C., about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., about 40° C., about 45° C., or about 50° C. In particular embodiments, the crosslinking reaction is carried out at about 20-40° C.
The crosslinking reaction may be carried out at a pH according to the judgment of those of skill in the art. For example, it is well-known in the art that crosslinking agents are effective at crosslinking at a particular pH or ranges of pH. In certain embodiments, the crosslinking reaction is carried out at a pH of about 6-12, about 7-12, about 7-11, about 7-10, or about 7.2-10. In other embodiments, the crosslinking reaction is carried out at a pH of about 6, about 7, about 7.2, about 9, about 10, about 11, or about 12.
The crosslinking reaction may be carried out for a period of time according to the judgment of those of skill in the art. In certain embodiments, the crosslinking reaction is carried out for about 1 minute, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 16 hours, about 20 hours, about 24 hours, about 40 hours, about 48 hours, or about 72 hours.
The concentration of DNA aptamer used in the crosslinking reaction may be a concentration according to the judgment of those of skill in the art. In certain embodiments, the concentration of the DNA aptamer is about 0.00005-0.0005%, about 0.0001-0.001%, or about 0.00025-0.0025%.
Additional aspects and embodiments of DNA aptamers and protein targets described herein can be found in the art using ordinary skill and routine experimentation. For example, additional aptamers, aptamers configured to bind other targets, and protein targets can be found by searching publicly-available databases such as the Apta-Index™, which is freely and publicly available on the World Wide Web at https://www.aptagen.com/apta-index/. Searches can be performed, for example, by aptamer chemistry, target category, length, and other restrictions. Aptamers can then be found and tested using routine skill in the art.
The collagen starting material used for producing crosslinked collagen material of the present invention can be a collagen or collagens of any type. In certain embodiments, the crosslinked collagen material of the present invention is produced from a collagen starting material comprising a fibril forming collagen. Fibril forming collagens include type I, type II, type III, type V, and type XI collagens. In other embodiments, the crosslinked of the present invention is produced from a collagen starting material comprising a fibril associated collagen. Fibril associated collagens include type IX, type XII, type XIV, type XVI, type XIX, and type XXI collagens. In other embodiments, the crosslinked collagen material of the present invention is produced from a collagen starting material comprising a sheet forming collagen. Sheet forming collagens include type IV, type VIII, and type X collagens. In yet other embodiments, the crosslinked collagen material of the present invention is produced from a collagen starting material comprising a beaded filament collagen or an anchoring fibril collagen. Beaded filament collagens and anchoring filament collagens include type VI collagen and type VII collagen, respectively. Other collagen types useful in the present methods include type XIII, type XV, type XVII, type XVIII, type XX, type XXII, type XXIII, type XXIV, type XXV, type XXVI, type XXVII, and type XXVIII collagen. (See Haralson and Hassell, Extracellular Matrix, A Practical Approach, 8-11, Oxford University Press, 1995, the contents of which is hereby incorporated by reference in its entirety.) In a particular embodiment, a fibril forming collagen (i.e., type I, type II, type III, type V, or type XI collagen) is the collagen starting material used to produce crosslinked collagen according to the methods of the present invention.
In one embodiment, the collagen starting material useful for producing crosslinked collagen material is recombinant collagen. In another embodiment, the collagen starting material useful for producing crosslinked collagen material is recombinant human collagen. The use of any single type of recombinant collagen (e.g., recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, etc.) or any mixture of more than one type of recombinant collagen (e.g., a mixture of recombinant type I collagen and recombinant type III collagen) as the collagen starting material for producing a crosslinked collagen material is specifically contemplated by the present invention. Recombinant collagens and methods of their production have been described in, e.g., International Publication Nos. WO 2006/052451 and WO 1993/007889, each of which is hereby incorporated by reference in its entirety.
Production of other collagens suitable for use in the present compositions and methods can be specifically engineered using molecular biology techniques known to one of skill in the art. Such collagens can be modified by, e.g., an alteration in the polypeptide coding sequence, including deletion, substitutions, insertions, etc., to increase resistance to degradation. For example, recombinant collagens with alterations in the amino acid sequence at specific protease cleavage sites can be produced. Accordingly, in one embodiment, the present invention provides novel compositions comprising collagen, wherein the collagen is a recombinant Type III collagen.
The methods of the present invention are useful for producing crosslinked collagen materials using recombinant collagen (e.g., recombinant human collagen) as the collagen starting material. Unlike naturally-derived collagens, recombinant collagens lack intermolecular and intramolecular crosslinks that, if present, help stabilize the collagen material (including collagen fibrils) under conditions suitable for various crosslinking reactions, including, for example, basic pH conditions (e.g., pH≥8) or increased temperature (e.g., temperature≥40° C.). Under such conditions, recombinant collagens and, in particular, recombinant collagen fibrils made from recombinant collagens, are unstable, resulting in fibril dissolution and triple helix melting.
The present invention provides crosslinked collagen materials. In some embodiments, the invention provides crosslinked recombinant collagen suitable for implantation into a human or animal body. Such a crosslinked recombinant collagen implant is suitable for medical or cosmetic use. Typically, crosslinked recombinant collagen according to the invention is implanted or injected into various regions of the skin or dermis, depending on the particular application or cosmetic procedure, including dermal, intradermal, and subcutaneous injection or implantation. The crosslinked collagen materials of the present invention can also be injected or implanted superficially, such as, for example, within the papillary layer of the dermis, or can be injected or implanted within the reticular layer of the dermis. Materials for injection or implantation into the skin, in particular for cosmetic benefit, are often referred to in the art as “dermal fillers”. Accordingly, in one embodiment, a dermal filler, typically a cosmetic dermal filler, comprising crosslinked recombinant collagen according to the invention is provided.
The crosslinked collagen materials of the present invention may be used to produce implantable collagen compositions. Production of implantable collagen compositions has been described in, e.g., International Publication No. WO 2006/052451, the contents of which is hereby incorporated by reference herein in its entirety. In certain embodiments, the present invention provides implantable collagen compositions, comprising at least one crosslinked collagen material. The crosslinked collagen material can be any crosslinked collagen of the invention, for instance crosslinked “fibril forming” collagen materials prepared by one of the methods described herein. In one aspect, the implantable collagen composition comprises crosslinked recombinant type III collagen material.
The crosslinked collagen materials of the present invention can be formulated or used at any concentration useful to those of skill in the art. In certain embodiments, the formulations of the materials of the invention comprise 0.03-0.3 mg/ml, 1-10 mg/ml.
It is understood that the compositions of the present invention can include additional components suitable to the particular formulation. For example, in certain embodiments, the implantable compositions of the present invention are intended for injection and are formulated in aqueous solutions. The compositions can be formulated to include pharmaceutically acceptable carriers and excipients. Such carriers and excipients are well-known in the art and can include, e.g., water, phosphate buffered saline (PBS) solutions, various solvents, and salts, etc., for example, physiologically compatible buffers including physiological saline buffers such as Hanks solution and Ringer's solution.
The amount of crosslinked collagen material appropriately included in a particular formulation is determined as standard in the art for such formulations, and is dictated by the intended use. In certain embodiments, the present invention provides implantable compositions comprising crosslinked collagen material wherein the collagen material is in aqueous solution at a concentration between about 0.03 to about 10 mg/ml.
The crosslinked collagen materials provided herein can be used in any method known or contemplated by those skilled in the art. In particular, the present crosslinked collagen materials can be used in any of the numerous medical and cosmetic applications, including tissue augmentation procedures, in which collagen is currently used. The present crosslinked collagen materials are suitable for use in tissue augmentation procedures. Use of the present crosslinked collagen materials in cosmetic as well as in medical procedures is specifically provided.
In one aspect, the present invention provides implantable compositions containing crosslinked collagen materials suitable for use in soft tissue augmentation procedures. The present compositions can be implanted or injected into various regions of the skin or dermis, depending on the particular application or cosmetic procedure, including dermal, intradermal, and subcutaneous injection or implantation. The crosslinked collagen materials of the present invention can also be injected or implanted superficially, such as, for example, within the papillary layer of the dermis, or can be injected or implanted within the reticular layer of the dermis.
In addition to soft tissue augmentation, use of the crosslinked collagen materials for hard tissue augmentation is provided by the present invention. The present crosslinked collagen materials are useful in various hard tissue augmentation applications, including, for example, as a bone-void filler, dental implant, etc.
Cosmetic uses of the crosslinked collagen materials of the present invention include treatment of fine lines, such as fine superficial facial lines, wrinkles, and scars, as well as treatment of pronounced lines, wrinkles, and scars. In some aspects, the crosslinked collagen materials of the present invention are used for other cosmetic uses, including treatment for or reducing transverse forehead lines, glabellar frown lines, nasolabial fold, vermilion border, periorbital lines, vertical lip lines, oral commissure, etc., as well as defining the lip border. The crosslinked collagen materials of the present invention are also useful for correcting contour deformities and distensible acne scars, or for treating other tissue defects, such as, for example, atrophy from disease or trauma or surgically-induced irregularities.
In certain embodiments, the crosslinked collagen materials of the present invention are used for surgical procedures involving tissue augmentation, tissue repair, or drug delivery. In some aspects, the crosslinked collagen materials are used for tissue augmentation in conditions such as urinary incontinence, vasicoureteral reflux, and gastroesophageal reflux. For example, crosslinked collagen materials of the present invention may be used to add tissue bulk to sphincters, such as a gastric or urinary sphincter, to provide proper closure and control. In instances of urinary incontinence, such as stress incontinence in women or incontinence following a prostatectomy in men, the crosslinked collagen materials of the invention may be provided to further compress the urethra to assist the sphincter muscle in closing, thus avoiding leakage of urine from the bladder.
Similarly, gastroesophageal reflux disease (GERD, also known as peptic esophagitis and reflux esophagitis) is a disorder that affects the lower esophageal sphincter, the muscle connecting the esophagus with the stomach. GERD occurs when the lower esophageal sphincter is incompetent, weak, or relaxes inappropriately, allowing stomach contents to flow up into the esophagus (i.e., reflux). Malfunction of the lower esophageal sphincter muscles, such as that resulting from muscle tonal loss, can lead to incomplete closure of the lower esophageal sphincter, causing back up of acid and other contents from the stomach into the esophagus. Poor response to dietary modification or medical treatment may require surgery to correct the dysfunction. In one embodiment, crosslinked collagen materials of the present invention are used in such procedures and, for example, are injected into the area of the esophageal sphincter to provide bulk to the lower esophageal sphincter.
In other embodiments, the crosslinked collagen materials of the invention are used to fill or block voids and lumens within the body. Such voids may include, but are not limited to, various lesions, fissures, diverticulae, cysts, fistulae, aneurysms, or other undesirable voids that may exist within the body; and lumens may include, but are not limited to, arteries, veins, intestines, Fallopian tubes, and trachea. For example, an effective amount of the present material may be administered into the lumen or void to provide partial or complete closure, or to facilitate repair of damaged tissue.
In other aspects, tissue repair is achieved by providing the crosslinked collagen material of the present invention to an area of tissue that has been diseased, wounded, or removed. In some embodiments, crosslinked collagen materials of the invention are used to fill in and/or smooth out soft tissue defects such as pockmarks or scars. In such cases, a formulation of the present invention is injected beneath the imperfection. The improved persistence of the present crosslinked collagen materials would be beneficial, e.g., by reducing the number and frequency of treatments required to obtain a satisfactorily result. In certain embodiments, the crosslinked collagen materials are used for intracordal injections of the larynx, thus changing the shape of this soft tissue mass and facilitating vocal function. Such use is specifically provided for the treatment of unilateral vocal cord paralysis. Further, the present invention provides use of the crosslinked collagen materials in mammary implants, or to correct congenital anomalies, acquired defects, or cosmetic defects.
The present crosslinked collagen materials can also be used in various surgical or other procedures for remodeling or restructuring of various external or internal features, e.g., plastic surgery for corrective or cosmetic means, etc.
In any of the embodiments described above, the present crosslinked collagen materials may be used for drug delivery, for example, to deliver drugs to an injection site. The drugs can be delivered in a sustained manner from an in vivo depot formed by the crosslinked collagen upon injection of an implantable composition of the present invention. Drugs delivered in this manner may thus enhance tissue repair, and could provide additional therapeutic benefit.
In additional embodiments, the invention further contemplates incorporation of cells into the crosslinked collagen materials to provide a means for delivering cells to repopulate a damaged or diseased tissue or to provide products synthesized by the cells to the tissues surrounding the injection site.
In any of the embodiments described above, the crosslinked collagen materials of the present invention may be delivered or administered by any suitable method known or contemplated by those of skill in the art. The invention specifically contemplates delivery by injection, e.g., using a syringe. In some embodiments, the crosslinked collagen materials may additionally contain a biocompatible fluid that functions as a lubricant to improve the injectability of the formulation. The crosslinked collagen materials of the invention can be introduced into the tissue site by injection, including, e.g., intradermal, subdermal, or subcutaneous injection.
Collagen loaded with plasmid DNA (pDNA) vectors has been shown to facilitate cellular uptake of the vector. At the same time, calcium phosphate nanoparticles coated with pDNA vectors are a classical strategy for non-viral transfection. As described herein, a DNA-collagen complex material made with a pDNA vector that has then been subsequently mineralized with nanoparticles of calcium phosphate may impart greater or comparable transfection efficiency to each separate strategy.
Methods of transfection using crosslinked collagen materials as described herein can be utilized to introduce exogenous agents (for example nucleic acids, plasmid vectors, viral vectors, and the like) into cells in need thereof. Cells in need thereof can be mammalian cells, for example osteoblasts.
In an embodiment, the calcium phosphate has been precipitated by mixing aqueous solutions of sodium chloride, sodium phosphate dibasic, and calcium chloride with up to these final concentrations after mixing. 36 mm NaCl, 8 mm Na2HPO4, 15 mm CaCl2.
One embodiment of the present invention provides kits comprising the crosslinked collagen materials of the invention. For example, the present invention provides kits for augmenting or replacing tissue of a mammal. The kits comprise one or more crosslinked collagen materials of the present invention in a package for distribution to a practitioner of skill in the art. The kits can comprise a label or labeling with instructions on using the crosslinked collagen material for augmenting or replacing tissue of a mammal according to the methods of the invention. In certain embodiments, the kits can comprise components useful for carrying out the methods such as means for administering a crosslinked collagen material such as one or more syringes, canulas, catheters, needles, etc. In certain embodiments, the kits can comprise components useful for the safe disposal of means for administering the crosslinked collagen material (e.g. a ‘sharps’ container for used syringes). In certain embodiments, the kits can comprise crosslinked collagen material in pre-filled syringes, unit-dose or unit-of-use packages.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Gennaro, A. R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J. G., Limbird, L. E., and Gilman, A. G., eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Weir, D. M., and Blackwell, C. C., eds. (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, C. R., and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag.
In embodiments according to the present disclosure, applications of the present disclosure include but are not limited to:
Models of drug discovery or other in vitro assays for bone and/or calcification/mineralization.
Aspects of the present disclosure also have dental aspects as teeth are mineralized tissue, and DNA of complexes are described herein can bind to a mineral (for example calcium phosphate or hydroxyapatite) to create more of a bone mimetic material.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
The effect of premixing aptamer and collagen versus pre-conjugate of aptamer followed by addition of collagen was examined.
scrambled=5AmMC6/TAA AAC GCG CTT AAG CTG GTG TTA CTC GAG CGG TCT TCT ATT GAA ATA ATT TCT GAA GGC ACA CGA CAT ATG ATC TTC AG (SEQ ID NO:1). 5AmMC6 specifies a terminal amino group with 6 carbon spacer was conjugated to the 5′ end of the oligonucleotide sequence.
Mixtures of scrambled aptamer (1 μM) were mixed with rat tail type I collagen (0.3 mg/mL). Mixtures were at 10, 30, 50% volume fraction collagen. Mixtures were incubated at room temperature for 24 hours prior to surface conjugation. Solutions were used to conjugate to surface with 20 μM sulfo-SANPAH and incubated at room temperature for 24 hours. Surfaces were also conjugated with 1 and 2 μM solutions of scrambled aptamer and 20 μM sulfo-SANPAH and then incubated with 0.3 mg/mL and 0.6 mg/mL solutions of rat tail type I collagen.
As show in
With pre-conjugation with aptamer, there was no discernable fiber formation at all treatments. Surface wetting character observed to be changed from that of only an aptamer conjugated surface, which indicates collagen has adsorbed to the surface. The aptamer needs to have free mobility for fiber formation.
In 96 well plate, mixtures of scrambled aptamer (1 μM) were mixed with rat tail type I collagen (0.3 mg/mL). Mixtures were at 10% increments from 0% to 100% volume fraction collagen with 2 replicates. Turbidity measured over time immediately following mixing for six hours at 5 minute increments at 400 nm using a BioTek plate reader in absorbance mode. Data was fit with 3rd order interactive regression model with factors of time, collagen fraction, and aptamer type (
Turbidity showed little change with time (
In 96 well plate, mixtures of scrambled aptamer (1 μM) were mixed with rat tail type I collagen (0.3 mg/mL). Mixtures were at 10% increments from 0% to 100% volume fraction collagen with 2 replicates. Mixtures were incubated at room temperature for 24 hours. 10 μL aliquots of 30 and 70% volume fraction collagen were taken and placed on slides. They were then stained with ethidium bromide homodimer.
Fibers were stained for DNA at high and low concentrations of collagen. Structurally fibers looked fuzzy at 30% collagen and more whispy/defined at 70% collagen (
In 96 well plate, mixtures of scrambled aptamer (1 μM) were mixed with rat tail type I collagen (0.3 mg/mL). Mixtures were at 25% increments from 0% to 100% volume fraction collagen with 1 replicate. Solutions were incubated at room temperature for 2 hours and then SYBR Safe DNA stain was added in a 2:1 ratio to the aptamer-collagen solutions and incubated for 30 minutes in the dark at room temperature. The solutions were centrifuged at 2000 g for 5 minutes and the supernatant was added to a 96 well plate giving 2 repeats per solution. Fluorescent intensity was measured by exciting at 488 nm and reading emission at 520 nm.
Standard curves were linear. The percent DNA bound increased with increased amount of collagen in solution (
Calculated using mFold hosted by The RNA Institute at the University of Albany SUNY. Conditions for calculations were 25° C., 10 mM [Na+], oligomer corrected.
Three thermodynamically stable configurations were found. Two had hairpins, and 1 had 2 hairpins (
At first, ssDNA oligomer length appeared to have no effect when measured as the amount of bound DNA per available collagen on a mass per mass basis (
Fibers formed with varying density and size distribution for different volume fractions of collagen, favoring volume fractions of collagen that equated to mass fractions of DNA in solution in the range of 8-30%. This corresponded to DNA-collagen binding greater than 0.05 μg bound ssDNA/μg collagen. However, as shown by the 90 nucleotide ssDNA, there is an optimal range for fiber formation. For a mass fraction of DNA in solution of ˜45%, no fibers were observed; instead, a few faint ssDNA rich globs were present potentially the result of ssDNA self-aggregation and/or a lack of sufficient collagen in solution
As shown in
As shown in
These results showed that the amount of mineral bound to the dna-collagen fibers can be dependent on the sequence. Later experiments have shown that the surface coverage for fibers is the same for different dna sequences of the same length. These results are referring to
Many of the most structurally robust complexes in the body are composed of mineralized tissues such as tendon, cartilage, dentin, and bone. Mineralized tissues have been a sustained challenge in tissue engineering fields due to their enhanced structural properties at relatively low densities. Our group has recently discovered that DNA aptamers form fibral complexes with collagen type I. There has also been evidence found
Aspects of methods of the present disclosure are shown in
Mineralization progression is shown in
Quantification of mineralization presented in the graphical representation above (
DNA is a highly polyanionic biomolecule that interacts with both collagen and hydroxyapatite. Harnessing these complexes, the combination of a hydroxyapatite templating DNA aptamer and type I collagen rapidly synthesizes mineralized self-assembled DNA aptamer-collagen complex fibers and 3D gels. These complexes are hierarchical, interwoven, curly nanofibrils resembling native extracellular matrix, which can mineralize an interpenetrating nanocrystalline hydroxyapatite phase. On demand mineralization is possible enabling temporal control of the process. Surprisingly, mineralization is independent of DNA sequence allowing for other DNA aptamers to be used in the synthesis of these mineralized complexes affording them even greater biofunctionality without loss of mineralization potential. In response to this promising biomaterial platform, primary human osteoblasts differentiate towards an osteocyte-like lineage important for biomaterial osseointegration. These fibers and gels have promise not only as osteoconductive coatings and scaffolds, but as coatings and scaffolds for any tissue using this new class of biofunctional materials.
Paramount to the challenges facing bone tissue engineering is recapitulating the hierarchical structure of bone.1 Biomimetic approaches have been pioneered to achieve aligned, intrafibrillar hydroxyapatite mineralization of collagen gels utilizing polyanionic species, namely polyaspartate.2-5 This method relies on anionic species to form an amorphous calcium phosphate precursor phase, which can penetrate collagen fibril D-spacings by capillary action, termed the polymer-induced liquid-precursor (PI LP) process.3
Interestingly, DNA is a highly polyanionic biomolecule that interacts with both collagen and hydroxyapatite. In the 1970s, DNA was shown to bind to type I, II, and IV collagens and later in the late 1990s shown to form self-assembled aggregates ranging from nanoparticles to micron sized fibers.6-9 DNA-collagen complexes have since been utilized for gene delivery applications focusing on nanoparticle sized aggregates for efficient cellular endocytosis as well as plasmid DNA eluting collagen scaffolds.10-12 In fact, this complex protects DNA from enzymatic attack and is a possible mechanism for preserving DNA in ancient bone.12,13 In the same vein, DNA and calcium phosphate non-specifically bind together, which has been utilized for gene delivery by the endocytosis of calcium phosphate nanoparticles coated with plasmid DNA.14-17 Few groups have combined these three components to improve gene delivery efficiency with some success; however, these complexes have not been applied as strategies for tissue engineering.18-20
These interactions are ubiquitous for nucleic acids and other structural forms of DNA, namely DNA aptamers—engineered sequences of single stranded DNA (ssDNA) designed to interact with a specific target.9,21-25 Recently, using precipitation systematic evolution of ligands by exponential enrichment a series of hydroxyapatite templating DNA aptamers (HAP aptamers) have been reported to facilitate mineral formation and to bind hydroxyapatite.26,27 Likewise, our group has shown that ssDNA on the length scale of DNA aptamers binds and forms ssDNA- collagen complex fibers with promising utility for mediating healthy vascular cell behavior.28 Hierarchical, fibrous architectures endow biopolymers with their unique chemomechanical properties, which are necessary for regulating cellular phenotype.29 Thus, being able to recapitulate these biophysical cues is paramount to engineering extracellular matrix (ECM) mimetics. Herein, we report for the first time the combination of a hydroxyapatite templating DNA aptamer and collagen to form mineralized self-assembled DNA aptamer-collagen complex fibers and 3D gels. These fibers and gels have promise not only as osteoconductive coatings and implants, in vitro 3D bone and calcified tissue models, but as coatings and scaffolds for any tissue using this simple, versatile biomaterial platform to engineer mimetic ECM.
Previously, we have shown that self-assembled ssDNA-collagen complex fibers are dependent on ssDNA sequence length and the ratio of ssDNA to collagen in solution.28 Likewise, because DNA has an affinity to non-specifically bind with HAP, for all studies we used a random sequence ssDNA of the same length as the HAP aptamer as a reference to determine the effect of the DNA aptamer as compared to the effect of purely ssDNA, respectively.15 DNA aptamer-collagen complex self-assemblies were synthesized by mixing different volume fractions (0-100%) of 0.3 to 3.0 mg/mL rat tail tendon type I collagen (Corning) diluted in sterile deionized water with either 1 to 10 μm random sequence ssDNA (5′-TAA AAC GCG CTT AAG CTG GTG TTA CTC GAG CGG TCT TCT A-3′; SEQ ID NO:12) or 1 to 10 μM HAP aptamer (5′-CAG GTG GGC GCG CTG TCG TGG GTG CTC GGG TGC GGT TGG G-3′; SEQ ID NO:13) (Integrated DNA Technologies) both diluted in sterile deionized water.27 DNA oligomer properties are presented in Table 1. Complexes spontaneously formed upon mixing and were incubated at room temperature overnight followed by storage in solution at 4° C. until needed.
The amount of ssDNA in the DNA aptamer-collagen complex self-assembled fibers for different volume fractions of type I collagen and ssDNA oligomer was assessed fluorometrically by measuring the intensity of a fluorescent DNA stain. Solutions of different volume fractions were diluted at a 1:2 volume ratio of fiber solution to SYBR Safe DNA stain (Invitrogen). The stain concentrate was diluted first at a 1:10,000 volume ratio in deionized water. Then the mixtures were incubated protected from light for 30 minutes at room-temperature. After incubation, the mixtures were centrifuged at 2000 g for five minutes. Supernatant was plated in duplicate into black walled 96 well microplates and fluorescence intensity was measured by exciting at 488 nm and detecting at 520 nm using a UV-Vis Synergy H1 plate reader (BioTek). ssDNA in the supernatant was quantified using a standard curve fit with a 4-parameter logistic regression to correlate fluorescent intensity to ssDNA concentration. Individual standard curves were prepared for each ssDNA to account for any differences in DNA stain interaction with the two ssDNA sequences due to their secondary structure. Bound ssDNA for the different volume fractions was calculated as the difference of the initial amount of ssDNA in the solution and the amount of ssDNA measured in the supernatant.
Fibers were immobilized to untreated polystyrene well plates (Eppindorf) using sulfo-SANPAH (Proteochem) heterobifunctional crosslinker. Briefly, well plates were treated with 20 μM sulfo-SANPAH diluted in deionized water and irradiated with 365 nm UV light for 10 minutes to activate the nitrophenyl azide group of the crosslinker for bonding to the polystyrene surface. Then the wells were rinsed three times with sterile deionized water and incubated with the fiber solutions overnight at room temperature. During incubation, the sulfo-NHS ester from the sulfo-SANPAH is able to non-specifically react with primary amines present on the DNA aptamer-collagen complex self-assembled fibers. Following incubation, the wells were rinsed three times with sterile deionized water and stored in deionized water at 4° C. until needed.
Fiber morphology for the different volume fractions of type I collagen and DNA aptamer was qualitatively assessed by phase contrast and fluorescence microscopy using immobilized fibers. For fluorescence microscopy, immobilized fibers were stained with SYBR Safe DNA stain (Invitrogen) diluted at a 1:10,000 volume ratio in deionized water. The fibers were incubated with the stain protected from light for at least 30 minutes and then imaged using an epifluorescence Nikon TE-2000U inverted microscope.
Immobilized fibers were mineralized by incubating overnight at room-temperature with 500 μL of mineralizing solution having a final concentration of 25 mM NaCl (Sigma Aldrich), 8 mM Na 2 HPO4 (Sigma Aldrich), 15 mM CaCl2 (Sigma Aldrich) added sequentially. Then the wells were rinsed with deionized water and stored at 4° C. in deionized water until needed.
Mineralized fibers were formed in a one-step process by preparing two precursor solutions: 1) DNA aptamer and CaCl2 and 2) collagen, NaCl, Na2 HPO4 together. These two solutions were then mixed together to give an effective final concentration of 10 mM CaCl2, 36.67 mM NaCl, 4 mM Na2HPO4, 0.1-0.9 μM aptamer, and 0.03-0.27 mg/mL collagen with a final volume of 250 μL. Mineralized gels were formed in a one-step process by preparing two precursor solutions: 1) DNA aptamer and CaCl2 and 2) collagen, NaCl, Na2 HPO4 together. These two solutions were then mixed together to give an effective final concentration of 10 mM CaCl2, 36.67 mM NaCl, 4 mM Na2HPO4, 1-9 μM aptamer, and 0.3-2.7 mg/mL with a final volume of 500 μL.
Mineralized fibers and gels were formed in a two-step process by preparing fibers and gels as previously described in Section 2.1. Then the fibers and gels were incubated in 250 μL and 500 μL of mineralizing solution with a final concentration 10 mM CaCl2, 36.67 mM NaCl, 4 mM Na2HPO4, respectively.
Mixtures were incubated at room-temperature and removed from the mineralization solution after 1, 3, and 6 days. This was done by centrifugation to pellet the assemblies, rinsing with deionized water, and then resuspending in 100% molecular biology grade ethanol (Fisher Scientific) thereafter for storage at room-temperature until analysis by either x-ray diffraction, transmission electron microscopy, or scanning electron microscopy. Resuspension in ethanol was done to facilitate sample preparation for those analysis techniques.
After mineralization, immobilized fibers were stained with 500 μL of 40 mM alizarin red stain (Sigma Aldrich) for 30 minutes at room temperature. Then washed with deionized water three times. Stained fibers were imaged by phase contrast microscopy. Then, 250 μL of 10% (w/v) cetylpyridinium chloride (Sigma Aldrich) was added to release the calcium-bound stain and agitated on an orbital shaker for 5 minutes. The solution was collected and centrifuged at 10,000 g for 10 minutes. 100 μL aliquots of the supernatant were plated in duplicate and read at 550 nm using a Synergy H1 Spectrophotometer. Absorbance was converted to alizarin red stain concentration using a standard curve fit with a 4-parameter logistic regression model.
The change in crystallinity of one- and two-step mineralized gels incubated for 1, 3, and 6 days in mineralization solution was assessed by X-ray diffraction (XRD) using a Panalytical Xpert-Pro system. Gels were placed on a zero-background holder and irradiated by monochromatized Cu Kα X-ray radiation from an Empyrean Cu LFF DK406691 fixed anode X-ray tube, operated at 45 kV and 40 mA, together with a diffractometer scan step size of 2θ=0.0167°, and a dwell time of 0.127 s step-1, over a 28 range of 10-60°. Diffraction patterns were processed at the same time in Spectragryph v1.2.10 to perform background removal and smoothing using a 2nd order scattering baseline followed by a 100 interval, rectangular moving average filter.
DNA aptamer-collagen complex fiber ultrastructure was assessed by negative staining fibers with phosphotungstic acid. Fibers were synthesized by mixing 0.3 mg/mL rat tail tendon type I collagen (Corning) diluted in sterile deionized water (20% volume fraction of mixture) with either 1 μM random ssDNA or HAP aptamer diluted in sterile deionized water (80% volume fraction of mixture). Fibers were allowed to form for 1 hour at room-temperature and then centrifuged at 2000g for 5 minutes. The supernatant was removed and replaced with 1% phosphotungstic acid aqueous staining solution (Electron Microscopy Sciences) at pH 7.0. Fibers were incubated in the staining solution for 1 hour at room temperature and then pipetted onto lacey carbon copper grids (Electron Microscopy Sciences). The grids were then air-dried. Stained fibers were imaged by transmission electron microscopy (TEM). Fiber diameters were measured in Gwyddion 2.51 using line profiles defined perpendicular to the fiber longitudinal axis. Mean fiber diameters were compared using an unpaired t test with Welch's correction with an alpha-level of 0.05 in GraphPad Prism 8.3.0 (538).
Crystallites from one- and two-step mineralized fibers incubated for 1, 3, and 6 days in mineralization solution were imaged and assessed by TEM and selected area electron diffraction (SAED). Fiber solutions in ethanol were pipetted onto lacey carbon copper grids (Ted Pella) and the remaining ethanol was allowed to evaporate.
All imaging was performed using a FEI Tecnai F20 S/TEM operated between 80-200 keV in brightfield (BF) and SAED modes. Electron diffraction patterns were indexed in ImageJ (National Institute of Health).
Gel morphology of one- and two-step mineralized gels incubated for 6 days in mineralization solution was assessed by scanning electron microscopy (SEM). Gels were placed and air-dried on circular cover glass and the cover glass was then adhered to SEM stubs using double-sided tape. Gels were then sputter coated with Au/Pd (7 kV, 10 mA, 30 mTorr, 60 s). SEM micrographs were taken using a Tescan MIRA3 field emission SEM with an operating voltage between 0.2-30 keV. The presence of calcium and phosphate in the gel was confirmed by energy dispersive X-ray spectroscopy (EDS) using a mounted EDAX Octane Pro EDS system. EDS spectra were collected at three separate points across the sample.
Human osteoblasts (HObs) (Cell Applications, Lot #3258, 52-year-old black female) were grown on tissue culture polystyrene in a humidified cell culture incubator kept at 37° C. and 5% CO2. Cells were used between passage number 4-6. HObs were cultured in fully supplemented Osteoblast Growth Medium (Cell Applications). Media was exchanged every 2-3 days.
20% volume fraction collagen solution fibers were formed for both the random ssDNA and HAP aptamer as previously described in Section 2.1. These fibers were immobilized to untreated polystyrene 24 well well-plates (Eppindorf) and mineralized in the same manner as previously described in Section 2.3, 2.5. HObs were seeded at 5,000 cells/cm in each well as well as a tissue-culture polystyrene control (Corning) in triplicate. HObs were cultured for 3 days without media renewal. Phase images were taken daily. After 3 days, cells were rinsed in phosphate-buffered saline and fixed in 4% formalin for 15 minutes. Then rinsed three times with 5 minutes of agitation on an orbital shaker between rinses and stored in phosphate-buffered saline at 4° C. until immunocytochemistry was performed.
Immunocytochemistry was performed to visualize HObs morphology and protein expression. Cells were permeabilized with 0.1% Triton-X 100 in phosphate-buffered saline (PBS) for 10 minutes, then rinsed 3 times with PBS, and blocked for 30 minutes with 1% bovine serum albumin (BSA) in PBST (0.1% Tween 20 in PBS). Cells were then incubated at 4° C. overnight with a primary antibody for osteopontin (abcam, ab69498) at a 1:200 dilution in 1% BSA in PBST. Then the cells were rinsed 3 times with 1% BSA in PBST and incubated for 1 hour at room-temperature in the dark with a secondary antibody (abcam, ab150080) at a 1:200 dilution in 1% BSA in PBST. Following incubation, the cells were rinsed 4 times with 1% BSA in PBST and mounted using ProLong Diamond with DAPI (Invitrogen). In a similar manner to stain for cytoskeletal F-actin, after the permeabilization step cells were incubated with Texas-Red phalloidin (Invitrogen) following the manufacturer's instructions and mounted using ProLong Diamond with DAPI (Invitrogen). Fluorescence microscopy was conducted using an epifluorescence Nikon TE-2000U inverted microscopy.
Gels were prepared by mixing type I rat tail collagen to a final concentration of mg/mL and either the random ssDNA or HAP aptamer to a final concentration of 8 μM in complete Osteoblast Growth Medium (Cell Applications) to a final volume of 500 μL. HObs were seeded at 62,500 cells at passage 6. Gels were prepared in individual wells of a 24 well ultra-low attachment plate (Corning). The gels with embedded cells were then incubated overnight to promote cell attachment before mineralization (
Upon mixing dilute solutions of the HAP aptamer and type I collagen, DNA aptamer-collagen complexes spontaneously and rapidly self-assembled in aqueous solution. Fibers formed (
We next investigated whether these fibers could bind calcium phosphate mineral after forming. To do this, we immobilized the fibers to a polystyrene substrate using the heterobifunctional crosslinker sulfo-SANPAH. Fibers were immobilized to ease rinsing of the fibers during later processing and to remove any unincorporated ssDNA and collagen. We then incubated the fiber functionalized surface in a solution of NaCl and Na2HPO4 and initiated mineralization by the addition of CaCl2. Within minutes, visible calcium phosphate precipitated. Both the random ssDNA and the HAP aptamer bound mineral to their fibers, which was visualized by staining for Ca using alizarin red stain (
Interested by this competition and the potential interference of calcium phosphate precipitate on fiber formation, we investigated whether the formation of fibers and their mineralization would occur in a one-step synthesis process by mixing a solution of HAP aptamer and CaCl2 with a solution of collagen, NaCl, and Na2HPO4. As with the two-step synthesis (i.e. fibers formed and then mineralized), fiber formation readily occurred (<10 minutes) and these fibers displayed a bound mineral phase.
Not only DNA aptamer-collagen complex fibers, but also 3D gels were able to be produced. By increasing the ssDNA and collagen concentrations by 10 times, we achieved the spontaneous and rapid formation of DNA aptamer-collagen gels (
To visualize the fiber ultrastructure, unmineralized fibers were negatively stained using phosphotungstic acid and imaged by TEM. This stain is widely used for visualizing the D-spacing of aligned collagen fibrils.14 Unexpectedly, solid, compact fibrils were not observed. But rather we saw masses of very thin “spaghetti-like” fibrils consisting of highly intermixed, curved chains (
To characterize the mineralization of the fibers and gels in more detail, a combination of XRD, TEM, SAED, SEM, and EDS was performed. XRD patterns of two-step mineralized gels for both the random ssDNA (
Hydroxyapatite and octacalcium phosphate (OCP) have extremely similar d-spacings making it difficult to determine crystalline phase exclusively from diffraction patterns for randomly oriented crystallites.3,32 SAED patterns for mineralized fibers formed using both the random ssDNA (
3Values reported by Olszta et al. for natural equine bone, HAP (JCPDS 9-432), and OCP (JCPDS 79-0423)3
SEM of 6 day mineralized gels revealed a topographically rich surface akin to that of native extracellular matrix (ECM) with pits, pores, and striations (
We cultured primary human osteoblasts (HObs) on immobilized DNA aptamer-collagen complex fibers in both the mineralized and unmineralized state for each ssDNA sequence to gauge their response to the fiber biophysical cues. Remarkably, the HObs readily attached and within 24 hours began differentiating to an osteocytic lineage indicated by their extensive dendritic processes (
Going further, we were interested in HObs behavior in a 3D environment made using our DNA aptamer-collagen complex gels. Gels were synthesized by embedding the cells in the collagen solution followed by the addition of the random ssDNA or the HAP aptamer to initiate gelation. Cells took on the same dendritic morphology as for the 2D environment as well as forming an interconnected network throughout the gel (
We are the first to report the synthesis and utility of DNA aptamer-collagen complexes for their use in bone tissue engineering. It should be appreciated the simplicity and rapidity for synthesizing these ECM-mimetics. Simply put, they are made by mixing two solutions: a DNA aptamer solution with a collagen solution. The synthesis time is seconds to minutes. These complexes can be stored in a dry-state or aqueous-state in physiological buffer at room-temperature for extended periods of time.12 Moreover, these complexes are customizable being non-specific for DNA aptamer sequence such that DNA aptamers designed as agonists or antagonists could be used as well as being non-specific for other collagens such that atelocollagen, type II collagen, and type IV collagen could be used.6-9,24,38 These properties are paramount to achieving off-the-shelf, one-step, intra-operative tissue engineering solutions 39. Being multimodal (particles, fibers, gels) enables this biomaterial platform to support a breadth of applications from nucleic acid delivery (particles), coatings (fibers), and scaffolds (gels).
Importantly, is the promise of supplementing these architectures with other native biomolecules or additives to engineer more mimetic and functional ECM.40-42 We have started to do this by using a hydroxyapatite templating DNA aptamer to incorporate nanocrystalline hydroxyapatite into the complex for producing a biomimetic mineralized ECM. Nanocrystalline HAP mineralization occurred for short, monodisperse ssDNA irrespective of sequence. The mineralization also was extrafibrillar leading to fibers covered in randomly oriented HAP. Self-assembled peptide amphiphile nanofibers have been mineralized with the HAP c-axis aligned in the nanofiber longitudinal direction, such that our DNA aptamer-collagen complex fibrils may also be able to achieve such an arrangement by aligning the nanofibrils from their naturally curled state.43 In the context of bone tissue engineering this ubiquity for short, monodisperse ssDNA to mineralize as a DNA-collagen complex enables DNA aptamers for targeting other species to be used to form the complex without losing mineralizing potential as both the HAP aptamer and the random ssDNA complexes promoted HAP mineralization. Hence, future investigations are required to evaluate other DNA aptamer sequences for the maintenance of their secondary structures in mineralized DNA aptamer-collagen complexes.
TEM revealed our DNA aptamer-complexes to be hierarchical “spaghetti-like” fibrils (curled, intermixed nanofibrils at multiple length scales) with mean diameter of ˜7 nm. This would suggest the nanofibrils are on the scale of individual complex structures based on the model for the dsDNA-collagen complex being a central dsDNA double helix surrounded by five collagen triple helices 30. However, the nanofibril curls were across 10s and not 1000s of nanometers, which is contrary to the rigidity of a collagen triple helix with a length of 300 nm.30 Though, collagen triple helices are unstable at body temperature and for rat tail tendon type I collagen the denaturation point is as low as 28° C.44 Paired with our TEM observations of fibril curvature and mean diameter, we believe our DNA aptamer-collagen nanofibrils are not exclusively formed with triple helix collagen but have type I collagen monomeric chains to support the observed extreme fibril curvature. More detailed modeling of short ssDNA-collagen complex interactions needs to be completed to resolve the arrangement of the species that form them.
Mineralization was achieved as easily as the fiber and gel synthesis by either a one-step or two-step process. One-step mineralization enables the complete production of the mimetic ECM at once whereas the two-step process enables temporal control of the mineralization process. This temporal control enables this platform for evaluating the effects of progressive tissue calcification, for instance in the context of heart valvular and blood vessel calcification. Mineralization completed within 24 hours indicated by both XRD patterns and TEM micrographs. Protein-based PI LP mineralization requires appreciably more time to complete, suggesting that ssDNA-based mineralization works at an accelerated rate.3,5,45 Our DNA aptamer-collagen complexes demonstrate ECM mimetic nanotopographic pits, pores, and “poorly crystalline” HAP crystallites critical to successful osseointegration of biomaterials.39
Especially exciting is that DNA aptamer-collagen complexes rapidly promoted osteoblast differentiation towards an osteocytic phenotype. Osteocytes regulate bone remodeling and thus inducing differentiation to this cell type is ideal for facilitating osseointegration of the scaffold.42 Differentiation is a multistep process in which osteoblasts migrate into mineralized matrix and produce an interconnected network of dendritic processes.37,46,47 Because this effect was advanced by immobilized DNA aptamer-collagen complex fibers, this modality could be a successful osteoconductive coating for both orthopedic and dental implants leading to a quicker recovery and favorable integration of the implant. Combined with the proven mineralized cell-assembled tissue structures, DNA aptamer-collagen complex gels could be used as cell-laden large defect fillers. There is a significant need for in vitro models to aid in drug discovery. This platform could be used as an in vitro model for tissue calcification by utilizing other cell types such as vascular smooth muscle cells instead of osteoblasts to model vascular calcification. Only recently, has a biomimetic mineralized cell-laden model been developed focusing on mesenchymal stem cell differentiation.45 Future investigations of our system should be used to evaluate mesenchymal stem cell differentiation to compare the efficacy of our cell-assembled mineralized DNA aptamer-collagen complexes to this aim. Similarly, there is a need for addressing hard/soft interfaces paramount to effective connective tissue regeneration.48,49 Such interfaces could be formed by utilizing our two-step mineralization process with a concentration gradient.50 At the same time, the one-step synthesis and mineralization process affords DNA aptamer-collagen complexes injectability for point-of-site administration.51-53 Pertinent to tissue engineering, is potential for developing cell-assembled prevascularization by co-culture of endothelial and osteogenic cells.39 Osteoblasts exert an angiogenic microenvironment by their secretion of vascular endothelial growth factor.54 Likewise, osteoblasts produce nitric oxide, which supports a healthy, vasodilatory endothelial cell phenotype.55 Because of the demonstrated rapidly-formed cell-assembled tissue structures, we are hopeful this platform could quickly generate pre-vascularized mineralized bone tissue using peripheral blood derived endothelial progenitor cells as an autologous endothelial cell source and adipose-derived or bone marrow derived mesenchymal stem cells as an autologous osteogenic cell source.39 Nonetheless, further investigations are necessary to show the potential for DNA aptamer-collagen based-biomaterials to make these translational hopes a reality.
Here we show the rapid and facile synthesis of DNA aptamer-collagen nanofibril complexes as a new class of biofunctional materials for tissue engineering applications. Adding a mineralization solution before or after complex synthesis generates an interpenetrating hydroxyapatite phase affording the system temporal control of the mineralization process. These nanofibrous complexes remarkably resemble native extracallular matrix architecture with pits, pores, and striations. Moreover, formation and mineralization were independent of DNA aptamer sequence enabling a diversity of DNA aptamers to be used for targeting species of interest including cells, biomolecules, or ions without the loss of properties. Bone cells actively remodeled and matured supporting the mineralized variant of these complexes as a highly osteoconductive biomaterial. Although this work focuses on bone tissue engineering, DNA aptamer-collagen complexes should be viewed as a new class of biofunctional materials with broad applicability and potential to improve therapeutic outcomes in biomedical applications.
DNA is a highly polyanionic biomolecule that interacts with both collagen and hydroxyapatite. Harnessing these complexes, the combination of a hydroxyapatite templating DNA aptamer and type I collagen rapidly synthesizes mineralized self-assembled DNA aptamer-collagen complex fibers and 3D gels. These complexes are hierarchical, interwoven, curly nanofibrils resembling native extracellular matrix, which can mineralize an interpenetrating nanocrystalline hydroxyapatite phase. On demand mineralization is possible enabling temporal control of the process. Surprisingly, mineralization is independent of DNA sequence allowing for other DNA aptamers to be used in the synthesis of these mineralized complexes affording them even greater biofunctionality without loss of mineralization potential. In response to this promising biomaterial platform, primary human osteoblasts differentiate towards an osteocyte-like lineage important for biomaterial osseointegration. These fibers and gels have promise not only as osteoconductive coatings and scaffolds, but as coatings and scaffolds for any tissue using this new class of biofunctional materials.
Vascularization of engineered tissue is one of the hallmark challenges of tissue engineering. Leveraging our newly developed self-assembled DNA aptamer-collagen complex-based biomaterial platform, we used a VEGF-R2 targeting aptamer and its receptor agonist divalent assembly as the DNA component to form complex fibers. Human umbilical vein endothelial cells (HUVECs) quickly remodeled these fibers into tubulogenic structures over 72 hours. Moreover, DNA-collagen complexes composed of the divalent assembly promoted enhanced expression of von Willebrand factor (vWF), angiopoietin-2 (ANGPT-2), and matrix metalloproteinase-2 (MMP-2) by HUVECs as measured by either immunocytochemistry or ELISA. Endothelial cell phenotype was thought to be directed by both biochemical cues afforded by the agonist behavior of the divalent aptamer assembly as well as by the biophysical cues afforded by the complex fiber nanotopography. Collectively, these results support the development of an angiogenic endothelial cell phenotype stimulated by the DNA aptamer-collagen fibers. Thus, the combination of engineered DNA aptamer nanotechnology and DNA-collagen complexation phenomena is a promising biofunctional natural scaffold material system for tissue engineering and regenerative medicine applications.
Nucleic acids and proteins interact to change each other's function. One such interaction is the complexation of DNA and collagen.1-12 Fifty years ago, it was discovered that both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) readily bind to the cellular basement membrane.1,2,5 Specifically, DNA binds to type I, II, and IV collagens and not to fibronectin.1,2,5 DNA physical properties were major contributors to this complexation event. DNA length mediated the process favoring shorter DNA while being independent of DNA strandedness.9,12 These early investigations; though, were conducted using enzyme-linked immunosorbent assays (ELISA) and thus overlooked the possibility for observing any self-assembled structures resulting from the interaction of these two biomacromolecules.1,2,5,9,12 Not until about 30 years later were
DNA-collagen complexes observed to self-assemble into fibrils.13 Thus, began a renewed interest in the DNA-collagen complexation. Especially at the time for delivering cyclic plasmid DNA for gene transfection. For this application, nanosized DNA-collagen particles rather than fibers were more efficient and thus the fibrous assemblies were not heavily investigated thereafter.14-16 The types of structures could be tuned from nanoparticles to fibers by varying the relative amounts of DNA to collagen.14-18 From these earlier works it was found that dsDNA structure (linear or cyclic), molecular weight distribution, and purity all influenced fibril formation.19 All of these studies focused exclusively on large (>1,000 base pairs), random, dsDNA sequences; despite, the past ELISA data showing that collagen has an avidity for short (<200 base pairs).9,12
Already, DNA-collagen complexes have been used for delivery of plasmids and antisense oligonucleotides, for wound dressings, and for antimicrobial coatings.16,20,21 Though, these applications have not made use of recent advances in DNA nanotechnology. DNA has moved from being purely a genetic encoder to being a highly versatile, engineered biomolecule of tremendous utility.22,23 One such form of DNA is as short ssDNA aptamers, which are increasingly being used for their high affinity, stability, and versatility.24 In fact, engineering DNA aptamers as assemblies has enabled their ability to act as both receptor agonists and antagonists.25,26 Thus, there are many opportunities for fabricating highly biofunctional composite materials by incorporating DNA aptamers in the assembly of DNA-collagen complexes.
Only recently though have these assemblies been proposed as a versatile platform for tissue engineering and regenerative medicine.27 Within an aqueous environment, monodisperse ssDNA rapidly and spontaneously complexed with collagen forming stable fibers that mimic the architecture of the extracellular matrix.27 In fact, endothelial cells in direct contact with these complex fibers demonstrated enhanced expression of angiogenic markers.27 Given the challenge of vascularizing engineered tissue, this natural material system shows promise as an emerging scaffold biomaterial.28,29
Vascular endothelial growth factor (VEGF) is a highly potent growth factor principally responsible for stimulating angiogenesis by activating endothelial cells.30,31 We have shown that a vascular endothelial growth factor receptor 2 (VEGF-R2) targeting DNA aptamer sequence when assembled as two oligomers tethered together is able to activate the receptor leading to angiogenic behavior by endothelial cells.25 By taking advantage of engineered DNA aptamer nanotechnology and DNA-collagen complexation phenomena, we show that a VEGF-R2 agonist DNA aptamer divalent assembly interacts with collagen to form complex nanofibers for stimulating an angiogenic endothelial cell phenotype.
A VEGF-R2 targeting aptamer sequence was used in both a monovalent (single sequence) and divalent form (two single sequences tethered by a (PEG)6 linker). For all studies, we used a random sequence ssDNA of the same length and GC content as a control to determine the effect of the targeting and functionality of the DNA aptamer as compared to the effect of purely ssDNA, respectively.32 The VEGF-R2 targeting aptamer monovalent sequence is: ‘5-GAT GTG AGT GTG TGA CGA GCT ACG ACG TCT GGT GTA ATT TAT AAA GAC ACT GTG TAT ATC AAC AAC AGA ACA AGG AAA GG-3’ (SEQ ID NO:14) .The random ssDNA oligomer monovalent sequence is: ‘5-TAA TGA GAA GTA TGT GTA GAG TCA ATG AGA TAC GCA ATT GGG AAG ACA AGA GTA TTG ACT CGG ACT GAG TAC AAT CGT CC-3’ (SEQ ID NO:15). The VEGF-R2 targeting aptamer divalent assembly sequence is: ‘5-GAT GTG AGT GTG TGA CGA GCT ACG ACG TCT GGT GTA ATT TAT AAA GAC ACT GTG TAT ATC AAC AAC AGA ACA AGG AAA GG 3’-(PEG)6-3′ GG AAA GGA ACA AGA CAA CAA CTA TAT GTG TCA CAG AAA TAT TTA ATG TGG TCT GCA GCA TCG AGC AGT GTG TGA GTG TAG-5′ (two SEQ ID NO:14 linked with a PEG6 linker). The random ssDNA oligomer divalent assembly sequence is: ‘5-TAA TGA GAA GTA TGT GTA GAG TCA ATG AGA TAC GCA ATT GGG AAG ACA AGA GTA TTG ACT CGG ACT GAG TAC AAT CGT CC-(PEG)6-TAA TGA GAA GTA TGT GTA GAG TCA ATG AGA TAC GCA ATT GGG AAG ACA AGA GTA TTG ACT CGG ACT GAG TAC AAT CGT CC-3’ (two SEQ ID NO:15 linked with a PEG6 linker). DNA oligomer properties are presented in Table 3. ssDNA-collagen self-assembled fibers were synthesized by mixing different volume fractions (0-100%) of 0.3 mg/mL rat tail tendon type I collagen (Corning) with 1 μM of each ssDNA oligomer (Integrated DNA Technologies) both diluted in sterile deionized water. Fibers spontaneously formed upon mixing and were incubated at room temperature overnight.
Fibers were immobilized to untreated polystyrene well plates (Eppindorf) using sulfo-SANPAH (Proteochem) heterobifunctional crosslinker. Briefly, well plates were treated with 20 μM sulfo-SANPAH diluted in deionized water and irradiated with 365 nm UV light for 10 minutes to activate the nitrophenyl azide group of the crosslinker for bonding to the polystyrene surface. Then the wells were rinsed three times with sterile deionized water and incubated with the fiber solutions overnight at room-temperature. During incubation, the sulfo-NHS ester from the sulfo-SANPAH is able to non-specifically react with primary amines present on the DNA aptamer-collagen self-assembled fibers. Following incubation, the wells were rinsed three times with sterile deionized water and stored in deionized water at 4° C. until needed.
Fiber morphology for the different volume fractions of type I collagen and DNA aptamer was qualitatively assessed by phase and fluorescence microscopy using immobilized fibers. For fluorescence microscopy, immobilized fibers were stained with SYBR Safe DNA stain (Invitrogen) diluted at a 1:10,000 volume ratio in deionized water. The fibers were incubated with the stain protected from light for at least 30 minutes. To quantify surface coverage, full well images were taken with a Keyence BZ-X700 fluorescence microscope. Utilizing ImageJ software, the fibers were then identified using the threshold tool and the area coverage for each image was determined using the measure tool.
Green fluorescent protein expressing human umbilical vein endothelial cells (GFP-HUVECs) (Angio-Proteomie) and non-transfected human umbilical vein endothelial cells (HUVECs)(LifeLine Cell Technologies) were grown on tissue culture polystyrene at 37° C. and 5% CO2. Cells were used between passage number 5-8. GFP-HUVECs were cultured in proprietary fully supplemented Endothelial Growth Medium (Angio-Proteomie) and non-transfected HUVECs were cultured in fully supplemented VascuLifee VEGF Endothelial Medium (LifeLine Cell Technologies), containing recombinant human (rh) fibroblast growth factor, ascorbic acid, hydrocortisone hemisuccinate, fetal bovine serum, L-glutamine, rh insulin-like growth factor, rh epidermal growth factor, rh VEGF, heparin sulfate, gentamicin, and amphotericin B. Media was exchanged every 2-3 days.
Downstream angiogenic markers were evaluated using immunocytochemistry. Fibers formed from volume fractions of 0-100% type I collagen and DNA aptamer were conjugated to untreated polystyrene 24-well well plates or untreated 6 well plates as previously described using sulfo-SANPAH. GFP-HUVECs were seeded at 7,500 cells/cm2 in fully supplemented Endothelial Growth Medium. HUVECs were seeded at 3,000 cells/cm2 in VascuLife® VEGF Endothelial Medium without VEGF supplement. After 3 days of culture without media refresh, the cells were formalin fixed for 15 minutes. Cells were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). Then wells were blocked with 1% bovine serum albumen (BSA) in 0.1% Tween 20 in PBS (PBST). Following blocking, cells were incubated overnight at 4° C. with primary antibody for von Willebrand factor (vWF) (abcam, ab6994). The primary antibody was diluted in PBST at a ratio of 1:200. Afterwards, cells were incubated for 1 hour at room temperature with an AlexaFluor 594 secondary antibody (abcam, ab150080) and mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratories) or ProLong Diamond Antifade Mountant with DAPI (Invitrogen). Images were taken with a Nikon TE-2000U epi-fluorescence microscope using identical exposure settings across treatments. Well plate and secondary antibody controls did not show any non-specific fluorescence.
Secretion of angiogenic markers by HUVECs was measured by enzyme linked immunosorbent assay (ELISA). HUVECs were seeded onto DNA-collagen fibers immobilized within tissue culture polystyrene wells at a density of 3,000 cells/cm2 in VascuLife® Endothelial Medium (Lifeline Cell Technologies) without VEGF supplement. Media was collected after 3 days of culture and stored at −80° C. until assayed. Cell culture media was centrifuged at 10,000 g and the media supernatant was collected and assayed. Quantification of the HUVECs secretion of vWF, angiopoietin-2 (ANGT-2), and matrix metalloproteinase-2 (MMP-2) was quantified using RayBio® Human vWF ELISA kit (RayBiotech), RayBio® Human Angiopoietin-2 ELISA kit (RayBiotech), RayBio® Human Matrix Metalloproteinase-2 ELISA kit (RayBiotech) following the manufacturer's instructions.
Statistical analyses were conducted using GraphPad Prism 8.3.0 (538). Comparisons between treatments were evaluated by unpaired t test with an alpha level of 0.05 denoting statistical significance.
We first tested a VEGF-R2 targeting DNA aptamer and a random sequence ssDNA oligomer of the same length and GC content for complexation with type I collagen. Upon mixing dilute solutions of DNA aptamer and collagen, not only nanofibrils, but large self-assembled fibers (>10 μm) formed rapidly (<10 minutes) and spontaneously (
Collagen fibers did not form in the absence of DNA, when each was replaced with an equivalent volume of deionized water in the mixtures. To better visualize the fibers, we immobilized them onto untreated tissue-culture polystyrene using sulfo-SANPAH and labeled them using a fluorescent DNA stain. As expected, fibers formed for the same volume fractions of collagen for both the DNA aptamer and random sequence oligomer. Interestingly, fibers were not fully immobilized to the surface such that fibers were observed extending several microns vertically from the surface, which swayed with the motion of the surrounding fluid much like a kelp forest in the ocean. Previously, we demonstrated fiber formation is dependent both on the relative amounts of ssDNA to collagen in solution as well as the length of the ssDNA.27 Native tissue basement membranes are diverse structures consisting of micron to submicron fibers, pits, and holes33,34. These structures act as biophysical cues to direct cellular function35,36. Fibers provide anisotropy to support contact guided cellular alignment as well as 3D geometries that lead to more physiological cell behvior.37,38 Likewise, nanoscale features are known to regulate cell shape, polarity, migration, proliferation, and differentiation.39 Being able to synthesize these types of features with relative ease out of natural ECM proteins is an important step towards developing functional engineered matrices. No fibers were observed for collagen volume fractions greater than 50%. Staining revealed a heterogenous fiber size distribution, which depended on collagen volume fraction (
Owing to their 3D nanotopography and biomacromolecular composition, we probed the functional utility of our DNA aptamer-collagen fiber self-assemblies for tissue engineering. To begin, we evaluated the remodeling behavior of green fluorescent protein expressing human umbilical vein endothelial cells (GFP-HUVECs) in response to VEGF-R2 targeting aptamer-collagen fibers. GFP-HUVECs readily attached and grew to surfaces with immobilized aptamer-collagen fibers. Morphologically, GFP-HUVECs looked the same for aptamer-collagen fibers formed using the VEGF-R2 targeting aptamer and the random sequence ssDNA oligomer. Over 72 hours, the cells remodeled, proliferated, and progressively engulfed the larger fibers (
Previously, we demonstrated receptor agonist behavior using a divalent form of this VEGFR2 targeting aptamer.25 In this form, two separate aptamer sequences are physically tethered together, and it was suggested that this enabled the dimerization of the VEGF-R2 receptor leading to its activation. In the context of DNA-collagen complexes, their structure could act as a mediator to bring individual aptamer oligomers within close enough proximity to elicit receptor activation in an analogous manner as proposed for the divalent assembly. To gauge this possibility, we looked at phenotypic differences for endothelial cells in contact with the DNA aptamer-collagen fibers and between fiber formulation by immunostaining for the endothelial cell marker, von Willebrand factor (vWF). In culture, endothelial cells have been shown to demonstrate elevated vWF expression upon stimulation by VEGF.51,52 Endothelial cells growing on the fibrillar DNAcollagen complexes (10%, 30% vol. fraction collagen) displayed greater expression of vWF compared to those cells adjacent to the fibers for both fibers formed using the VEGF-R2 targeting aptamer and the random sequence ssDNA oligomer as indicated by differences in fluorescent intensity (
Spurred by these results for the monovalent form of the aptamer, we turned to the divalent form to investigate its action on endothelial cells when presented as part of the fibrillar DNA-collagen complex. Unlike with the monovalent form, upon mixing the divalent assembly with collagen not only fibers, but large macroscopic aggregates formed that were visible in solution by the naked eye (
This greater degree of agglomeration was thought to be due to the (PEG)6 linker physically tethering the two ssDNA oligomer sequences such that the divalent assembly acted as a type of crosslinker between individual complex fibers. As we had done previously, we immobilized these newly formed fibers to tissue culture polystyrene and cultured HUVECs on these functionalized surfaces. Additionally, we did this in the absence of soluble, exogenous VEGF to mitigate any competing effects with the divalent aptamer for receptor activation. Immunostaining of HUVECs cultured on fibers formed using the VEGF-R2 targeting aptamer in a divalent assembly displayed enhanced vWF expression compared to HUVECs cultured on fibers formed using the random sequence also assembled into a divalent assembly (
Von Willebrand factor has been linked to angiogenic processes in endothelial cells by inhibiting VEGF-R2 signaling through αvβ2 integrin binding, though VEGF-R2 activation does facilitate vWF exocytosis.52,54 However, the role of vWF in angiogenesis has been found to be tissue- and stimulus-specific with it being depending on the situation either stimulating of angiogenesis in its absence or its presence being a requisite for angiogenesis.55 vWF is also a major glycoprotein used for maintaining hemostasis.56 It is found extracellularly in both the plasma and surrounding ECM.56,57 Its increased expression and the observed angiogenic remodeling behavior could in part be that the endothelial cells are modifying these fibers into more of their specific niche. Equally, vWF controls angiopoietin-2 (ANGPT-2) levels in endothelial cells by promoting ANGPT-2 storage in Weibel-Palade bodies and inhibiting ANGPT-2 synthesis.58 Decreased intracellular vWF leads to the release of ANGPT-2, which upon endothelial cell activation synergizes with VEGF-R2 signaling to destabilize blood vessels and promote angiogenesis.58 Given this association, we measured HUVEC ANGPT-2 secretion, and found that the inclusion of the divalent aptamer assembly in the DNA-collagen fibers promoted an increase in the secretion of ANGPT-2. (
Here we showed the rapid and facile synthesis of DNA aptamer-collagen complex fibers as a new class of biofunctional materials for tissue engineering and regenerative medicine applications. Previous work by our group has demonstrated the utility of ssDNA-collagen as a biofunctional material platform. This report expands on that work by demonstrating that in addition to random ssDNA, specific targeting sequences in the form of DNA aptamers or aptamer assemblies can also be used and their functionality or bioactivity can be maintained. Fibers formed using DNA aptamers and collagen show exciting potential to confer biophysical cues derived from their fibrous structure while also providing specific signaling by DNA aptamers to cells. Much like we showed using our receptor agonist variant of a VEGF-R2 targeting aptamer, other DNA aptamers could be used to synthesize complex, adaptive microenvironments to direct cell phenotype. The DNA aptamer-collagen complex offers the potential for greater depth for both DNA- and collagen-based biomaterials through the unique union of these two biomacromolecules.
Vascularization of engineered tissue is one of the hallmark challenges of tissue engineering. Leveraging our newly developed self-assembled DNA aptamer-collagen complex based biomaterial platform, we used a VEGF-R2 targeting aptamer and its receptor agonist divalent assembly as the DNA component to form complex fibers. Human umbilical vein endothelial cells (HUVECs) quickly remodeled these fibers into tubulogenic structures over 72 hours. Moreover, DNA-collagen complexes composed of the divalent assembly promoted enhanced expression of von Willebrand factor (vWF), angiopoietin-2 (ANGPT-2), and matrix metalloproteinase-2 (MMP-2) by HUVECs as measured by either immunocytochemistry or ELISA. Endothelial cell phenotype was thought to be directed by both biochemical cues afforded by the agonist behavior of the divalent aptamer assembly as well as by the biophysical cues afforded by the complex fiber nanotopography. Collectively, these results support the development of an angiogenic endothelial cell phenotype stimulated by the DNA aptamer-collagen fibers. Thus, the combination of engineered DNA aptamer nanotechnology and DNA-collagen complexation phenomena is a promising biofunctional natural scaffold material system for tissue engineering and regenerative medicine applications
We are the first to show that short DNA aptamers complex with type I collagen for the formation of self-assembled fibers and gels. We investigated the synthesis of these complexes and the biological response of vascular and osseous cells to them. Sequences (15-160 nucleotides) were random or engineered to bind to vascular endothelial growth factor receptor 2 (VEGF-R2) or hydroxyapatite and assembled as either monovalent or divalent. Fibers formed rapidly (<30 minutes) and spontaneously at 25° C. This fibrillogenesis supports the simple, expeditious manufacture of extracellular matrix-like suprastructure 3D gels and coatings. Fiber formation was independent of DNA sequence, but dependent on DNA length and the relative amount of DNA to collagen in solution. Transmission electron microscopy revealed fibers are physical entanglements of DNA aptamer-collagen nanofibrils with diameters<10 nm. Human osteoblasts remodeled fibers and gels showing favorable osteocytic morphological features. Human umbilical vein endothelial cells formed tubulogenic-like structures and remodeled fibers. Immunofluorescence revealed endothelial-specific marker expression (vWF, VE-cadherin) was enhanced locally for cells attached to the fibers containing the VEGF-R2 binding aptamer agonist assembly. Conceivably, these complexes could be used as a biomaterial in which the collagen provides a substrate for cellular adhesions, fibrous topography, and DNA in the form of a DNA aptamer for targeting a specific small molecule, protein, or cell. Especially exciting is the promise of forming advanced biomimetic extracellular matrices (ECM) containing intercalated bioactive DNA aptamers by combining these fibers with other ECM components. Ultimately, the ssDNA-collagen complex offers greater depth to DNA aptamer engineering.
Collagen and single stranded DNA (ssDNA) complex to self-assemble into fibers depending on the length of the ssDNA and the relative amounts of collagen and ssDNA in solution. When monodisperse, random sequences of ssDNA in the range of 15 to 90 nucleotides and type I collagen were mixed together at room-temperature, we report for the first-time fibers several tens of microns in length and as large as 10 μm in diameter. Fiber formation was rapid and spontaneous requiring no further treatment after mixing. Most notably, more ssDNA oligomers were incorporated into the fibers formed using shorter ssDNA oligomers. Endothelial cells formed angiogenic-like structures using the fibers with elevated expression of von Willebrand factor for cells in direct contact with the fibers. These fibers open the door to future applications in the administration and functionality of ssDNA.
DNA and proteins interact with each other to modify their respective functions. One such interaction is the complexation of DNA and collagen, first discovered in 1976 while investigating the accumulation of DNA and anti-DNA antibody complexes in the tissue of patients with autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.1-12 From these investigations, it was identified that both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) readily bind to the collagenous component of the glomerular basement membrane.1,2,5 Specifically, DNA binds to type I, II, and IV collagens and not to fibronectin or reference protein, bovine serum albumin.1,2,5 In fact, complexes of DNA and anti-DNA antibodies require DNA first to bind to the basement membrane and then to interact with the anti-DNA antibody in order to accumulate at the basement membrane rather than as preformed DNA-anti-DNA complexes in solution.1,9 Important to these findings was the effect of DNA structure on DNA-collagen complexation. DNA length mediated the process favoring shorter DNA while being independent of DNA strandedness.9,12 These investigations; though, were all conducted using enzyme-linked immunosorbent assays.1,2,5,9,12 Missing from these studies was demonstration of collagen's ability to undergo fibrillogenesis-collagen triple helix self-assembly into fibrils. No observation of fibrillogenesis or the structure of this interaction was made at the time. It was not until 1997 that Kitamura et al. observed self-assembled DNA-collagen fibrils using salmon milt dsDNA and salmon type I collagen.13 The authors found these fibrils formed rapidly and spontaneously with distinct cross-banding patterns.13 Thus, began a renewed interest in the DNA-collagen complex, especially as a vehicle for gene delivery applications using cyclic plasmid DNA. However, this application favors nanosized DNA-collagen particles rather than fibers.14-16 Kaya et al. revealed that dsDNA structure, either linear or cyclic, modified fibril formation as did the DNA molecular weight distribution and purity.17 Further characterization indicated that the relative amounts of dsDNA and collagen influenced fibril formation.18,19 This complex has been utilized further as a component for wound dressings, antimicrobial coatings, and protein analysis devices.20-22 Paralleling this work, Svintradze et al. developed a theoretical model for the dsDNA-collagen complex.23 The interaction between dsDNA and collagen triple helices was defined by the joining of the hydration shells for each molecule to form aggregates of a central DNA double helix surrounded by five collagen triple helices.23,24 These studies have focused exclusively on large (>1,000 base pairs), random, dsDNA sequences; despite, collagen's demonstrated avidity for short (<200 base pairs) DNA irrespective of strandedness.9,12
We found that short (<100 nucleotides), monodisperse ssDNA oligomers complex with type I collagen to form self-assembled fibers. Upon mixing dilute solutions of ssDNA and collagen, not only fibrils (<1 μm), but large self-assembled fibers (>10 μm) formed rapidly (<120 minutes) and spontaneously (
Previously, fibrils have been observed for more concentrated mixtures of dsDNA and collagen, without mention of such aggregation into large fiber bundles.13,17-19 Gene delivery applications favored nanoparticle-sized complexes and neglected further investigation of larger aggregates formed using plasmid DNA and atelocollagen.14 Likewise, decreased DNA-collagen fibril formation time has been observed using a salmon DNA solution of ˜25% ssDNA and ˜75% dsDNA as compared to 100% dsDNA, suggesting ssDNA more rapidly interacts with collagen.17 A coacervation effect has been observed and modeled for a dsDNA-gelatin A system with excess gelatin A and constant amount of DNA.29 Following charge neutralization of this complex, the addition of more gelatin A led to an overcharging effect and phase-separation of the complex from solution. A similar coacervation effect, may explain the formation of these large ssDNA-collagen fiber self-assemblies
To further understand ssDNA-collagen fiber formation as a function of oligomer length, we combined several short, monodisperse, random sequence ssDNA oligomers (15, 33, 45, 90 nucleotides) with type I collagen. (
We found fiber formation depends on the length of the ssDNA and on the relative amount of ssDNA in solution (
We believe the variation in fiber formation for the different mixtures of ssDNA and
collagen is due to differences in ssDNA-collagen binding of the oligomers. Previous reports found in vivo DNA-collagen binding favored shorter DNA (160-200 nt); however, ssDNA on the scale of DNA aptamers (15-100 90 nucleotides) has not been previously investigated.12 To test in this specific range, we measured the binding of our short, monodisperse, random sequence ssDNA oligomers (15, 33, 45, 90 nt) with type I collagen using a fluorometric binding assay. We observed that ssDNA oligomer binding peaked at ˜0.15 μg ssDNA/μg collagen regardless of the oligomer length, which occurred between 12-30% mass fraction of DNA in solution. (
nt ssDNA oligomer displayed reduced binding as the mass fraction of DNA in solution increased beyond its maximum binding of ˜18%. This trend proceeded to such an extent that for a ˜45% mass fraction of DNA in solution (50% volume fraction collagen), no fibers were observed; instead, a few faint ssDNA rich aggregates were present potentially the result of ssDNA self-aggregation and/or a lack of sufficient collagen in solution. With this boundary determined, our data suggests any ssDNA oligomer sequence can form these ssDNA-collagen fibers so long as the relative amount of ssDNA to collagen in solution is within the appropriate range.
To better reveal the effect of ssDNA oligomer length on the observed fibrillogenesis, we then calculated the amount of bound ssDNA per available collagen on a mole per mass basis (
of ssDNA in solution, and the 90 nt ssDNA oligomer had a maximum binding of 4.9±0.4 pmol bound ssDNA/μg collagen for a 17.83% mass fraction of ssDNA in solution. When the value for maximum binding from (
As a preliminary study, we cultured green fluorescent protein expressing human umbilical vein endothelial cells (GFP-HUVECs) on immobilized fibers formed using an 80 nucleotide random ssDNA sequence. Because endothelial cells express contact guided behavior for engineered topographies and electrospun fibers, we were interested in how they would respond to the biophysical cues conferred by our ssDNA-collagen fibers.31 The cells readily attached and interacted with the fibers for all fiber forming conditions. Of note, the cells appeared to consolidate the fibers into larger structures with branches and tubules after three days of culture (
The work we present herein is an early report on the dynamics of ssDNA- collagen
interaction that yields self-assembled nano- and micro-fibers, as well as fiber bundles that appear when cells are present on the fibers. This work is a departure from other work in that we report the dependence of ssDNA size on fiber formation. These self-assembled fibers show great promise as a strategy to fabricate advanced biomimetic extracellular matrices (ECM) containing intercalated bioactive DNA aptamers by combining these fibers with other ECM components. Another potential application for the use of these ssDNA-collagen fibers is in biomaterial functionalization. There is a great interest in recent years to functionalize biomaterial surfaces with origami-based DNA nanotechnology, for applications such as drug delivery. This material platform would also enhance cell adhesion through the incorporated collagen and the fibrous structure in which the collagen would provide additional physical stimuli. Lastly, in this work we demonstrate the formation of fibers utilizing random ssDNA sequences on the order of size similar to ssDNA aptamers. Given that the fiber formation appears to be sequence independent, there exists the possibility of using functional DNA aptamers in this fiber matrix. DNA aptamers are characterized for their high affinity and specificity to targets such as ions, molecules, and proteins, their smaller size and low immunogenicity.36-38 These advantages have led DNA aptamers to being used as antagonists, agonists, delivery agents, and sensors.36-39 Fibers formed using a biofunctional DNA aptamer and collagen shows great potential to confer biophysical cues derived from the fibrous structure while also delivering a DNA aptamer to cells with site-specificity. While the maintenance of aptamer structure is paramount for their overall function, this aspect requires thorough characterization in the context of DNA aptamer-collagen fibers, which is our current focus. Ultimately, the ssDNA-collagen complex offers the potential for greater depth to DNA aptamer engineering.
Nucleic acid-collagen complex (NACC) fibers and hydrogels self-assemble when single-stranded DNA (ssDNA) and type I collagen are mixed together (James, B. D., et al, ACS Biomater. Sci. Eng. 2020, 6, 213-218 (2019)). Hydrogels are used as constructs to engineer tissues. However, collagen hydrogels have poor mechanical properties. This is due to the random alignment of the fibers and the high-water content of the gels (Ahearne, Y. Y et al, Topics in Tissue Engineering, Vol. 4. Eds. N Ashammakhi, R Reis, & F Chiellini (2008)). While collagen fibers show changes in orientation due to mechanical deformation, elastin fibers tend to remain uniformly distributed (Chow, M. J. et al, Biophysical Journal Vo. 106, 12, 2684-2692 (2014)). In the extracellular matrix (ECM) elastin fibers impart a compressive intrinsic stress on collagen. Therefore, if elastin is added to the NACC, then the mechanical properties of the complexes will be altered.
The NACCs were prepared by mixing 1 μM ssDNA with 0.3 mg/mL type I collagen. A separate solution was prepared adding 0.3 mg/mL elastin to the NACCs. The solutions were made with a 1:1 and 1:2 concentration ratio of elastin to collagen. A random 80 nucleotide ssDNA sequence was used because the sequence of the ssDNA does not affect the formation of the NACC fibers (James, B. D., et al, ACS Biomater. Sci. Eng. 2020, 6, 213-218 (2019)). To best visualize the fibers, they had to be immobilized onto a glass slide. This was done by treating a glass slide with (3-aminopropyl)triethoxysilane and then immobilizing the fibers using sulfo-SANPAH. The fibers were stained with SYBR Safe DNA stain to highlight the ssDNA in the fibers during fluorescence imaging. The Young's modulus of these fibers was measured by atomic force microscopy (BioAFM). A Bruker/JPK NanoWizard 4 BioAFM was used for these experiments. Fibers were measured in QI mode and force curves were fit to a Hertz model to extract the Young's modulus. When using a higher concentration of ssDNA (10 μM), collagen, (3.0 mg/mL) and elastin (3.0 mg/mL) NACC gels were formed. The storage modulus and the loss modulus of these gels were measured using an Anton Paar modular compact rheometer. The data collected was compared to that of NACC without elastin. Lastly, a DNA binding assay was conducted to assess the effect of elastin on the amount of ssDNA that binds with the collagen (James, B. D., et al, ACS Biomater. Sci. Eng. 2020, 6, 213-218 (2019)).
As measured by BioAFM, the Young's modulus was lower in the fibers with the elastin than those without (
Collagen and elastin naturally interact in the ECM. By being able to adjust the mechanical properties of the hydrogels with the inclusion of elastin, different components can be mimicked such as a singular alveolar wall (Dunphy, S. E., Journal of the Mechanical Behavior of Biomedical Materials, Volume 38, 251-259 (2014)). This takes advantage of the natural interactions between elastin, DNA, and collagen, and allows for a simpler method to integrate elastin to NACCS. Furthermore, by adding DNA to these complexes, specific biomolecules or cells could be targeted using a DNA aptamer. The nucleic acid elastin collagen complexes with allow for further development and applications of hydrogels by being able to adjust the properties of the gels.
To assess the impact of varying DNA sequences, 80 nucleotide DNA sequences were studied with a range of specific nucleotides, guanine and cytosine (G-C), as these exhibit stiffer behavior. Rheological assessment of NACC gels with a range of G-C content (%) confirms that the NACCs exhibit gel-like behavior. The gel-like behavior begins as soon as the DNA and collagen are mixed, with gel formation occurring within 10-15 min (
When considering the impact of DNA concentration on overall NACC mechanical properties, a study was conducted comparing 10 μM and 100 μM DNA concentration at three different sequences containing a range of G-C concentration. Rheology frequency Sweep curves, show that the mechanical properties of NACC gels were unchanged at both 10 μM and 100 μM DNA concentration when fabricated with DNA whose sequence contained 10% G-C content (
Another DNA property under investigation is the impact of DNA length on NACC mechanical properties. Data indicate that DNA length may contribute to overall gel properties. Frequency sweep data shows the modulus change as a function of DNA length (
In summary, these data show that the addition of DNA to collagen results in a tunable hydrogel NACC system, with variable mechanical properties and gel formation kinetics. By tuning DNA properties such as increasing the concentration of the rigid G-C interaction results in a range of gel modulus values.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application is a continuation-in-part of International Application No. PCT/US2021/072653, filed Dec. 1, 2021, which claims benefit of U.S. Provisional Application No. 63/120,998, filed Dec. 3, 2020, and U.S. Provisional Application No. 63/158,546, filed Mar. 9, 2021, which are hereby incorporated herein by reference in their entireties.
This invention was made with Government Support under Grant No. 1453098 awarded by the National Science Foundation and Grant No. F31HL147445 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63120998 | Dec 2020 | US | |
| 63158546 | Mar 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/072653 | Dec 2021 | US |
| Child | 18313064 | US |
