DESCRIPTION

3D PRINTING WITH NUCLEIC ACID ADHESIVES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/877,174 filed on September 12, 2013, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The invention was made with government support under Grant No. 1-ROl- GM094933 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] Embodiments of the disclosure concern at least the fields of 3D printing, microparticles and microparticle manufacture, self-assembly processes, and nucleic acid technology.

BACKGROUND OF THE INVENTION

[0004] Nucleic acid hybridization has previously been used for the programmed assembly of materials (Seeman, 2010). However, the objects that have been constructed are frequently restricted to the nanoscale, with few examples of nucleic acid hybridization leading to the production of visible materials (Macfarlane, et al, 2011; Zheng, et al , 2009). Moreover, even when visible objects are generated, the cost of using DNA for their generation is exorbitant; for example, millimeter crystals were generated from gold nanoparticles measuring 7.2 nm in diameter that were functionalized with ~30 molecules of 10 nm long DNA (Macfarlane, et al, 2011). If these nanoparticles had been assembled into a colloidal gel with a density of 1% v/v, it would have cost on the order of $2500 per liter (excluding the price of the gold). Clearly, there is a need for new materials that can be generated in larger quantities at a reduced expense. BRIEF SUMMARY OF THE INVENTION

[0005] Embodiments of the disclosure include 3D printing methods wherein a shaped structure is produced from extrusion of a composition, such as a colloidal gel, wherein the composition comprises self-assembled microparticles. Examples of 3D printing parameters, such as rates of printing, extrusion, and coordination therebetween are contemplated herein.

[0006] Embodiments of the disclosure include methods of producing a shaped colloidal gel, comprising extruding any composition as contemplated herein into a shape, including a desired shape. In specific embodiments, the shape is tailored for the application of such a shaped structure into an individual, such as a mammal, including a human. In specific embodiments, the composition is extruded via a 3D printer.

[0007] In specific embodiments, there is a colloidal gel that comprises a plurality of microparticles that interact via nucleic acid hybridization. In particular embodiments, a single microparticle comprises a plurality of nucleic acids having a first sequence that is

complementary to a second sequence of a second nucleic acid, a plurality of which are located on a second microparticle. The colloidal gel has properties that are able to be manipulated at least based on the length and content of the nucleic acids (or length of a linker thereto) and on the types and sizes and combinations of sizes of microparticles.

[0008] The present invention overcomes limitations in the prior art by providing compositions of microparticles that can associate with each other through specific DNA-DNA interactions. In certain preferred embodiments, the microparticles can self-assemble into a colloidal gel. These materials may be generated at reduced costs as compared to nanoparticle compositions. In some embodiments, the microparticle compositions may be used for cell culture and/or an implantable or injectable cell scaffold material, e.g., to treat a wound or a disease in a subject, such as a mammal or a human.

[0009] In specific embodiments, a shaped structure comprising a colloidal gel is produced under conditions that allow control of internal structures at the microscale; such microscale properties may be programmed by microparticles in the colloidal gel and through their nanoscale DNA interactions between microparticles.

[0010] As shown in further details below, these compositions may be used for the generation of colloidal gels of microparticles at reduced costs. For example, by using certain polystyrene particles functionalized with an equivalent DNA density, the DNA price may be reduced significantly as compared to DNA functionalized nanoparticles. This price difference may provide a significant advantage for applications of DNA as a "smart glue" for the production of larger or human scale objects, particularly as compared to the use of nanoparticles comprising more expensive materials such as gold.

[0011] An aspect of the present invention relates to a composition comprising: a plurality of first and second microparticles, wherein the first microparticle is coated with or covalently attached to a plurality of a first sequence, wherein the second microparticle is coated with or covalently attached to a plurality of a second sequence, wherein the first and second sequences can hybridize under stringent conditions; wherein the first and second microparticles are from about 0.5 μιη to about 500 μιη in diameter. In some embodiments, the first or second microparticle is or contains polystyrene, poly(lactic-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polylactic acid (PLA), gelatin, hydroxyapatite, collagen, hyaluronic acid, or a biologically derived polymer. In some embodiments, the first and second microparticle are not gold, platinum, or silica. In some embodiments, the first and second microparticle are polystyrene, poly(lactic-co-glycolic acid) (PLGA), polymers comprising polyethylene glycol, or gelatin. The first and second microparticles may be about 1-100 μιη in diameter, more preferably about 1-25 μιη in diameter, or more preferably about 5-10 μιη in diameter. The composition or microparticle may comprise a growth factor such as, e.g., FGF-2 (bFGF), nerve growth factor (NGF), or platelet derived growth factor (PDGF). The composition or

microparticle may comprise a morphogen. In some embodiments, the microparticle comprises a cell capture moiety such as, e.g., an antibody, a peptide, or an aptamer. The composition may further comprise a collagen, hyaluronic acid (hyaluronan, HA), fibrin, a fibrin glue, an extracellular matrix compound, a proteoglycan, fibronectin, or laminin. In some embodiments, the proteoglycan is heparan sulfate, condrotin sulfate, or keratin sulfate. The cell-capture moiety may be an antibody or an aptamer. In some embodiments, the cell-capture moiety is anti-VEGF antibody, a cell surface receptor antibody, or an anti-pan-Cadherin antibody. In some embodiments, the composition comprises both a growth factor and an extracellular matrix (ECM) compound. In some embodiments, the composition is further defined as a colloidal gel. The first and second sequences may be complementary. The first and second sequences may be from 10 to 100 nucleotides long, from 20 to 80 nucleotides long, from 25 to 50 nucleotides long, or from 35 to 45 nucleotides long. In some embodiments, a spacer moiety may link the hybridizing DNA to the microparticle. The spacer sequence may be comprised from DNA or other synthetic material (e.g. polyethylene glycol). In some embodiments, the composition further comprises a growth medium for cells. The composition may further comprise vz ^' able cells or cells dissociated from an intact tissue. In some embodiments, the composition further comprises chondrocytes, pluripotent cells, hepatocytes, or stem cells. In particular embodiments, the composition comprises a carrier, such as one that may include components appropriate for DNA hybridization and cell growth. This may include salts, growth factors, nutrients, and/or other gel components cross-linked or un-ross-linked, for example.

[0012] Another aspect of the present invention relates to an implant material comprising a composition of the present invention and a pharmacologically acceptable excipient. The implant material may be formulated for parenteral injection or surgical implantation.

[0013] Yet another aspect of the present invention relates to a method of producing a shaped colloidal gel, comprising extruding a composition of the present invention into a shape. In some embodiments, the composition is extruded via a 3D printer.

[0014] Embodiments of the disclosure include the 3D printing of particle -based gels. Specific embodiments encompass amorphous colloidal gels that would maintain a shaped structure following printing from a 3D printer.

[0015] In specific embodiments, nucleic acid sequences on separate microparticles (such as first and second sequences on first and second microparticles, respectively) are complementary. In specific embodiments, the first and second sequences are from 10 to 500 nucleotides long; from 20 to 80 nucleotides long; from 10 to 50 nucleotides long; from 25 to 50 nucleotides long; or from 35 to 45 nucleotides long. In specific embodiments, the nucleic acid sequences on the microparticles are labeled, such as with a fluorophore, radioisotope, or chromophore, for example, although in particular embodiments the microparticles are directly labeled.

[0016] Embodiments include an implant material comprising a composition as contemplated herein and a pharmacologically acceptable excipient. In specific embodiments, the implant material is formulated for parenteral injection or surgical implantation.

[0017] In some embodiments, there is a method of producing a shaped structure, comprising the step of extruding by three-dimensional (3D) printing a composition (which may comprise a colloidal gel) comprising a plurality of self-assembled microparticles into said shaped structure, wherein the plurality of microparticles interact through nucleic acid hybridization. In particular embodiments, the 3D printing utilizes a printer head that has a motion rate that is synchronized with an extrusion rate from the printer head, and an example of an extrusion rate is in the range of 0.1-100 ί/βεΰ per extrusion orifice. The 3D printing may utilize a printer head configured for utilization of a suspension provided to the printer head by a programmable syringe pump or a programmed droplet generator. In particular embodiments, the printing and extrusion from the printer are initiated simultaneously.

[0018] Compositions utilized in methods described herein may comprise a carrier, in specific embodiments. In particular cases, the composition comprises one or more compounds selected from the group consisting of cells, growth factors, morphogens, drugs, antibodies, peptides, proteins, aptamers, collagen, hyaluronic acid (hyaluronan, HA), fibrin, a fibrin glue, an extracellular matrix compound, a proteoglycan, fibronectin, laminin, extracellular matrix (ECM) compound, cell growth medium, synthetic hydrogel and natural hydrogel. Release of the compounds from the shaped structure may be by a controlled release, in specific embodiments.

[0019] In particular embodiments of the shaped structure, the dimensions of the shaped structure may be in the range of 1-100 centimeters. The topology of the shaped structure may be controlled at the nanometer scale, the micrometer scale, and the centimeter scale. In specific embodiments, the topology of the shaped structure is controlled at the nanometer scale through manipulation of the nucleic acid hybridization, thereby mediating interaction between microparticles in the plurality. In some embodiments, the topology of the shaped structure is controlled at the micrometer scale through manipulation of microparticle clusters that form nucleic acid-dependent substructures. In certain embodiments, the manipulation comprises manipulation of the size of the particles, of the stoichiometry of different sizes of particles, and/or of the length of a linker that links nucleic acid to the particle. In specific embodiments, the topology of the shaped structure is controlled at the centimeter scale by manipulation of the shape of the composition via 3D printing and/or by manipulation of the porosity of the microparticles in the composition.

[0020] In embodiments of the disclosure, the particles are present in the composition at a range of 1-90% weight/volume. Methods as contemplated herein may comprise the step of generating the self-assembled microparticles. In specific embodiments, the self- assembled microparticles comprise a plurality of first and second microparticles that interact by nucleic acid hybridization. The self-assembled microparticles may be further defined as comprising a plurality of one or more additional pairs of microparticles in which each member of a pair interact by nucleic acid hybridization. [0021] In specific cases, a first microparticle comprises a plurality of first nucleic acid sequences that are each hybridizable to one of a plurality of second nucleic acid sequences comprised on the second microparticle. Such nucleic acid sequences may be hydrophobically modified. In specific embodiments, the nucleic acid sequences are attached to the microparticle by a linker. The nucleic acid sequences may be attached to the microparticle by chemical modification or terminal transferase extension. In certain aspects, the nucleic acid sequences may be extended by polymerization on the microparticle. In particular embodiments, the density of microparticles in the composition is dependent upon the number of nucleic acid sequences on the microparticles, the length of nucleic acid sequences on the microparticles, or both. Porosity of the microparticles in the composition may be manipulated by selection of specific sizes of microparticles. In a specific embodiment, a high number proportion of large particles to small particles in the composition results in a size of the pores in the composition greater than the diameter of the large particles, and in some cases, a number proportion of large particles to small particles is approximately 1 : 1. In other embodiments, a low number proportion of large particles to small in the composition results in a size of the pores in the composition with similar diameter to the particles themselves, and a number proportion of large particles to small particles may be smaller than 1 :3.

[0022] In embodiments concerning generation of the self-assembled microparticles, a method may comprise exposure of microparticles to UV light, exposure to a particular temperature, and/or exposure to particular cross-linking conditions. [0023] Contemplated methods may further comprise the step of providing the shaped structure to an individual in need thereof. In specific embodiments, the shaped structure is configured to act as an implant, scaffold, or tissue for the individual.

[0024] Embodiments of the disclosure include shaped structures produced by any method contemplated herein. [0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0027] FIGS. 1A-C: (A) Dispersed, single particles appear in the non- complementary case. (B) In the complementary case, X (which was imaged using a green label) and X* (which was imaged using a red label) particles form clusters. (C) Microspheres with orthogonal specificities form specific clusters: X/Y (imaged using a red label) bind to Y*(imaged using a purple label), and X*(imaged using a green label), to form two appropriate clusters containing all three.

[0028] FIGS. 2A-B: Aggregation can be measured by optical density. (A) In the non-complementary case, microspheres form a cloudy suspension that scatters light (as seen in the image of the cuvette). The optical density (OD) increases monotonically with microparticle concentration. (B) The complementary particles show a divergent behavior at high bead- concentrations. A dense aggregate forms and falls out of suspension. Inset shows lower concentration for aggregate formation from dTDT-generated A/T beads.

[0029] FIGS. 3A-I: (A) 3D print of a cone design was executed with DNA-cross- linked colloidal gel. (B) An extruded figure rendered in the same material. (C) The same material bearing HEK cells expressing GFP; microparticle-CY5 shown, which was imaged using the red channel. (D) Schematic shows two parallel assembly processes. (E) Digital image shows an aggregate formed by parallel assembly of X/X* and Y/Y*. (F) Confocal microscope image of co-assembled X/X* (imaged using a green label) and Y/Y* (imaged using a red label) shows specific assembly into segregated clusters. (G) Schematic non-complementary particles that do not interact. (H) Digital image shows no aggregation. (I) Confocal microscope image of non- complementary particles in a monolayer without evident clustering.

[0030] FIGS. 4A-E: (A) ABS thermoplastic was extruded to extrude demonstrate the 3D object pattern. (B) Non-complementary particles generate a disperse cloud of particles. (C-E) 3D print of a cone design was executed with DNA-cross-linked colloidal gel. The synchronization of the extrusion rate to the motion rate of the print head is critical; images show the results of extruding at 1.3, 1.7, and 2.1 μΐ/sec, respectively.

[0031] FIGS. 5A-F: (A) The schematic shows how to pairs of particle types bearing DNA complementary only to each other should form a binary mixture of red and green clusters. (B) The aggregate assembles as with particles bearing one pair of complementary types. (C) Red and green clusters can be seen at the margins of the self-assembled aggregate using confocal microscopy. (D-F) Corresponding experiments with non-complementary particles show no assembly.

[0032] FIGS. 6A-E: (A) Schematic shows how small, green particles were mixed with large, red particles bearing complementary DNA . (B) Simulation predicted that the morphology of the assembled aggregate should become more granular as the number fraction of small particles exceeds the binding capacity of the large particles. (C) Schematic drawing shows how the inventors assembled large and small particles with a carrier gel into a large aggregate of the sphere and sectioned that sphere for microscopy. (D-E) Fluorescence micrographs confirm that the generated structure becomes more porous granular as the fraction of small particles increases.

[0033] FIGS. 7A-C: (A) Self Assembly of particles generated from acrylamide and decorated by X (green) and X* (red) DNA. (B) Non-complementary particles do not assemble. (C) Complementary particles and cells can be co-suspended and then extruded into an aggregate within a well plate.

[0034] FIGS. 8A-E: (A) Low magnification bright-field survey of cells extruded with non-complementary particles. False-color green overlay is fluorescence from hydrolyzed fluorescein diacetate. (B) High magnification brightfield/fluorescence overlay image of the edge of the confluent lawn of cells shows high fluorescein fluorescence. (C) Low magnification brightfield/fluorescence overlay survey of complementary particles shows a brighter, deeper mass of cells. (D) High magnification brightfield/fluorescence overlay image focused within the self-assembled mass. (E) Zoomed fluorescence-only image indicates that multicellular colonies formed within the mass.

[0035] FIGS. 9A-B: (A) Schematic shows a typical surface functionalization protocol. A oligonucleotide modified with fluorophore is incubated with a second

oligonucleotide bearing a reactive group such as a primary amine for attachment chemistry. These two species are annealed at 80° C for five minutes in a buffered salt solution. The annealed complex is then incubated with the particles in the presence of EDC (l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide) at room temperature for several (up to 18) hours. (B) different bead types can be generated by annealing different oligonucleotides with different sequences. Complementary bead types (e.g. X and X*) can be made by adding complementary DNA to the surfaces..

[0036] FIGS. 10A-E: (A) Schematic shows how acrylamide and DNA were encapsulated within microbubbles as a w/o emulsion and then polymerized. (B) Acrydite- modified DNA that was encapsulated within the emulsion bubbles was also modified with cholesterol in one case. (C) This leads to a distribution of DNA to the outer edge of the final particle as seen in imaging flow cytometry. (D) In the control case, cholesterol was omitted from the DNA complex. (E) Imaging flow cytometry of the non-cholesterol particles shows a homogeneous distribution of DNA.

DETAILED DESCRIPTION OF THE INVENTION

I. Examples of Definitions

[0037] "Colloidal gel" as used herein refers to a composition of microscopically dispersed insoluble particles suspended in a liquid which, by their inter-particle interactions, assume a gel like consistency.

[0038] "Identical" or "identity" as used herein in the context of two or more nucleic acids or polypeptide sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0039] "Complement" or "complementary" as used herein to refer to a nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

[0040] "Nucleic acid" or "oligonucleotide" or "polynucleotide" used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions. Nucleic acids on the microparticles are preferably single stranded; nonetheless it may be possible to use double stranded nucleic acids, or nucleic acids that contain portions of both double stranded and single stranded sequence, in various embodiments. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[0041] A nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are incorporated by reference. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids. The modified nucleotide analog may be located for example at the 5'-end and/or the 3'-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non- naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7- deaza-adenosine; O- and N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The 2'- OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH ₂ , NHR, NR ₂ or CN, wherein R is Ci-C ₆ alkyl, alkenyl or alkynyl and halo is F, CI, Br or I. Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g. , a hydroxyprolinol linkage as described in Krutzfeldt et al. (2005); Soutschek et al. (2004); and U.S. Patent

Publication No. 20050107325, which are incorporated herein by reference. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g. , to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. Additional examples of synthesized bases or nucleosides include, e.g., glycol based analogs (Zhang et al, 2005), C-glycosides and base analogs (Kool, 2002), peptide nucleic acids (Nielsen et al , 1999), and other sugar moiety analogs (Leumann, 2002). [0042] "Stringent hybridization conditions" used herein may mean conditions under which a first nucleic acid sequence will hybridize to a second nucleic acid sequence, such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10 °C. lower than the thermal melting point (T _m ) for the specific sequence at a defined ionic strength pH. The T _m may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T _m , 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 °C. for shorter sequences {e.g., about 10-50 nucleotides) and at least about 60 °C. for longer sequences {e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 °C, or, 5x SSC, 1% SDS, incubating at 65 °C, with wash in 0.2x SSC, and 0.1% SDS at 65 °C.

[0043] "Substantially complementary" used herein may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions.

[0044] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0045] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0046] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

II. General Embodiments

[0047] The present disclosure provides, in various aspects, microparticles that can self-assemble into a matrix or network of particles through specific DNA-DNA interactions on the surface or exterior of the microparticles. As shown in the below examples, DNA- functionalized microparticles may self-assemble into a colloidal gel, and the gel may be extruded with a 3D printer, e.g., at centimeter size scales. Unlike conventional 3D printed objects, the extruded materials have internal, microscale properties that are programmed exclusively by nanoscale DNA interactions. By controlling the assembly of materials from the molecular level, to the micron, to the macroscale, aspects of the present invention address one of the standing challenges for self-assembling materials (Wang, et ah, 2011). Using DNA smart glues to leverage functional microparticles assembly may provide significant advantages relative to charge-based assembly (Wang, et al., 2011; Smay, et al., 2002). Indeed, because micro- and nanoparticles have previously been engineered to have a range of important properties from controlled release of growth factors and morphogens (Patel, et al., 2008) to cell capture (Wan, et al., 2012) to drug targeting (Tan, et al., 2010), the ability to rationally integrate these features in self-assembling colloidal gels revolutionizes the construction of smarter biomaterials. In some embodiments, two different species of microparticles associate together via complementary DNA-DNA interactions; in further embodiments, a third or fourth, etc. , species of microparticle that associates with the first or second microparticle via complementary DNA-DNA interactions may be further included in the composition. III. 3D Printing

[0048] In embodiments herein, there is three dimensional (3D) printing of a particle-based composition into a desired shaped structure. In particular embodiments, there is extrusion by 3D printing of a microparticle-based colloidal gel into a desired shaped structure. In specific embodiments, the microparticles in the colloidal gel interact through nucleic acid hybridization. In specific embodiments, following self-assembly of the nucleic acid-based microparticles in a colloidal gel, there is extrusion of the gel by 3D printing a shaped structure from the colloidal gel.

[0049] The parameters for the 3D printing may be manipulated according to one or more conditions for the particle-based compositions and/or according to one or more desired outcomes. In specific cases, the 3D printer employs a printer head configured for utilization of a suspension of the particle-based composition provided to the printer head by a programmable syringe pump. In specific cases, the 3-D printer employs a droplet generator {i.e. inkjet techno logy). In particular embodiments, the suspension comprising the composition lacks a need to add an element to avoid shear (such as by addition of a fluid) because the self-assembled colloidal gel behaves as a shear-thinning fluid that does not clog the tubing.

[0050] In specific embodiments, the 3D printing utilizes a printer head that has a motion rate that is synchronized with an extrusion rate from the printer head. This may be determined by software, or the rate may be determined empirically. This synchronization may be determined by an empirically derived formula or by a ratio derived from fluid dynamics theory, for example. In certain cases, the extrusion rate is in the range of r individual extrusion apparatus. Examples of extrusion rates include 1.3 or 2.1 μΙΥβεα In particular embodiments, printing and extrusion from the printer are initiated simultaneously.

[0051] The shape generated by the printer may be dictated by a desired use for the shaped structure, and in specific embodiments the printing of the shape is controlled by computer. In specific embodiments, the shape is suitable for use as an implant, such as for tissue engineering purposes. In specific embodiments, the shape may be based on a 3-D scanned model of an organ to be replaced or repaired.

IV. Microparticles, Compositions Comprising Same, and Assembly Thereof

[0052] Contemplated herein is a printed macroscale object that is held together solely by DNA interactions. In particular embodiments, such printed colloidal gels held together by DNA adhesives are programmable at three distinct scales: (i) at the nanometer scale, specific DNA interactions mediate individual particle :particle interactions, (ii) At the micrometer scale, particle clusters form DNA-dependent substructures. This micron-scale topology can be further controlled by using different sizes and stoichiometries of oligonucleotide-derivatized beads (Tang et al, 2012). The length of the DNA linker also impacts the morphology of substructures, because long linkers produce larger, stronger clusters, (iii) Finally, the shape of the object and its material properties, such as porosity, can now be patterned at the centimeter scale by 3D printing and the composition of the colloidal mixtures, respectively. Using this novel system, one can further characterize how specific changes in sequence length, composition, and complementarity (as examples) impact biological/morphological outcomes that can be seen and manipulated in a solid.

[0053] In embodiments, the self-assembled colloidal gel is useful as a tissue scaffold material. Particles of the appropriate size and surface composition have been generated from both gelatin (Patel et al, 2008) and PLGA/HLA(Wang et al, 2011). The chemistry of DNA attachment is applicable to such biocompatible and biologically active material. The ability to control the macro-scale shape, the micro-topology by DNA-computation mediated self- assembly, and the underlying chemistry by surface modification and material choice is a unique combination of features for tissue engineering. In certain embodiments, at least two overall strategies to generating replacement tissue may be utilized: a cell oriented approach and a material-oriented approach. Several recent studies have explored the possibility of 3D-printing cells into tissue-like morphology (Mironov et al, 2003). As of yet, this approach is not as mature as the use of decellularized donor tissue that has been evaluated in human clinical trials (el- Kassaby et al., 2008; Baptista et al., 2009). A material-oriented approach attempts to mimic the decellularized organs in the chemical and physical structure of the extracellular matrix (ECM). This can be achieved by building scaffolds from biopolymers like hyaluronic acid and collagen as they are found in native ECM. Biomaterials have been made porous to approximate the physical environment in decellularized tissue by electrospinning (Yang et al, 2005) or using inverted colloidal crystals (Zhang et al, 2005). This seems to have performance benefits as a tissue scaffold, but the assembly/fabrication conditions are not compatible with living cells so such scaffolds must be seeded with cells post-synthesis. By using DNA to self-assemble the micro-morphology, cell-oriented and material-oriented approaches to bioprinting may be integrated.

[0054] As contemplated herein in the field of bio printing with these self- assembled colloidal gels, cell growth can be affected by the self-assembled environment. In particular, within the context of the acrylamide-based colloidal gels (for example) it is observed that mammalian cancer cells form spheroid colonies, which is very different behavior from their adherent spreading morphology on a polystyrene plate. Furthermore, this is not an effect of the polyacrylamide as the non-assembled acrylamide particles have no apparent impact. The same technique that was developed to create microspheres coated with DNA for the purpose of self- assembly is applicable to poly acrylic acid or divinyl polyethylene glycol, both of which have also been used as biomaterials, in specific embodiments.

[0055] It is clear that self-assembly affects the behavior of cells trapped within the self-assembled matrix. In specific embodiments, one can control the self-assembly process using the properties of DNA hybridization and/or DNA circuitry in order to characterize the effects of different self-assembly processes on the structure and how that structure might affect cells growing within it.

[0056] In specific embodiments, 3D printing of the assembled colloidal gel compels several parameters to be within certain bounds. For example, experiments indicate that a critical density of spheres within the composition is useful. The exact number density of particles in suspension depends upon the number and length of DNA hybridization events between the particles, in specific embodiments. The inventors explored a number of techniques to generate different lengths of single-stranded DNA on the surface of the particles. [0057] The inventors explored several methods to modify the aggregation. They modified the temperature, but under at least certain conditions this had little effect on the critical concentrations at which the particles assembled. The inventors also tried cross-linking the DNA with UV light (~10 mw/cm ² for several minutes) in the presence and absence of a chemical cross-linker (psoralen, millimolar concentration), but at least in these conditions it did not increase the stability or change the kinetics of the assembly appreciably. In specific

embodiments, for example under other cross-linking conditions or with additional components, temperature or UV light enables aggregation or changes the aggregation state.

[0058] The surface-density of the DNA at the surface of the particle is one factor for manipulation, in certain embodiments. Particles generated with DNA internal to the structure showed markedly less propensity to aggregate, at least under particular conditions. Particles made of similar material with DNA located at the periphery of the particle showed a much higher degree of aggregation, in certain aspects. The inventors achieved localization of the DNA to the particle surface using a hydrophobically-modified DNA included in the emulsion-based particle generation process (see, e.g., Example 6).

A. Examples of Microparticle Materials and Sizes

[0059] A variety of materials may be used to generate microparticles of the present invention. For example, the microparticles may be polystyrene, poly(lactic-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polylactic acid (PLA), gelatin, collagen, hyaluronic acid, or similar biologically derived polymers. Methods for the generation of microparticles are known in the art and include, e.g., emulsion solvent evaporation as described in Rosea et al. (2004), which is incorporated by reference herein in its entirety. In some embodiments, the

microparticles are not gold, platinum, or silica.

[0060] In specific embodiments the microparticles are generally spherical, although they may be of other shapes. In specific embodiments, other shapes might include rods, cubes, or rectangular prisms generated by photolithography or by means of self organization {e.g. groups within self-organized surfactant micro-tubes). In some cases, the majority of nucleic acid sequences on the particles are on the surface of the microparticle. In particular cases, there are more nucleic acid sequences on the surface of the microparticle than there are nucleic acid sequences interior to the microparticle. In specific embodiments, the nucleic acid sequences are not evenly distributed throughout the microparticle volume. [0061] A variety of sizes of microparticles may be used with the present invention. For example, microparticles may be about 0.5-500 μιη in diameter, or about , 1-500, 0.5-400, 1- 400, 1-300, 0.5-250, 1-200, 1-100, 10-500, 10-250, more preferably 1-25, 2-10, 5-10, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,12, 13 ,14 ,15 ,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 μι ίη diameter, or any range derivable therein. A composition may comprise microparticles of substantially the same size or, in some embodiments, microparticles of a variety of sizes of differing sizes may be used. It is anticipated that the physical properties of the resulting composition may be altered by altering the size of the microparticle. For example, assembled particles with different sizes may result in more linear clusters, whereas assembled particles may result in improved rigidity and/or less linear clusters.

[0062] This disclosure demonstrates that by using multiple binary pairs of complementary DNA types to assemble particles, particle-particle and ultimately material assembly can be programmed in a rational manner (e.g. as opposed to charge-based assembly). Given the progress that has been made in the DNA nanotechnology community (Zhang et al. , 2013; Wang and Ding, 2013; Zahid et al, 2013; and Linko and Dietz, 2013), such

programmability provides an interesting avenue for creating new materials. For example, one could sequentially add specific particle types such that clusters are formed and then bridged (a hierarchical assembly process). One could use logical conditional operators, such as AND gates based on conformation switching (Park et al, 2013), to locally determine if assembly is appropriate and permitted. More advanced DNA computation might also allow one to algorithmically determine the assembly process at the nano- to microscales (as has been demonstrated for tile-based assembly (Mao et al, 2000)). Light can be used to modify the behavior of DNA reaction networks and form two-dimensional patterns (Chirieleison et al, 2013); such techniques could also be used to selectively alter the morphology of three- dimensional colloidal gels (Lin et al, 2004).

B. Application of Nucleic Acids to the Microparticles

[0063] The inventors utilized chemical modification, rolling circle amplification (RCA), and terminal transferase extension as means of generating nucleic acid on the surface of the particles. Chemical modification can generate DNA links between 1 and 100 bases, in at least some cases. The inventors tried varying the lengths of the synthetic oligonucleotide on the surface. Modest increases in the lengths of the DNA may affect the strength of the assembly, but do not seem to affect the kinetics of the assembly process, in at least certain aspects. Rolling circle amplification can generate extremely long amplicons (kilobases) but requires priming of the surface with a circular template. It was considered that this had low efficiency because rolling circle amplification did not produce successful aggregation, in at least some

embodiments. Terminal transferase generates homopolymers (e.g. poly- A or poly-T) of lengths exceeding several hundred bases. Of these techniques, chemical modification and terminal transferase generated rapid aggregation at reasonable concentrations, in at least some embodiments.

[0064] Successful assembly has thus been accomplished with synthetic DNA of examples of lengths of 20nt or 40 nt, as well as with poly-A/poly-T of enzymatic origin with a mixture of lengths of range of about 100-500nt. Rolling circle amplification generates single- stranded DNA with lengths of many hundreds or thousands of bases, in specific embodiments. Aggregation with surface-bound RCA products was unsuccessful, which in particular embodiments is because of low surface density.

[0065] The microparticles may be coated with a plurality of a nucleic acid sequence. Several methods are available for covalently attaching a nucleic acid to a

microparticle. For example, a carboxylic acid present on the nucleic acid may be reacted with EDC ((N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide) to covalently attach the nucleic acid to a microparticle. In some embodiments, a microparticle, such as coupled to amine-DNA by coupling with EDC. Methods that may be used to coat or attach a nucleic acid such as DNA to the surface of a microparticle include methods as described, e.g., in Christiaens et al. (2006), and Sehgal et al. (1994). In some embodiments, a carboxylate-amine coupling via reaction with EDC or a heterobifunctional crosslinker may be used, e.g., as described in Sehgal et al. (1994). Nonetheless, it is anticipated that a wide variety of linkers may be used to link a plurality of nucleic acids to a microparticle such as, e.g., a sulfo-SMCC or disulfide linkage as described in Hermanson G. T. (In Bioconjugate Techniques (Third edition); Academic Press: Boston, 2013; pp. 867-920) which is incorporated by reference herein in its entirety. Additional methods for loading microparticles with nucleotide strands or oligonucleotides which may be used with the present invention include, e.g., those provided in US 2008/00274454 and US 2010/0099858.

[0066] The microparticles, the oligonucleotides or both may be functionalized in order to attach the oligonucleotides to the microparticles. Such methods are known in the art. For example, the reaction may involve carbonate-based chemistry that are used to attach DNA to any carboxylic acid or amidation, e.g., as described in Sehgal, 1994. In some embodiments, aldehyde chemistry may be used to conjugate a DNA to a microparticle. A variety of approaches may be used in various embodiments as described, e.g., in Zammatteo, et ah, 2000. In some embodiments, a transglutaminase ("meat glue") reaction may be used, e.g., as described in Lin, et al, 2006.

[0067] A variety of nucleic acid lengths may be used. Generally, larger complementary nucleic acids on separate microparticles will result in increased rigidity of the materials, due to increased hydrogen bonding between complementary nucleic acids on the separate microparticles. Nucleic acid lengths that may be used can range, e.g., from 10-1000 nucleotides or more, or from 20-1000, 10-750, 20-750, 10-500, 20-500, 10-100, 10-90, 20-80, 20-70, 20-60, 30-50, 30-60, 30-90, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, or any range derivable therein. While in some embodiments, the nucleotides bound to the microparticle are not modified, one or more modified (e.g., chemically modified) nucleic acid may be included in the nucleic acid sequences.

[0068] In some preferred embodiments, the nucleic acid sequences on the two sequences will be complementary or substantially complementary. Nonetheless, differences may exist between the sequences and still allow for hybridization under stringent conditions. The two nucleic acids may be substantially complementary or they may have, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, or 99% sequence complementarity. In some embodiments, the melting temperature (T _m ) of the interaction between the association of the two sequences is at least 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C, or any range derivable therein.

C. Additional Biological Agents for the Composition

[0069] In certain embodiments, the composition (including a colloidal gel composition), comprises one or more additional biological agents to provide activity within the colloidal gel itself, within the 3D-printed shaped structure, and/or following delivery to an individual in need thereof.

1. Growth factors

[0070] Microparticle compositions of the present invention may comprise a pharmacologically effective amount of a growth factor. The growth factor may promote growth or differentiation of cells. In some embodiments, the growth factor may promote pluripotent cells or substantially undifferentiated cells to remain in a dedifferentiated state. Growth factors that may be used with the present invention include, e.g., FGF-2 (bFGF), nerve growth factor (NGF), or platelet derived growth factor (PDGF), epidermal growth factor (EGF), brain derived neurotrophic factor (BDNF), and other proteins of the same or similar evolutionary families.. In various embodiments, one or more growth factors may either be included within a microparticle or in a composition comprising the microparticles.

2. Extracellular Matrix Compounds

[0071] In some embodiments, a microparticle composition of the present invention may comprise a cellular growth medium and/or one or more extracellular matrix (ECM) compound, such as an ECM protein, proteoglycan, or polysaccharide. The ECM protein may be a proteoglycan such as, e.g., heparan sulfate, condrotin sulfate, or keratin sulfate. The ECM polysaccharide may be a non-proteoglycan polysaccharide such as, e.g., hyaluronic acid

(hyaluronan, HA). In some embodiments, the ECM protein is a fiber, such as a collagen or elastin. The collagen may be fibrillar cartilage (type I, II, III, V, XI cartilage), facit cartilage (type IX, XII, XIV), short chain cartilage (type VIII, X), basement membrane cartilage (type IV), or type VI, VII, or XIII cartilage. In some embodiments, the ECM compound is fibronectin or laminin. Generally, in some embodiments, it may be preferable to mix the larger ECM compounds with microparticles of the present invention; however, in some embodiments, it may be feasible to provide smaller ECM compounds on or in the microparticles. 3. Cells

[0072] In embodiments of the disclosure, one or more types of cells may be included in the composition (particularly the colloidal gel composition). The cells may be of any kind, including differentiated or non-differentiated. Non-differentiated cells may be comprised in the composition with one or more agents that facilitate differentiation of the cells into a desired type {ex vivo and/or in vivo). In specific embodiments, the cells are chondrocytes, pluripotent cells, hepatocytes, somatic cells, or stem cells. In specific embodiments, the cells are obtained from an individual in which the shaped structure generated by a method contemplated herein is delivered. In specific embodiments, the cells in the colloidal gel composition are included with a suitable cell growth medium which may also include growth factors or small molecules designed to promote the appropriate level of proliferation. [0073] The types of cells included in the composition may be selected based on the desired application of the 3D-printed shaped structure. For example, upon 3D printing of a shaped bone graft or implant, the composition may comprise bone cells or cells that can be differentiated ex vivo or in vivo into bone cells. 4. Other Compounds

[0074] In some embodiments, a microparticle composition of the present invention further comprises fibrin, a fibrin glue, hydroxyapatite, crushed cancellous bone, polyethylene glycol polymer, or a media comprising extracellular matrix components such as, e.g.,

MATRIGEL (BD Biosciences; San Jose, CA). The other component may act as a

chemoattractant, promote cellular growth, and/or promote regeneration of a tissue. In particular embodiments, the composition comprises one or more drugs, such as one or more antibiotics, cancer drugs, analgesics, diuretic, anti-diabetic, proton pump inhibitor, calcium channel blocker, ACE inhibitor, thyroid drug, statin, and so forth.

[0075] In some embodiments, an additional therapeutic compound is included in the microparticle composition. The additional therapeutic compound may be, e.g., an antibiotic, an anti-inflammatory compound (e.g., a COX inhibitor, a COX 2 inhibitor, a corticosteroid, caffeine, a natural or synthetic small molecule, etc.), or an active nucleic acid or aptamer. In some embodiments, an experimental compound may be included in a microparticle composition to test the properties (e.g., toxicity, possible therapeutic effects, etc.) of the compound. For example, in some embodiments, the experimental compound may be included in a microparticle composition of the present containing a cellular growth medium and cells, and properties of the cells (e.g., toxicity, apoptosis, cellular growth, etc.) may be subsequently measured after incubation of the cells with the experimental compound for some period of time (e.g., 1-12 hours, 1-7 days, 1-6 weeks, etc.). In some embodiments, the additional compound may either be included within a microparticle or in a composition comprising the microparticles.

V. Examples

[0076] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

EXPERIMENTAL METHODS [0077] Preparation of DNA-modified microspheres. Polystyrene microspheres

(2.3 μιη; Bangs Labs, Fishers, IN) were purchased and coupled to amine-DNA (IDT, Coralville, IA) by EDC coupling (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, Sigma- Aldrich, St Louis, MO). Some 50 μΐ of the particles (10% w/v solids) were washed three times by centrifugation and resuspended in 100 mM MES buffer pH 4.5 (Sigma- Aldrich). The inventors then mixed 500 pMol of 5' amine-modified common linker "C" (composed of domain 1) with 550 pMol of an appropriate DNA oligonucleotide (e.g. for bead type X, fluorescein- modified X DNA had a sequence corresponding to domains 1 *-2-3) also in MES buffer. The fluorophore (if any) noted in the main text and is added only to the 5' terminus during synthesis. This mixture was heated to 80 °C for 4 minutes then cooled at 0.1 °C per second to room temp. The annealed DNA was then added to the particles. The particles were shaken at 1200 rpm and 5 μΐ of 1M EDC was added slowly with shaking. The reaction proceeded for 12-18 hours. The particles were then washed two times by centrifugation and resuspended in 0.4 M

tetraethylammonium bicarbonate buffer (TEAB, Sigma) with 0.1% Tween (Sigma) and 10 μΜ sodium azide (Life Technologies, Grand Island, NY). Sequence Information: (X; SEQ ID NO: l), CTT CTA TTA CTG AAT AAG ACG AGA ATA CTA AAC CTC CCT CGT CAG TGA GCT AGG TTA GAT GTC G; (X*; SEQ ID NO:2), GAG GGA GGT TTA GTA TTC TCG TCT TAT TCA GTA ATA GAA GGT CAG TGA GCT AGG TTA GAT GTC G; (Y; SEQ ID NO:3), GTT AAT GGC ACA AAG TTT TAG GAG GGA GGT CAG TGA GCT AGG TTA GAT GTC

G; (Y*; SEQ ID NO:4), CTC CCT CCT AAA ACT TTG TGG TCA GTG AGC TAG GTT AGA TGT CG; (C; SEQ ID NO:5), 5' Amine - CGA CAT CTA ACC TAG CTC ACT GAC.

[0078] Flow cytometry of DNA-assembled clusters. DNA coated particles (as described above) were mixed at a one-to-one ratio. For instance, bead type X (which displays single-stranded domains 2,3) were mixed with bead type X* (which displays single-stranded domain 3*,2*). These bead mixtures were allowed to assemble at high concentrations (1%) for approximately 1 minute, and then diluted to a final concentration (0.01%) in PBS (phosphate buffered saline, Fisher Scientific, Waltham, MA). This final sample was loaded into the flow cytometer (Imagestream X, Amnis, Seattle).

[0079] Measurement of Critical Volume Percentage for large-scale aggregation. A cuvette with 500 of PBS was placed into a thermal mixer shaker set to 750 RPM. Microparticles varying complement report non-complementary DNA were mixed at the appropriate concentration. Microparticles were then slowly pipetted into the cuvette and shaking was immediately initiated. Particles were allowed to shake for three minutes and the optical density was measured. The concentration of particles at which the optical density decreased rather than increased was noted. [0080] Terminal Deoxynucleotidyl Transferase extension of DNA. 2.3 um beads were conjugated to the common linker, amine-labeled DNA as described above. This DNA was then extended with Terminal Deoxynucleotidyl Transferase (DNTT, Thermo Fisher, Waltham, MA) according to manufacturer's specifications. Briefly, the beads and enzyme were suspended in the provided reaction buffer along with ATP or TTP. The reaction was allowed to progress at 37° overnight.

[0081] Tissue culture and incorporation of GFP-labeled cells into the colloidal gel: complementary bead types were generated as described above. HEK-393T cells expressing GFP were cultured according to standard protocols. Briefly cells were cultured on Corning T75 flasks in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher) supplemented with FBS (fetal bovine serum) to 5%, 100 ug/ml penicillin and 100 μg/ml streptomycin. Cells were passaged by detaching with .25% trypsin-EDTA. Freshly detached cells were rinsed with PBS then mixed with bead type X (generated as described above). Bead type X* was then added to the suspension. Cell/bead slurry was then slowly pipetted into a micro-well pre-filled with media and allowed to incubate at 37°C with 5% carbon dioxide. Cells within the colloidal aggregate were observed every day thereafter with a fluorescence microscope. Confocal microscopy was performed on day five as described below.

[0082] Microscopic imaging of the internal structure of the aggregates.

Particle slurry containing complementary DNA coated particles were prepared at 4% w/v in a 1%) w/v solution of alginic acid in PBS. To this was added 0.1%> bromophenol blue as a contrast agent for sectioning. Various ratios of the two particle types were pipetted and mixed by gentle vortexing. A hanging drop of ~2 μΐ of the resulting aggregate was dropped into excess 1M calcium chloride. After gelling, the excess calcium chloride was aspirated away and replaced with tissue freezing medium (TFM). The gel spheres embedded in TFM were frozen at -20 °C and sectioned with a cryostat (Microm HM 550, Thermo-Fisher, Waltham, MA) then mounted and imaged on an inverted fluorescence microscope (1X51 , Olympus, Tokyo). [0083] 3D printing of DNA-assembled aggregates. A computer-controlled syringe pump (SIAlab.com, Seattle, WA) was connected to a polyethylene tube which was connected to the 3D printer head. The polyethylene tube was purged thoroughly with PBS. Particle slurry containing complementary or non-complementary DNA coated particles was aspirated into the polyethylene tubing (50 μΙ_, at 40% w/v). The particles were then extruded at a carefully controlled rate tuned to match the rate of motion of the printer. Printing and extrusion word initiated simultaneously.

[0084] Confocal microscopy of DNA-assembled aggregates. A sample of particles (complementary or on complementary) was pipetted into a well slide containing approximately 100 of PBS. Complementary particles produced a large (millimeter sized) aggregate at the bottom of the well. Non-complementary microparticles produced a thin layer of particles at the bottom of the well. The slide was then loaded onto a confocal microscope (SP2 AOBS, Leica, Germany). The inventors attempted to image the large aggregate and found that the high scattering of the assembled particles did not permit us to see deeply into the aggregate. The inventors chose for that reason to image at the margin of the assembled aggregate. [0085] Generation of DNA-coated particles from acrylamide by suspension polymerization. In suspension polymerization, the polymerization initiator is soluble in the discrete phase and monomer microdroplets polymerize to form the polymer particles of similar size. Indeed, it is critical that the polymerization form the particles in such a manner as to preserve the morphology of the micro-droplets, as the cholesterol-directed segregation of the DNA to the edges occurs within the micro-droplet while it is still a free monomer solution. The inventors followed protocol similar to that demonstrated in Yamazaki et al. , 2003 This is distinct from emulsion polymerization. In emulsion polymerization, initiated monomers grow into stabilized nano- and micro-particles in the continuous phase. They diffuse slowly from the larger droplets to feed the growing chains within the continuous phase. The growing particles are stabilized by surfactants. Unlike dispersion polymerization, the size of particles generated by emulsion polymerization does not correlate to the size of the monomer droplets that are initially formed (Arshady, et ah, 1992).

[0086] Chill 1 ml oil, 1% Span-80, 1 steel ball bearing. Make 20% acrylamide (500 μΐ 40% acryhbis, 50 μΐ 20x PBS, 450 μΐ 1M NaCl). Anneal C-linker with assembly DNA (1 nMol each in 10 μΐ of PBS). Chill 100 μΐ of acrylamide mix. Add appropriate DNA. Add 2 μΐ TEMED. Reserve 8 μΐ of 10% APS in a separate tube. Add acrylamide mix to APS, pump twice, add to oil, homogenize 4 min at 42 RPM. Purge 5x with argon. Stir at RT for ~20 min. Spin down, wash 3x with ethanol, dry in speedvap. Reconstitute in PBS. Test assembly and run the Amnis Imaging flow cytometer. EXAMPLE 2

MICROSPHERE CLUSTER ASSEMBLY BY DNA HYBRIDIZATION

[0087] In order to demonstrate DNA-mediated assembly of objects, the inventors first functionalized 2.3 μιη polystyrene particles with fluorescent oligonucleotides, and examined the ability of particles bearing two different oligonucleotides to hybridize to one another via imaging flow cytometry (Tan, et ah, 2012) (FIG. 1).

[0088] When the oligonucleotides were non-complementary, only approximately 0.03%) of all detection events contained aggregates with both types of particles. However, of these virtually all were false positives; both particles shared the same image, but were not bound to one another. In contrast, when the oligonucleotides were complementary 33% of the imaged events were bound clusters. To examine higher order clustering three sets of beads were generated using combinations of two different DNA oligonucleotides bearing fluorescent labels: X/Y-red beads, X*-green beads and Y*-purple beads (where * indicates a complement; false colored for the purposes of imaging). These three bead types were mixed at a 1 : 1 : 1 ratio, and again analyzed by imaging FACS. No X*-Y* assemblies were found (FIG. 1C) but the suspension contained representatives of all other cluster types (X/Y bound to Y*, X/Y bound to X*, and there were higher order clusters containing all three). Assembly appeared to be efficient, as some 50%> of all measured objects were self-assembled clusters; in non- complementary controls, only about 1% of events represented apparent clusters. EXAMPLE 3

PARAMETERS FOR LARGE-SCALE AGGREGATION VIA HYBRIDIZATION

[0089] At high concentrations of complementary particles, amorphous aggregates form and fall out of suspension (even with mild shaking). This observation suggested a simple assay for oligonucleotide-mediated aggregation. At low concentrations, both complementary and non-complementary particles disperse into suspension and yield visible light scattering that can be easily measured with a densitometer (FIG. 2A). At a critical concentration,

complementary particles form an aggregate that sinks to the bottom of the cuvette and the optical density falls to zero (FIG. 2B). The critical concentration at which the trend towards increasing optical density reverses is thus a metric for the strength of aggregation. In the case of short, surface immobilized DNAs (40 base-pairs or so), aggregates formed virtually instantaneously at between 20-40% w/v.

[0090] The length and base-composition of the surface DNA has an impact on cluster formation (Tang, et αί, 2012) and also on the critical concentration at which large-scale aggregation occurs. In order to generate longer, surface-bound DNA molecules, the inventorsour used the immobilized oligonucleotides as primers for un-templated extension by

deoxynucleotidyl transferase (DNTT), which can produce DNAs with a range of distributions, including upwards of 100+ poly-A or poly-T stretches (el-Kassaby, et ah, 2008). When such poly-A and poly-T particles produced by DNTT are allowed to assemble, they form aggregates at 5% w/v concentrations or lower (FIG. 2B, inset).

EXAMPLE 4

3D-PRINTED OBJECTS FROM HYBRIDIZING COLLOIDAL GELS

[0091] The fact that microparticle aggregates precipitated from solution indicated that it might be possible to form solid objects based on DNA:DNA interactions. To this end, it was attempted to extrude complementary particles at high concentrations with a 3D printer and thereby form an amorphous colloidal gel that would hold its printed shape. While the Replicator 3D printer (Makerbot Industries, Brooklyn, NY) was originally developed for printing thermoplastics, it was modified for printing particle-based gels by directing the print head to utilize a suspension provided via a programmable syringe pump. By actuating the print head while controlling the dispense rate of the syringe pump, the 3D printer directed the extrusion of the colloidal gel into 3D (FIG. 4B) and 2D shapes (FIG. 4B, inset). In these instances, the colloidal gel was held together by 40 base-pairs of complementarity between two bead types (see Materials and Methods). Like other self-assembled colloidal gels, it is considered that the colloids behave as shear-thinning fluids (Smay, et al, 2002) that do not clog the tubing and that settle into a more solid form in the absence of shear.

[0092] The extruded gels held their form with slump heights (height without any visible gravitational collapse (Manley, et al, 2005) of greater than >6 mm under high salt conditions (see FIG. 3B). Under physiological salt conditions, the printed object holds its shape up to a slump height of >2 mm (FIG. 3C, inset). In contrast, non-complementary particles simply disperse into suspension and do not form a colloidal gel.

[0093] These results are proof of concept that DNA-connectors can mediate complex assembly of a macro scale object with internal properties defined by the nanoscale interactions. Initial studies also indicate that these aggregates are tissue-culture compatible; HEK cells expressing GFP readily proliferated within the proliferation within agglomerate (FIG. 3C). The initial studies with HEK 293 T cells expressing GFP indicated that these aggregates are tissue-culture compatible. Because the aggregates are highly opaque, one could not measure anything more precise than a positive GFP signal. The opacity issue is addressed further with hydrogel particles below.

[0094] Printed colloidal gels held together by DNA hybridization have three distinct scales impacted by rational design. At the nanometer scale, DNA hybridization (and, eventually, potentially DNA computation) mediates the interaction between particles. At the micrometer scale, clusters have a topology that is dependent on the underlying DNA-DNA interactions as has been shown by demonstrating that the substructure of the large aggregates is comprised of both clusters (see FIG. 3F). Additionally, this micron-scale topology can be rationally controlled by using different sizes and stoichiometries of oligonucleotide-derivatized beads. Finally, the object as a whole can be patterned at the centimeter scale by 3D printing.

[0095] The morphology of the colloidal gel can be altered through changes in the DNA that connects the particles and the choice of particle size. The morphology of small clusters can be altered and the results can be detected the results with flow cytometry (Manley, et al, 2005). Homogeneously sized beads can be used to form globular clusters. When particles with different sizes were assembled, there were linear clusters. Likewise, results in the present work show that the length of the DNA linker is a parameter that affects morphology; long linkers produce larger, stronger clusters, for example.

[0096] In specific embodiments, this self-assembled colloidal gel is useful as a tissue scaffold material. Initial assembly results with both PLGA and gelatin as the particle material has been obtained. These results show that self-assembling microparticles can be made from biocompatible or biologically active material. The ability to control the macro-scale shape, the micro-topology by DNA-computation mediated self-assembly, and the underlying chemistry by surface modification and material choice is a unique combination of features for future applications. [0097] In specific embodiments, the materials contemplated herein are used for generating a replacement tissue. For example, two overall strategies to generating replacement tissue include the following: a cell-oriented approach and a material-oriented approach. Several recent studies have explored the possibility of 3D-printing cells into tissue-like morphology (Yang, et al, 2005). As of yet, this approach is not as mature as the use of decellularized donor tissue that has been evaluated in human clinical trials (el-Kassaby, et al, 2008; Baptista, et al, 2009). A material-oriented approach attempts to mimic the decellularized organs in the chemical and physical structure of the extracellular matrix (ECM). This can be achieved by building scaffolds from biopolymers like hyaluronic acid and collagen as they are found in native ECM. Biomaterials have been made porous to approximate the physical environment in decellularized tissue by electrospinning (Yang, et al, 2005) or using inverted colloidal crystals (Zhang, et al, 2005). This seems to have performance benefits as a tissue scaffold, but the assembly/fabrication conditions are not compatible with living cells, so such scaffolds must be seeded with cells post- synthesis. By using DNA to self-assemble the micro-morphology, as with the present disclosure, cell-oriented and material-oriented approaches to bioprinting may be integrated. EXAMPLE 5

GENERATING SUBSTRUCTURE IN THREE-DIMENSIONAL COLLOIDAL GEL

[0098] While there have been a number of other biocompatible materials that can be printed in 3D, the advantage of DNA as a 'smart glue' for such materials is that in particular embodiments one can program sub-structures within the overall printed object. As a first example of this programmed substructure, one can mix binary pairs of complementary particles to form specific clusters within the three-dimensional matrix. This could not be accomplished with, for instance, charge-charge attraction of colloidal particles.

[0099] Two complementary pairs of DNA were generated on the surfaces of 4 bead types. One pair was coupled to fluorescein-DNA and the other pair to Cy5-DNA as shown in FIG. 5A. When these four bead types were mixed at a total concentration of 40% w/v, they formed a colloidal gel consistent other results with a single pair of complementary particle types. Upon examining the margins of this structure (FIG. 5B) with confocal microscopy, it was clear that the two assembly processes had produced independent clusters of red and green particles (FIG. 5C).

[0100] As a second method for generating controlled substructure within the self- assembled aggregate, the stoichiometry of large and small complementary particles was varied. This is shown in schematic in FIG. 6A. Simulations predict that as the fraction of small particles increases, the large particles will begin to saturate. FIG. 6B shows increasing numbers of independently nucleated, self-assembled granules in the product of self-assembly with high proportion of small particles. Simulations also show a granular, porous appearance at higher number fractions of small particles. To reveal this texture in experiment, there was assembled 4% w/v particles in a carrier solution of 1% alginic acid that was subsequently gelled with calcium chloride (FIG. 6C). The 4% w/v bead suspension was the maximum density of particles that could be reliably imaged with optical methods. Dense colloidal gels scatter light too efficiently to permit optical microscopy of the internal structure (though they can generate free standing structures without a carrier gel, as demonstrated above).

[0101] This colloidal gel was sectioned within its carrier gel using a cryostat. As predicted by simulation, the internal structure was found to depend on the ratio of small to large particles in the suspension (FIG. 6D-E). A high proportion of large particles produced large pores (100 μιη) surrounded by relatively uniform material. A high proportion of small particles produced a much higher proportion of micropores (10 μιη).

[0102] In specific embodiments, one can use more advanced DNA logic on the surface of these particles to mediate conditional assembly or algorithmically driven assembly. These processes have been explored for generating assemblies from nano-scale building blocks such as DNA origami tiles (Mao, et ah, 2000). Such nanoscale building blocks are highly precise and comparatively difficult to fabricate relative to colloidal particles. By using a "dumb" material as filler, one can leverage the scalability of bulk polymer chemistry with the programmability and specificity of "smart" DNA. Polystyrene particles can be acquired commercially with highly monodisperse size distributions making them attractive substrates for initial investigations. One can apply the same principles to other polymer chemistries as well. Hydrogels are particularly attractive as substrate material because they can release other chemicals or DNA into the surrounding solution (Kang, et αί, 2011). In specific embodiments, this allows for conditional assembly and disassembly in a time-dependent manner. Furthermore, hydrogels have a similar index of refraction to water. This allows for microscopy deeper into an assembled aggregate than with polystyrene particles.

EXAMPLE 6

CREATION OF DNA-COATED ACRYLAMIDE-BASED COLLOIDAL GEL

[0103] As noted above, polystyrene-based colloidal gels scatter light very efficiently and are highly opaque to light microscopy. Preliminary experiments indicated that mammalian tissue culture could be performed within these self-assembled structures, but microscopic observations within the mass were unable to be made. It was endeavored to recreate these results in a hydrogel that has an index of refraction much closer to that of water.

[0104] Acrylamide particles were generated by dispersion polymerization.

Briefly, acrylamide monomers in a aqueous solution were mixed with a DNA complex bearing three modifications: a double bond (acrydite), a fluorophore and a cholesterol modification. This DNA/acrylamide mixture was mixed with radical initiators and rapidly dispersed into mineral oil. Polymerization takes several minutes. During this time the cholesterol-modified DNA migrates to the oil water interface, concentrating at the margins of the polymerizing micro- droplets. Once polymerized, the microspheres are decorated at their surface with DNA which is optimal for self-assembly (FIG. 7A). Without the cholesterol modification, the DNA is evenly distributed throughout the particle volume and a smaller fraction is available for bridging between adjacent particles (see materials and methods).

[0105] These DNA coated acrylamide microspheres were washed extensively over the course of several days to remove any acrylamide monomer. Their self-assembly behavior was observed with a flow cytometer. DNA mediated self-assembly occurred with complementary DNA as shown by representative images in FIG. 7A. In the complementary case, many events (12.4%) were clusters of large, assembled particles. In the non-complementary case, only a single false positive event (shown in FIG. 7B) shared the same high-intensity red and green fluorescence as the complementary aggregates pictured. Only 1.3% of the events detected in the non-complementary showed any aggregation.

[0106] Complementary acrylamide particles can form the same type of colloidal gel demonstrated with polystyrene spheres. Complementary particles at 40% weight volume were co-suspended with cells and then extruded from a pipette tip to a well plate. The extruded object held its shape as shown in the blue-dyed aggregate indicated in FIG. 7C.

EXAMPLE 7

CELL BEHAVIOR WITHIN DNA-COATED ACRYLAMIDE-BASED COLLOIDAL GEL

[0107] Mammalian cells (A431 human epithelial carcinoma) were co-extruded with complementary or non-complementary acrylamide particles into a six well plate of tissue culture media and allowed to grow for four days. Because the non-complementary particles did not aggregate, they were largely washed away from the cells during staining (despite efforts to wash extremely gently). They can be seen in FIG. 8 as a shadow in the upper left. Virtually all observed cells adhered to the polystyrene floor of the well plate and grew into a confluent lawn. Fluorescein diacetate staining indicated that the outermost edge cells were particularly active with regard to the hydrolysis of the fluorogenic substrate by intracellular esterase (FIG. 8).

[0108] Cells growing within the mass of colloidal gel generated by complement report showed changes relative to cells grown either without any particles were with non- complementary particles. Firstly, the activation of fluorescein diacetate was much higher. Many punctate spots could be seen out of the plane of focus (FIG. 8C). Secondly the DNA assembled aggregate remained after washing. Because the hydrogel particles do not scatter light as effectively as the polystyrene particles, the cells were observed within the self-assembled mass (Figure 8D). It was especially interesting to note that cells formed spherical colonies within the context of the colloidal gel (FIG. 8E).

[0109] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the

compositions and methods of this invention have been described in terms of preferred

embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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