Patent Grant
11135414
Many factors affect gene expression in organisms. For instance, small RNAs, generally 20 to 25 nucleotides in length, have emerged as important regulators of eukaryotic gene expression. One class of small RNA is the short interfering RNA (siRNA). siRNA plays a role in the RNA interference (RNAi) pathway, and particularly in RNA silencing, a sequence-specific RNA degradation process that is triggered by double-stranded RNA (dsRNA). siRNAs are double-stranded with small 3′ overhangs and derive from longer dsRNA precursors that induce silencing. They serve as guides to direct destruction of target RNA and have been implicated as primers in the amplification of dsRNA via the activity of a cellular RNA dependent RNA polymerase.
Since their discovery, synthetic siRNA have been produced that may induce RNAi in mammalian cells through silencing or otherwise suppressing transcription of multiple genes. However, despite promising results, problems still exist with successful utilization of siRNA technology, among which delivery methods play a large role. Typically, siRNA have been delivered either by direct injection, electroproration, or by complexing with a transfecting agent. However, the siRNA remains actively present for only a matter of hours following delivery. In order to obtain longer lasting effectiveness, improved delivery methods must be found that may provide steady, long-term delivery of siRNA.
Transdermal delivery devices that provide a route for siRNA to be delivered in an active state at effective, steady concentrations over a period of time would be of great benefit. Many difficulties must be overcome to reach this goal. For instance, the human body has developed many systems to prevent the influx of foreign substances such as enzymatic degradation in the gastrointestinal tract, structural components that prevent absorption across epithelium, hepatic clearance, and immune and foreign body response.
Transdermal devices have been developed for sustained delivery of certain drugs including those for treatment of vertigo, contraception, and smoking addiction. In order to be successful, a transdermal device must deliver an agent across the epidermis, which has evolved with a primary function of keeping foreign substances out. The outermost layer of the epidermis, the stratum corneum, has structural stability provided by overlapping corneocytes and crosslinked keratin fibers held together by coreodesmosomes and embedded within a lipid matrix, all of which provide an excellent barrier function. Beneath the stratum corneum is the stratum granulosum, within which tight junctions are formed between keratinocytes. Tight junctions are barrier structures that include a network of transmembrane proteins embedded in adjacent plasma membranes (e.g., claudins, occludin, and junctional adhesion molecules) as well as multiple plaque proteins (e.g., ZO-1, ZO-2, ZO-3, cingulin, symplekin). Tight junctions are found in internal epithelium (e.g., the intestinal epithelium, the blood-brain barrier) as well as in the stratum granulosum of the skin. Beneath both the stratum corneum and the stratum granulosum lays the stratum spinosum. The stratum spinosum includes Langerhans cells, which are dendritic cells that may become fully functioning antigen-presenting cells and may institute an immune response and/or a foreign body response to an invading agent.
Unfortunately, transdermal delivery methods are presently limited to delivery of low molecular weight agents that have a moderate lipophilicity and no charge. Even upon successful crossing of the natural boundary, problems still exist with regard to maintaining the activity level of delivered agents and avoidance of foreign body and immune response.
The utilization of supplementary methods to facilitate transdermal delivery of active agents has improved this delivery route. For instance, microneedle devices have been found to be useful in transport of material into or across the skin. In general, a microneedle device includes an array of needles that may penetrate the stratum corneum of the skin and reach an underlying layer. Examples of microneedle devices have been described in U.S. Pat. No. 6,334,856 to Allen, et al. and U.S. Pat. No. 7,226,439 to Prausnitz, et al., both of which are incorporated herein by reference.
While the above describes improvement in the art, room for further improvement exists.
In accordance with one embodiment of the present invention, a device for delivery of an siRNA construct across a dermal barrier. The device comprises a microneedle and a plurality of nanostructures fabricated on a surface of the microneedle, the nanostructures being arranged in a predetermined pattern. An siRNA construct is in fluid communication with the microneedle.
In accordance with another embodiment of the present invention, a method for delivering an siRNA construct across a dermal barrier. The method comprises penetrating the stratum corneum with a microneedle. The microneedle comprises a plurality of nanostructures formed on a surface of the microneedle and arranged in a pattern. The siRNA construct is in fluid communication with the microneedle, the siRNA construct being transported across the stratum corneum following penetration of the stratum corneum by the microneedle.
In accordance with yet another embodiment of the present invention, a method for forming a device for delivery of an siRNA construct across a dermal barrier is disclosed. The method comprises fabricating an array of microneedles; fabricating a pattern of nanostructures on a surface of at least one of the microneedles; and associating an siRNA construct with the microneedles such that the siRNA construct is in fluid communication with the microneedles.
A full and enabling disclosure of the subject matter, including the best mode thereof, directed to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, which makes reference to the appended figures in which:
Reference now will be made in detail to various embodiments of the disclosed subject matter, one or more examples of which are set forth below. Each example is provided by way of explanation, not limitation. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made in the present disclosure without departing from the scope or spirit of the subject matter. For instance, features illustrated or described as part of one embodiment may be used on another embodiment to yield a still further embodiment. Thus, it is intended that the present disclosure covers such modifications and variations as come within the scope of the appended claims and their equivalents.
In general, a device for delivery of siRNA constructs is disclosed. More specifically, the device may include a plurality of microneedles at a surface and a pattern of structures fabricated on the microneedles. At least a portion of the structures are fabricated on a nanometer scale. The device is also associated with one or more siRNA constructs, for instance in a layer of the device or in a reservoir that is in fluid communication with the surface that includes the microneedles.
Without wishing to be limited to any particular theory, it is believed that the pattern of nanostructures, i.e., the nanotopography, of a device may improve delivery of siRNA while minimizing foreign body and immune response. Through utilization of a device, siRNA may be targeted for delivery to a specific site, e.g., a specific tissue or cell type in a specific delivery area, or may be delivered in a systemic fashion, for instance via the cardiovascular system.
The siRNA agents of the devices are sufficiently short that they do not trigger a deleterious non-specific interferon response in normal mammalian cells. Thus, the administration of a composition including one or more siRNA agents may be used to affect transcription of a target gene, while circumventing an interferon response as well as minimizing foreign body response. The siRNA of a device may generally include a duplexed region of less than 50, less than 40, or less than 30 nucleotide pairs, for instance between about 20 and about 25 pairs. In general, an siRNA polynucleotide comprises a double-stranded RNA (dsRNA) but is not intended to be so limited and may comprise a single-stranded RNA.
An siRNA may be formed according to any known process. For instance, an siRNA may be synthesized synthetically or via transcription of a DNA construct either in vivo or in vitro. In general, an siRNA molecule may be synthesized using methods, reagents and equipment available to one of skill in the art. By way of example, siRNA may be designed and engineered using computer software available commercially from various vendors, e.g., OligoEngine (Seattle, Wash.); Dharmacon, Inc. (Lafayette, Colo.); Ambion Inc. (Austin, Tex.); and QIAGEN, Inc. (Valencia, Calif.)). See also Elbashir et al., 2000 Genes & Development 15:188-200; Elbashir et al., 2001 Nature 411:494-98.
According to such methods, a cDNA sequence may be scanned for target sequences that have AA dinucleotides. Sense and anti-sense oligonucleotides may be generated to these targets that contain a G/C content of, for example, about 35 to 55%. These sequences may then be compared to others in the human genome database to minimize homology to other known coding sequences (e.g. by performing a BLAST search using the information available through the NCBI database).
An siRNA polynucleotide molecule may be generated by in vitro or in vivo transcription of suitable DNA sequences (e.g., polynucleotide sequences encoding a target polypeptide, or a desired portion thereof). The DNA may be incorporated into a vector with a suitable RNA polymerase promoter (such as for example, T7, U6, H1, or SP6 although other promoters may be equally useful). Endogenous RNA polymerases within a cell may mediate transcription in vivo or cloned RNA polymerase may be used for transcription in vivo or in vitro. For transcription from a transgene or an expression construct, a regulatory region may be used to transcribe siRNA strands.
A vector may be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470 to Nabel, et al.), or by stereotactic injection (see, e.g., Chen et al., Proc. Natl. Acad. Sci. USA 91:3054-3057, 1994). A DNA template may include two transcription units, one that produces a transcript that includes the sense strand of an siRNA agent and one that produces a transcript that includes the antisense strand of an siRNA agent. When the templates are transcribed following delivery, an siRNA agent may be produced.
Polynucleotides that comprise an siRNA may be derived from a single-stranded polynucleotide that comprises a single-stranded oligonucleotide fragment (e.g., of about 18-30 nucleotides) and its reverse complement, typically separated by a spacer sequence. Cleavage of the spacer may provide a single-stranded oligonucleotide fragment and its reverse complement, and they may anneal to form, optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3′ end and/or the 5′ end of either or both strands, a double-stranded siRNA polynucleotide. The spacer may be of a length that permits the fragment and its reverse complement to anneal and form a double-stranded structure (e.g., like a hairpin polynucleotide) prior to cleavage of the spacer, and optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3′ end and/or the 5′ end of either or both strands. A spacer sequence may therefore be any polynucleotide sequence that is situated between two complementary polynucleotide sequence regions which, when annealed into a double-stranded nucleic acid, result in an siRNA polynucleotide.
A formed siRNA polynucleotide may have blunt ends. Optionally, at least one strand of the siRNA polynucleotide may have one or more nucleotides overhanging at the 3′ end. For instance, each strand of an siRNA polynucleotide duplex may have a two-nucleotide overhang at the 3′ end. The two-nucleotide overhang may be a thymidine dinucleotide (TT) but may also comprise other bases, for example, a TC dinucleotide or a TG dinucleotide, or any other dinucleotide. The overhang dinucleotide may also be complementary to the two nucleotides at the 5′ end of the sequence of the polynucleotide that is targeted for interference. For a discussion of 3′ ends of siRNA polynucleotides see, e.g., WO 01/75164, to Tuschl, et al., which is incorporated herein by reference. A double-stranded siRNA structure may be formed by a single self-complementary RNA strand or two complementary RNA strands.
An siRNA polynucleotide may comprise other naturally occurring, recombinant, or synthetic single-stranded or double-stranded polymers of nucleotides (ribonucleotides or deoxyribonucleotides or a combination of both) and/or nucleotide analogues (e.g., an oligonucleotide or polynucleotide or the like, typically in 5′ to 3′ phosphodiester linkage).
Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. Accordingly, siRNA containing nucleotide sequences identical to a portion of the target gene may be preferred for inhibition. However, siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence may also be effective for inhibition and are encompassed herein. For instance, an siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside.
An siRNA compound may exhibit variability by differing (e.g., by nucleotide substitution, including transition or transversion) at one, two, three or four nucleotides from a particular sequence. These differences may occur at any of the nucleotide positions of a particular siRNA, depending on the length of the molecule, whether situated in a sense or in an antisense strand of the double-stranded polynucleotide.
The nucleotide difference may be found on one strand of a double-stranded polynucleotide, where the complementary nucleotide with which the substitute nucleotide would typically form hydrogen bond base pairing may not necessarily be correspondingly substituted.
Sequence identity may be optimized by alignment algorithms known in the art and calculating the percent difference between the nucleotide sequences. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript.
Following formation, an siRNA polynucleotide may be tested for the ability to interfere with the expression of the target polypeptide according to methods known in the art. The determination of the effectiveness of an siRNA polynucleotide includes not only consideration of its ability to interfere with the expression of the target polypeptide, but also whether the siRNA polynucleotide is toxic to the host cell. For example, a desirable siRNA would exhibit an RNA interference activity and would also not exhibit an unwanted biological consequence. An example of an unwanted biological consequence is apoptosis of a cell for which cell death is not a desired as a result of the introduction of the siRNA into the host cell.
The properties of an siRNA agent, including its pharmacological properties, may be influenced and tailored, for example by the introduction of ligands, e.g. tethered ligands, or vectors, e.g., viral vectors, to the siRNA agent. In addition, pharmacological properties of an siRNA may be improved by incorporating a ligand in a formulation of an siRNA when the siRNA agent has a tethered ligand.
A wide variety of ligands may be tethered to an siRNA agent or used as formulation conjugate or additive, e.g., to the carrier of a ligand-conjugated monomer subunit. Examples are described below in the context of a ligand-conjugated monomer subunit but it should be understood that ligands may be coupled at other points to an siRNA agent.
Ligands may be coupled, covalently or non-covalently, either directly or indirectly via an intervening tether to the carrier. A ligand or tethered ligand may be present on a ligand-conjugated monomer when the ligand-conjugated monomer is incorporated into the growing strand. In one embodiment, a ligand may be incorporated into a “precursor” ligand-conjugated monomer subunit after a “precursor” ligand-conjugated monomer subunit has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether, e.g., TAP—(CH2)nNH2 may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer subunit into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, may subsequently be attached to the precursor ligand-conjugated monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor ligand-conjugated monomer subunit tether.
A ligand may alter the distribution, targeting or lifetime of an siRNA agent into which it is incorporated. For instance, a ligand may provide an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body. A ligand may improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide.
A ligand may act as a therapeutic modifier, e.g., for enhancing uptake; a diagnostic compound or reporter group e.g., for monitoring distribution; a cross-linking agent; a nuclease-resistance conferring moiety; and/or a natural or unusual nucleobase; among other uses. Non-limiting examples may include lipophilic molecules, lipids, lectins, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins, carbohydrates (e.g., a dextran, pullulan, chitin, chitosan, synthetic (e.g., Oligo Lactate 15-mer) and natural (e.g., low and medium molecular weight) polymers, insulin, cyclodextrin or hyaluronic acid), proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. Other examples include folic acid or epithelial cell receptor ligands, such as transferin.
A ligand may be a naturally occurring or recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
A ligand may include a targeting group, e.g., a cell or tissue targeting agent, e.g., a thyrotropin, melanotropin, surfactant protein A, mucin carbohydrate, a glycosylated polyaminoacid, transferrin, bisphosphonate, polyglutamate, polyaspartate, or an Arg-Gly-Asp (RGD) peptide or RGD peptide mimetic.
A ligand may be a protein, e.g., glycoproteins, lipoproteins, e.g. low density lipoprotein (LDL), or albumins, e.g. human serum albumin (HSA), or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. A ligand may be a hormone or hormone receptor. They may also include non-peptidic species, such as cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose.
A ligand may be a substance, e.g., a drug, which may increase the uptake of the siRNA agent into a cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. A ligand may be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tetracyclin.
A ligand may be a lipid or lipid-based molecule that may bind a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand may allow for distribution of the conjugate to a target tissue, e.g., liver tissue, including parenchymal cells of the liver. Other molecules that may bind HSA may also be used as ligands. For example, neproxin or aspirin may be used. A lipid or lipid-based ligand may (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) may be used to adjust binding to a serum protein, e.g., HSA. A lipid based ligand may be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body.
Viral and non-viral vectors may be utilized as delivery vehicles in delivery of siRNA constructs as is known in the art, any of which may be incorporated herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a plasmid, which refers to a circular double stranded DNA loop into which additional nucleic acid segments may be ligated. Another type of vector is a viral vector, wherein additional nucleic acid segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to as recombinant expression vectors, or more simply expression vectors. In general, expression vectors of utility may be in the form of plasmids. In the present specification, plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, lentiviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. In one embodiment, lentiviruses may be used to deliver one or more siRNA molecules to a cell, e.g., a macrophage, T cell, dendritic cell, or hematopoietic stem cell.
The various components of a construct may be operably linked to one another, according to known practices. Within a vector, “operably linked” is intended to mean that a nucleotide sequence of interest is linked to regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in a target cell when the vector is introduced into the target cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the target cell, the level of expression of siRNA desired, and the like.
An siRNA construct may be incorporated into a delivery vehicle, e.g., a liposome, or a nano- or microparticle. For example, an siRNA may be encapsulated in a liposome as described in Liposome Technology, Vol. II, Incorporation of Drugs, Proteins, and Genetic Material, CRC Press. An siRNA, depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, may include phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
Depending on the particular sequence utilized and the dose of double-stranded siRNA material delivered, an siRNA may provide partial or complete loss of function for the target gene. A reduction or loss of gene expression in at least 99% of targeted cells has been shown for genes, e.g. U.S. Pat. No. 6,506,559 to Fire, et al. Lower doses of delivered material and longer times after administration of the selected siRNA may result in inhibition in a smaller fraction of cells.
A delivery device may be used to deliver siRNA to a gene for inhibition. For example, a gene that is essential for replication of a pathogen, transmission of a pathogen, or maintenance of an infection may be inhibited using a delivery device. As another example, genes of cells at risk for infection by a pathogen or already infected cells may be targeted. The target gene may be a pathogen or host gene responsible for entry of a pathogen into its host, drug metabolism by the pathogen or host, replication or integration of the pathogen's genome, establishment or spread of an infection in the host, or assembly of the next generation of pathogens. Methods of prophylaxis (i.e., prevention or decreased risk of infection), as well as reduction in the frequency or severity of symptoms associated with infection, are encompassed. A device may be used in combination with other treatment regimens, including virostatic and virotoxic agents, antibiotic agents, antifungal agents, anti-inflammatory agents, as well as combination therapies, and the like.
A device may be used for inhibiting gene expression of cancer-related genes. By way of example, an siRNA of a device may silence a gene of a cancer, including solid tumors and leukemias, including: apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), histiocytic disorders, leukemia (e.g., B cell, mixed cell, null cell, T cell, T-cell chronic, HTLV-II-associated, lymphocytic acute, lymphocytic chronic, mast cell, and myeloid), histiocytosis malignant, Hodgkin disease, immunoproliferative small, non-Hodgkin lymphoma, plasmacytoma, reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, cranio-pharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing, experimental, Kaposi, and mast cell), neoplasms (e.g., bone, breast, digestive system, colorectal, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, and urogenital), neurofibromatosis, and cervical dysplasia, and for treatment of other conditions in which cells have become immortalized or transformed. A device may be used in combination with other treatment modalities, such as chemotherapy, cryotherapy, hyperthermia, radial-ion therapy, and the like.
A device is not limited to any type of target gene or nucleotide sequence. However, the following classes of possible target genes are listed for illustrative purposes as target genes: developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogenes (e.g., ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYON, NRAS, PIM1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53, and WT1), and enzymes (e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hemicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases).
The device includes, in addition to the siRNA construct(s), microneedles upon which have been fabricated a plurality of nano-sized structures. As utilized herein, the term ‘fabricated’ generally refers to a structure that has been specifically designed, engineered, and/or constructed so as to exist at a surface of the device and is not to be equated with a surface feature that is merely an incidental product of a device formation process. Thus, there will be a predetermined pattern of nanostructures on the surface of the microneedles.
During use, the device, and specifically, the nano-sized structures on the surface of the microneedles, may interact with the dermal tissue and components thereof. This interaction may regulate or modulate (i.e., change) intracellular and/or intercellular signal transduction associated with cell/cell interactions, endocytosis, inflammatory response, and so forth. For instance, through interaction between the nanotopography on a surface and surrounding biological materials or structures, the device may regulate and/or modulate membrane potential, membrane proteins, and/or intercellular junctions (e.g., tight junctions, gap junctions, and/or desmasomes). This may encourage the transdermal delivery of the siRNA constructs. Moreover, the siRNA constructs may be delivered across the dermal barrier without instigating a foreign body or immune response.
The device may be constructed from a variety of materials, including metals, ceramics, semiconductors, organics, polymers, etc., as well as composites thereof. By way of example, pharmaceutical grade stainless steel, titanium, nickel, iron, gold, tin, chromium, copper, alloys of these or other metals, silicon, silicon dioxide, and polymers may be utilized in forming a device. Typically, the microneedles of the device are formed of a biocompatible material that is capable of carrying a pattern of nano-sized structures on a surface. The term “biocompatible” generally refers to a material that does not substantially adversely affect the cells or tissues in the area where the device is to be delivered. It is also intended that the materials do not cause any substantially medically undesirable effect in any other areas of a living subject utilizing a device. Biocompatible materials may be synthetic or natural. Some examples of suitable biocompatible materials, which are also biodegradable, include polymers of hydroxy acids such as lactic acid and glycolic acid polylactide, polyglycolide, polylactide-co-glycolide, copolymers with PEG, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone). Other suitable materials may include, without limitation, polycarbonate, polymethacrylic acid, ethylenevinyl acetate, polytetrafluorethylene, and polyesters. The various components of a device (e.g., the microneedles, the base, the top, drug contacting areas, etc.) may be non-porous or porous in nature, may be homogeneous or heterogeneous across the device with regard to materials, geometry, solidity, and so forth, and may have a rigid fixed or a semi-fixed shape.
The dimensions of the channel 16, when present, may be specifically selected to induce capillary flow of a drug compound. Capillary flow generally occurs when the adhesive forces of a fluid to the walls of a channel are greater than the cohesive forces between the liquid molecules. Specifically, capillary pressure is inversely proportional to the cross-sectional dimension of the channel 16 and directly proportional to the surface tension of the liquid, multiplied by the cosine of the contact angle of the fluid in contact with the material forming the channel. Thus, to facilitate capillary flow in the patch, the cross-sectional dimension (e.g., width, diameter, etc.) of the channel 16 may be selectively controlled, with smaller dimensions generally resulting in higher capillary pressure. For example, in some embodiments, the cross-sectional dimension of the channel typically ranges from about 1 micrometer to about 100 micrometers, in some embodiments from about 5 micrometers to about 50 micrometers, and in some embodiments, from about 10 micrometers to about 30 micrometers. The dimension may be constant or it may vary as a function of the length of the channel 16. The length of the channel may also vary to accommodate different volumes, flow rates, and dwell times for the drug compound. For example, the length of the channel may be from about 10 micrometers to about 800 micrometers, in some embodiments from about 50 micrometers to about 500 micrometers, and in some embodiments, from about 100 micrometers to about 300 micrometers. The cross-sectional area of the channel may also vary. For example, the cross-sectional area may be from about 50 square micrometers to about 1,000 square micrometers, in some embodiments from about 100 square micrometers to about 500 square micrometers, and in some embodiments, from about 150 square micrometers to about 350 square micrometers. Further, the aspect ratio (length/cross-sectional dimension) of the channel may range from about 1 to about 50, in some embodiments from about 5 to about 40, and in some embodiments from about 10 to about 20. In cases where the cross-sectional dimension (e.g., width, diameter, etc.) and/or length vary as a function of length, the aspect ratio can be determined from the average dimensions.
It should be understood that the number of microneedles shown in the figures is for illustrative purposes only. The actual number of microneedles used in a microneedle assembly may, for example, range from about 500 to about 10,000, in some embodiments from about 2,000 to about 8,000, and in some embodiments, from about 4,000 to about 6,000.
An individual microneedle may have a straight or a tapered shaft. In one embodiment, the diameter of a microneedle may be greatest at the base end of the microneedle and taper to a point at the end distal the base. A microneedle may also be fabricated to have a shaft that includes both a straight (untapered) portion and a tapered portion.
A microneedle may be formed with a shaft that is circular or non-circular in cross-section. For example, the cross-section of a microneedle may be polygonal (e.g. star-shaped, square, triangular), oblong, or any other shape. The shaft may have one or more bores and/or channels.
The size of individual needles may be optimized depending upon the desired targeting depth, the strength requirements of the needle to avoid breakage in a particular delivery location, etc. For instance, the cross-sectional dimension of a transdermal microneedle may be between about 10 nanometers (nm) and 1 millimeter (mm), or between about 1 micrometer (μm) and about 200 micrometers, or between about 10 micrometers and about 100 micrometers. The outer diameter may be between about 10 micrometers and about 100 micrometers and the inner diameter of a hollow needle may be between about 3 micrometers and about 80 micrometers. The tip typically has a radius that is less than or equal to about 1 micrometer.
The length of a microneedle will generally depend upon the desired application. For instance, a microneedle may be between about 1 micrometer and about 1 millimeter in length, for instance about 500 micrometers or less, or between about 10 micrometers and about 500 micrometers, or between about 30 micrometers and abut 200 micrometers.
An array of microneedles need not include microneedles that are all identical to one another. An array may include a mixture of microneedles having various lengths, outer diameters, inner diameters, cross-sectional shapes, nanostructured surfaces, and/or spacings between the microneedles. For example, the microneedles may be spaced apart in a uniform manner, such as in a rectangular or square grid or in concentric circles. The spacing may depend on numerous factors, including height and width of the microneedles, as well as the amount and type of any substance that is intended to be moved through the microneedles. While a variety of arrangements of microneedles is useful, a particularly useful arrangement of microneedles is a “tip-to-tip” spacing between microneedles of about 50 micrometers or more, in some embodiments about 100 to about 800 micrometers, and in some embodiments, from about 200 to about 600 micrometers.
Referring again to
The device may define a nanotopography on the surface of a microneedle in a random or organized pattern. The device may additionally define a nanotopography on the substrate surface from which the microneedle extends, though this is not a requirement.
A microneedle may include a plurality of identical structures formed on a surface or may include different structures formed of various sizes, shapes and combinations thereof. A predetermined pattern of structures may include a mixture of structures having various lengths, diameters, cross-sectional shapes, and/or spacings between the structures. For example, the structures may be spaced apart in a uniform manner, such as in a rectangular or square grid or in concentric circles. In one embodiment, structures may vary with regard to size and/or shape and may form a complex nanotopography. In one embodiment a complex nanotopography may define a fractal or fractal-like geometry.
As utilized herein, the term “fractal” generally refers to a geometric or physical structure having a fragmented shape at all scales of measurement between a greatest and a smallest scale such that certain mathematical or physical properties of the structure behave as if the dimensions of the structure are greater than the spatial dimensions. Mathematical or physical properties of interest may include, for example, the perimeter of a curve or the flow rate in a porous medium. The geometric shape of a fractal may be split into parts, each of which defines self-similarity. Additionally, a fractal has a recursive definition and has a fine structure at arbitrarily small scales.
As utilized herein, the term “fractal-like” generally refers to a geometric or physical structure having one or more, but not all, of the characteristics of a fractal. For instance, a fractal-like structure may include a geometric shape that includes self-similar parts, but may not include a fine structure at an arbitrarily small scale. In another example, a fractal-like geometric shape or physical structure may not decrease (or increase) in scale equally between iterations of scale, as may a fractal, though it will increase or decrease between recursive iterations of a geometric shape of the pattern. A fractal-like pattern may be simpler than a fractal. For example, it may be regular and relatively easily described in traditional Euclidean geometric language, whereas a fractal may not.
A microneedle surface defining a complex nanotopography may include structures of the same general shape (e.g., pillars) and the pillars may be formed to different scales of measurement (e.g., nano-scale pillars as well as micro-scale pillars). In another embodiment, a microneedle may include at a surface structures that vary in both scale size and shape or that vary only in shape while formed to the same nano-sized scale. Additionally, structures may be formed in an organized array or in a random distribution. In general, at least a portion of the structures may be nanostructures formed on a nano-sized scale, e.g., defining a cross-sectional dimension of less than about 500 nm, for instance less than about 400 nm, less than about 250 nm, or less than about 100 nm. The cross sectional dimension of the nanostructures may generally be greater than about 5 nanometers, for instance greater than about 10 nanometers, or greater than about 20 nanometers. For example, the nanostructures may define a cross sectional dimension between about 5 nanometers and about 500 nanometers, between about 20 nanometers and about 400 nanometers, or between about 100 nanometers and about 300 nanometers. In cases where the cross sectional dimension of a nanostructure varies as a function of height of the nanostructure, the cross sectional dimension can be determined as the average from the base to the tip of the nanostructures, or as the maximum cross sectional dimension of the structure, for example the cross sectional dimension at the base of a cone-shaped nanostructure.
A pattern that includes structures of different sizes may include larger structures having a cross-sectional dimension formed on a larger scale, e.g., microstructures having a cross-sectional dimension greater than about 500 nanometers in combination with smaller nanostructures. In one embodiment, microstructures of a complex nanotopography may have a cross-sectional dimension between about 500 nanometers and about 10 micrometers, between about 600 nanometers and about 1.5 micrometers, or between about 650 nanometers and about 1.2 micrometers. For example, the complex nanotopography of
When a pattern includes one or more larger microstructures, for instance, having a cross-sectional dimension greater than about 500 nanometers, determined either as the average cross sectional dimension of the structure or as the largest cross sectional dimension of the structure, the complex nanotopography will also include nanostructures, e.g., first nanostructures, second nanostructures of a different size and/or shape, etc. For example, pillars 102 of the complex nanotopography of
A nanotopography may be formed of any number of different elements. For instance, a pattern of elements may include two different elements, three different elements, an example of which is illustrated in
The spacing of individual elements may also vary. For instance, center-to-center spacing of individual structures may be between about 50 nanometers and about 1 micrometer, for instance between about 100 nanometers and about 500 nanometers. For example, center-to-center spacing between structures may be on a nano-sized scale. For instance, when considering the spacing of nano-sized structures, the center-to-center spacing of the structures may be less than about 500 nanometers. This is not a requirement of a topography, however, and individual structures may be farther apart. The center-to-center spacing of structures may vary depending upon the size of the structures. For example, the ratio of the average of the cross-sectional dimensions of two adjacent structures to the center-to-center spacing between those two structures may be between about 1:1 (e.g., touching) and about 1:4, between about 1:1.5 and about 1:3.5, or between about 1:2 and about 1:3. For instance, the center to center spacing may be approximately double the average of the cross-sectional dimensions of two adjacent structures. In one embodiment, two adjacent structures each having a cross-sectional dimension of about 200 nanometers may have a center-to-center spacing of about 400 nanometers. Thus, the ratio of the average of the diameters to the center-to-center spacing in this case is 1:2.
Structure spacing may be the same, i.e., equidistant, or may vary for structures in a pattern. For instance, the smallest structures of a pattern may be spaced apart by a first distance, and the spacing between these smallest structures and a larger structure of the pattern or between two larger structures of the pattern may be the same or different as this first distance.
For example, in the pattern of
Structures of a topography may all be formed to the same height, generally between about 10 nanometers and about 1 micrometer, but this is not a requirement, and individual structures of a pattern may vary in size in one, two, or three dimensions. In one embodiment, some or all of the structures of a topography can have a height of less than about 20 micrometers, less than about 10 micrometers, or less than about 1 micrometer, for instance less than about 750 nanometers, less than about 680 nanometers, or less than about 500 nanometers. For instance the structures can have a height between about 50 nanometers and about 20 micrometers or between about 100 nanometers and about 700 nanometers. For example, nanostructures or microstructures can have a height between about 20 nm and about 500 nm, between about 30 nm and about 300 nm, or between about 100 nm and about 200 nm, though it should be understood that structures may be nano-sized in a cross sectional dimension and may have a height that may be measured on a micro-sized scale, for instance greater than about 500 nm. Micro-sized structures can have a height that is the same or different from nano-sized structures of the same pattern. For instance, micro-sized structures can have a height of between about 500 nanometers and about 20 micrometers, or between about 1 micrometer and about 10 micrometers, in another embodiment. Micro-sized structures may also have a cross sectional dimension on a micro-scale greater than about 500 nm, and may have a height that is on a nano-sized scale of less than about 500 nm.
The aspect ratio of the structures (the ratio of the height of a structure to the cross sectional dimension of the structure) can be between about 0.15 and about 30, between about 0.2 and about 5, between about 0.5 and about 3.5, or between about 1 and about 2.5. For instance, nanostructures may have an aspect ratio falling within any of these ranges.
The device surface may include a single instance of a pattern, as shown in
The formation of nanotopography on a microneedle surface may increase the surface area of the microneedle without a corresponding increase in volume. Increase in the surface area to volume ratio is believed to improve the interaction of the microneedle surface with surrounding biological materials. For instance, increase in the surface area to volume ratio is believed to encourage mechanical interaction between the nanotopography and surrounding proteins, e.g., extracellular matrix (ECM) proteins and/or plasma membrane proteins. As utilized herein, the term “protein” generally refers to a molecular chain of amino acids that is capable of interacting structurally, enzymatically or otherwise with other proteins, polypeptides or any other organic or inorganic molecule.
In general, the surface area to volume ratio of a nanopatterned surface may be greater than about 10,000 cm−1, greater than about 150,000 cm−1, or greater than about 750,000 cm−1. Determination of the surface area to volume ratio may be carried out according to any standard methodology as is known in the art. For instance, the specific surface area of a surface may be obtained by the physical gas adsorption method (B.E.T. method) with nitrogen as the adsorption gas, as is generally known in the art and described by Brunauer, Emmet, and Teller (J. Amer. Chem. Soc., vol. 60, February, 1938, pp. 309-319), incorporated herein by reference. The BET surface area can be less than about 5 m2/g, in one embodiment, for instance between about 0.1 m2/g and about 4.5 m2/g, or between about 0.5 m2/g and about 3.5 m2/g. Values for surface area and volume may also be estimated from the geometry of molds used to form a surface, according to standard geometric calculations. For example, the volume may be estimated according to the calculated volume for each pattern element and the total number of pattern elements in a given area, e.g., over the surface of a single microneedle.
For a device that defines a fractal or fractal-like patterned nanotopography at a surface, the nanotopography may be characterized through determination of the fractal dimension of the pattern. The fractal dimension is a statistical quantity that gives an indication of how completely a fractal appears to fill space as the recursive iterations continue to smaller and smaller scale. The fractal dimension of a two dimensional structure may be represented as:
where N(e) is the number of self-similar structures needed to cover the whole object when the object is reduced by 1/e in each spatial direction.
For example, when considering the 2 dimensional fractal known as the Sierpenski triangle illustrated in
Thus, the Sierpenski triangle fractal exhibits an increase in line length over the initial two dimensional equilateral triangle. Additionally, this increase in line length is not accompanied by a corresponding increase in area.
The fractal dimension of the pattern illustrated in
In addition to or alternative to the examination of surface area to volume ratio and/or fractal dimension, the microneedles of the siRNA delivery devices may be characterized by other methods including, without limitation, surface roughness, elastic modulus, and surface energy.
Methods for determining the surface roughness are generally known in the art. For instance, an atomic force microscope process in contact or non-contact mode may be utilized according to standard practice to determine the surface roughness of a material. Surface roughness that may be utilized to characterize a microneedle may include the average roughness (RA), the root mean square roughness, the skewness, and/or the kurtosis. In general, the average surface roughness (i.e., the arithmetical mean height of the surface are roughness parameter as defined in the ISO 25178 series) of a surface defining a fabricated nanotopography thereon may be less than about 200 nanometers, less than about 190 nanometers, less than about 100 nanometers, or less than about 50 nanometers. For instance, the average surface roughness may be between about 10 nanometers and about 200 nanometers, or between about 50 nanometers and about 190 nanometers.
The microneedle surface may be characterized by the elastic modulus of the surface, for instance by the change in elastic modulus upon the addition of a nanotopography to the surface. In general, the addition of a plurality of structures forming nanotopography on the microneedle surface may decrease the elastic modulus of a material, as the addition of nano-sized structures on the surface will lead to a reduction in continuity of the surface and a related change in surface area. As compared to a similar microneedle formed according to the same process and of the same materials, but for the pattern of nanotopography on the surface, a microneedle including nanotopography thereon may exhibit a decrease in elastic modulus of between about 35% and about 99%, for instance between about 50% and about 99%, or between about 75% and about 80%. By way of example, the effective compression modulus of a nanopatterned surface can be less than about 50 MPa, or less than about 20 MPa. In one embodiment the effective compression modulus can be between about 0.2 MPa and about 50 MPa, between about 5 MPa and about 35 MPa, or between about 10 MPa and about 20 MPa. The effective shear modulus can be less than about 320 MPa, or less than about 220 MPa. For instance, the effective shear modulus can be between about 4 MPa and about 320 MPa, or between about 50 MPa and about 250 MPa, in one embodiment.
A microneedle including nanotopography thereon may also exhibit an increase in surface energy as compared to a similar microneedle that does not have the pattern of nanotopography thereon. For instance, the microneedle including a nanotopography formed thereon may exhibit an increase in surface energy as compared to a similar microneedle of the same materials and formed according to the same methods, but for the inclusion of the pattern of nanotopography on the surface. For instance, the water contact angle of a surface including a nanotopography thereon can be greater than about 80°, greater than about 90°, greater than about 100°, or greater than about 110°. For example, the water contact angle of a surface can be between about 80° and about 150°, between about 90° and about 130°, or between about 100° and about 120°, in one embodiment.
When forming nanostructures on the surface of the device, the packing density of the structures may be maximized. For instance, square packing (
The microneedles including a fabricated nanotopography on the surface of the microneedles may be formed according to a single-step process, i.e., the microneedles are formed with the nanostructures on the surface at the time of formation. Alternatively, a multi-step process may be used, in which a pattern of nanostructures are fabricated on a pre-formed microneedle. For example, an array of microneedles may be first formed and then a random or non-random pattern of nanostructures may be fabricated on the surface of the formed microneedles. In either the single-step or two-step process, the nano-sized structures may be fabricated on the microneedle surface or on a mold surface according to any suitable nanotopography fabrication method including, without limitation, nanoimprinting, injection molding, lithography, embossing molding, and so forth.
An array of microneedles may be formed according to any standard microfabrication technique including, without limitation, lithography; etching techniques, such as wet chemical, dry, and photoresist removal; thermal oxidation of silicon; electroplating and electroless plating; diffusion processes, such as boron, phosphorus, arsenic, and antimony diffusion; ion implantation; film deposition, such as evaporation (filament, electron beam, flash, and shadowing and step coverage), sputtering, chemical vapor deposition (CVD), epitaxy (vapor phase, liquid phase, and molecular beam), electroplating, screen printing, and lamination; stereolithography; laser machining; and laser ablation (including projection ablation).
An electrochemical etching process may be utilized in which electrochemical etching of solid silicon to porous silicon is used to create extremely fine (on the order of 0.01 μm) silicon networks that may be used as piercing structures. This method may use electrolytic anodization of silicon in aqueous hydrofluoric acid, potentially in combination with light, to etch channels into the silicon. By varying the doping concentration of the silicon wafer to be etched, the electrolytic potential during etching, the incident light intensity, and the electrolyte concentration, control over the ultimate pore structure may be achieved. The material not etched (i.e. the silicon remaining) forms the microneedles.
Plasma etching may also be utilized, in which deep plasma etching of silicon is carried out to create microneedles with diameters on the order of 0.1 μm or larger. Needles may be fabricated indirectly by controlling the voltage (as in electrochemical etching).
Lithography techniques, including photolithography, e-beam lithography, X-ray lithography, and so forth may be utilized for primary pattern definition and formation of a master die. Replication may then be carried out to form a device including an array of microneedles. Common replication methods include, without limitation, solvent-assisted micromolding and casting, embossing molding, injection molding, and so forth. Self-assembly technologies including phase-separated block copolymer, polymer demixing and colloidal lithography techniques may also be utilized in forming a nanotopography on a surface.
Combinations of methods may be used, as is known. For instance, substrates patterned with colloids may be exposed to reactive ion etching (RIE, also known as dry etching) so as to refine the characteristics of a fabricated nanostructure such as nanopillar diameter, profile, height, pitch, and so forth. Wet etching may also be employed to produce alternative profiles for fabricated nanostructures initially formed according to a different process, e.g., polymer de-mixing techniques.
Structure diameter, shape, and pitch may be controlled via selection of appropriate materials and methods. For example, etching of metals initially evaporated onto colloidal-patterned substrates followed by colloidal lift-off generally results in prism-shaped pillars. An etching process may then be utilized to complete the structures as desired. Ordered non-spherical polymeric nanostructures may also be fabricated via temperature-controlled sintering techniques, which form a variety of ordered trigonal nanometric features in colloidal interstices following selective dissolution of polymeric nanoparticles. These and other suitable formation processes are generally known in the art (see, e.g., Wood, J R Soc Interface, 2007 Feb. 22; 4(12): 1-17, incorporated herein by reference).
Other methods as may be utilized in forming a microneedle including a fabricated nanotopography on a surface include nanoimprint lithography methods utilizing ultra-high precision laser machining techniques, examples of which have been described by Hunt, et al. (U.S. Pat. No. 6,995,336) and Guo, et al. (U.S. Pat. No. 7,374,864), both of which are incorporated herein by reference. Nanoimprint lithography is a nano-scale lithography technique in which a hybrid mold is utilized which acts as both a nanoimprint lithography mold and a photolithography mask. A schematic of a nanoimprint lithography technique is illustrated in
To facilitate the release of the nanoimprinted substrate 32 including fabricated features 34 from the mold 30, as depicted in
A nanoimprinting process is a dynamic one which includes filling a mold followed by detachment of a formed polymer from the mold. To fill the mold features, the polymer temperature must be raised to a level high enough to initiate flow under the applied pressure. The higher the temperature, the lower the polymer viscosity, and the faster and easier the mold will fill. A higher pressure will also improve the fill rate and overall fill for better mold replication. To release the nanoimprinted substrate from the mold, the substrate temperature may be lowered to a point where the yield strength exceeds the adhesional forces exerted by the mold. By varying the temperature it is also possible to draw the polymer features during detachment to obtain different structures, for instance structures as illustrated in
The nanostructures may also be formed on the microneedle according to chemical addition processes. For instance, film deposition, sputtering, chemical vapor deposition (CVD), epitaxy (vapor phase, liquid phase, and molecular beam), electroplating, and so forth may be utilized for building structures on a surface.
Self-assembled monolayer processes as are known in the art may be utilized to form the structures on the microneedle surface. For instance, the ability of block copolymers to self-organize may be used to form a monolayer pattern on the surface. The pattern may then be used as a template for the growth of the desired structures, e.g., colloids, according to the pattern of the monolayer.
By way of example, a two-dimensional, cross-linked polymer network may be produced from monomers with two or more reactive sites. Such cross-linked monolayers have been made using self-assembling monolayer (SAM) (e.g., a gold/alkyl thiol system) or Langmuir-Blodgett (LB) monolayer techniques (Ahmed et al., Thin Solid Films 187: 141-153 (1990)) as are known in the art. The monolayer may be crosslinked, which may lead to formation of a more structurally robust monolayer.
The monomers used to form the patterned monolayer may incorporate all the structural moieties necessary to affect the desired polymerization technique and/or monolayer formation technique, as well as to influence such properties as overall solubility, dissociation methods, and lithographic methods. A monomer may contain at least one, and more often at least two, reactive functional groups.
A molecule used to form an organic monolayer may include any of various organic functional groups interspersed with chains of methylene groups. For instance a molecule may be a long chain carbon structure containing methylene chains to facilitate packing. The packing between methylene groups may allow weak Van der Waals bonding to occur, enhancing the stability of the monolayer produced and counteracting the entropic penalties associated with forming an ordered phase. In addition, different terminal moieties, such as hydrogen-bonding moieties, may be present at one terminus of the molecules, in order to allow growth of structures on the formed monolayer, in which case the polymerizable chemical moieties may be placed in the middle of the chain or at the opposite terminus. Any suitable molecular recognition chemistry may be used in forming the assembly. For instance, structures may be assembled on a monolayer based on electrostatic interaction, Van der Waals interaction, metal chelation, coordination bonding (i.e., Lewis acid/base interactions), ionic bonding, covalent bonding, or hydrogen bonding.
When utilizing a SAM-based system, an additional molecule may be utilized to form the template. This additional molecule may have appropriate functionality at one of its termini in order to form a SAM. For example, on a gold surface, a terminal thiol may be included. There are a wide variety of organic molecules that may be employed to effect replication. Topochemically polymerizable moieties, such as dienes and diacetylenes, are particularly desirable as the polymerizing components. These may be interspersed with variable lengths of methylene linkers.
For an LB monolayer, only one monomer molecule is needed because the molecular recognition moiety may also serve as the polar functional group for LB formation purposes. Lithography may be carried out on a LB monolayer transferred to a substrate, or directly in the trough. For example, an LB monolayer of diacetylene monomers may be patterned by UV exposure through a mask or by electron beam patterning.
Monolayer formation may be facilitated by utilizing molecules that undergo a topochemical polymerization in the monolayer phase. By exposing the assembling film to a polymerization catalyst, the film may be grown in situ, and changed from a dynamic molecular assembly to a more robust polymerized assembly.
Any of the techniques known in the art for monolayer patterning may be used. Techniques useful in patterning the monolayer include, but are not limited to, photolithography, e-beam techniques, focused ion-beam techniques, and soft lithography. Various protection schemes such as photoresist may be used for a SAM-based system. Likewise, block copolymer patterns may be formed on gold and selectively etched to form patterns. For a two-component system, patterning may also be achieved with readily available techniques.
Soft lithography techniques may be utilized to pattern the monolayer in which ultraviolet light and a mask may be used for patterning. For instance, an unpatterned base monolayer may be used as a platform for assembly of a UV/particle beam reactive monomer monolayer. The monomer monolayer may then be patterned by UV photolithography, e-beam lithography, or ion beam lithography, even though the base SAM is not patterned.
Growth of structures on a patterned monolayer may be achieved by various growth mechanisms, such as through appropriate reduction chemistry of a metal salt and the use of seed or template-mediated nucleation. Using the recognition elements on the monolayer, inorganic growth may be catalyzed at this interface by a variety of methods. For instance inorganic compounds in the form of colloids bearing the shape of the patterned organic monolayer may be formed. For instance calcium carbonate or silica structures may be templated by various carbonyl functionalities such as carboxylic acids and amides. By controlling the crystal growth conditions, it is possible to control the thickness and crystal morphology of the mineral growth. Titanium dioxide may also be templated.
Templated electroless plating techniques may be used to synthesize metals using existing organic functional groups. In particular, by chelating metal atoms to the carbonyl moieties of the organic pattern, electroless metal deposition may be catalyzed on the pattern, forming patterned metallic colloids. For instance, Cu, Au, Ni, Ag, Pd, Pt and many other metals plateable by electroless plating conditions may be used to form metal structures in the shape of the organic monolayer. By controlling the electroless plating conditions, it is possible to control the thickness of the plated metal structures.
Other ‘bottom-up’ type growth methods as are known in the art may be utilized, for example a method as described in U.S. Pat. No. 7,189,435 to Tuominen, et al., which is incorporated herein by reference, may be utilized. According to this method, a conducting or semiconducting substrate (for example, a metal, such as gold) may be coated with a block copolymer film (for example, a block copolymer of methylmethacrylate and styrene), where one component of the copolymer forms nanoscopic cylinders in a matrix of another component of the copolymer. A conducting layer may then be placed on top of the copolymer to form a composite structure. Upon vertical orientation of the composite structure, some of the first component may be removed, for instance by exposure to UV radiation, an electron beam, or ozone, degradation, or the like to form nanoscopic pores in that region of the second component.
In another embodiment, described in U.S. Pat. No. 6,926,953 to Nealey, et al., incorporated herein by reference, copolymer structures may be formed by exposing a substrate with an imaging layer thereon, for instance an alkylsiloxane or an octadecyltrichlorosilane self assembled monolayer, to two or more beams of selected wavelengths to form interference patterns at the imaging layer to change the wettability of the imaging layer in accordance with the interference patterns. A layer of a selected block copolymer, for instance a copolymer of polystyrene and poly(methyl methacrylate) may then be deposited onto the exposed imaging layer and annealed to separate the components of the copolymer in accordance with the pattern of wettability and to replicate the pattern of the imaging layer in the copolymer layer. Stripes or isolated regions of the separated components may thus be formed with periodic dimensions in the range of 100 nm or less.
The microneedle surface may include a random distribution of fabricated nanostructures. Optionally, the microneedle surface may include additional materials, in conjunction with the fabricated nanostructures. For example, the microneedle may have fabricated thereon an electrospun fibrous layer, and a random or non-random pattern of nanostructures may be fabricated on this electrospun layer.
Electrospinning includes of the use of a high voltage supplier to apply an electrical field to a polymer melt or solution held in a capillary tube, inducing a charge on the individual polymer molecules. Upon application of the electric field, a charge and/or dipolar orientation will be induced at the air-surface interface. The induction causes a force that opposes the surface tension. At critical field strength, the electrostatic forces will overcome surface tension forces, and a jet of polymer material will be ejected from the capillary tube toward a conductive, grounded surface. The jet is elongated and accelerated by the external electric field as it leaves the capillary tube. As the jet travels in air, some of the solvent may evaporate, leaving behind charged polymer fibers which may be collected on the surface. As the fibers are collected, the individual and still wet fibers may adhere to one another, forming a nonwoven web on the surface. A pattern of nanostructures may then be fabricated on the electrospun surface, for instance through an embossing technique utilizing a mold defining the desired nanostructures. Applying the mold to the microneedle surface at suitable temperature and pressure may transfer the pattern to the microneedle surface. A surface of random electrospun nano-sized fibers may further improve the desirable characteristics of a microneedle surface, e.g., one or more of surface area to volume ratio, surface roughness, surface energy, and so forth, and may provide associated benefits.
In addition to the nanostructures, the microneedle surface may be chemically functionalized for improved interaction with tissues or individual cells. For instance, one or more biomolecules such as polynucleotides, polypeptides, entire proteins, polysaccharides, and the like may be bound to the microneedle surface prior to use.
In some embodiments, the microneedle surface may include suitable reactivity such that additional desired functionality may spontaneously attach to the surface with no pretreatment of the surface necessary. However, in other embodiments, pretreatment of the structured surface prior to attachment of the desired compound may be carried out. For instance, reactivity of a structure surface may be increased through addition or creation of amine, carboxylic acid, hydroxy, aldehyde, thiol, or ester groups on the surface. In one representative embodiment, a microneedle surface including a pattern of nanotstructures formed thereon may be aminated through contact with an amine-containing compound such as 3-aminopropyltriethoxy silane in order to increase the amine functionality of the surface and bind one or more biomolecules to the surface via the added amine functionality.
Materials as may be desirably bound to the surface of a patterned device may include ECM proteins such as laminins, tropoelastin or elastin, Tropocollagen or collagen, fibronectin, and the like. Short polypeptide fragments may be bound to the surface of a patterned device such as an RGD sequence, which is part of the recognition sequence of integrin binding to many ECM proteins. Thus, functionalization of a microneedle surface with RGD may encourage interaction of the device with ECM proteins and further limit foreign body response to the device during use.
The siRNA construct for delivery via the device may be associated therewith according to any suitable methodology. For instance, a transdermal microneedle patch may be utilized for delivery of materials beneath the stratum corneum to the stratum spinosum or the stratum germinativum, or even deeper into the dermis. The siRNA, either free in the composition or held in a protected state within the composition, may be contained on the patch or fed to the patch so as to be transported across the stratum corneum in association with the microneedle, e.g., within the microneedle or at the surface of the microneedle.
The microneedle transdermal patch may include a reservoir, e.g., a vessel, a porous matrix, etc., that may store the siRNA construct and provide the siRNA construct for delivery. The device may include a reservoir within the device itself. For instance, the device may include a hollow, or multiple pores that may carry one or more siRNA constructs for delivery. The siRNA construct may be released from the device via degradation of a portion or the entire device or via diffusion of the agent from the device.
A formulation including one or more siRNA constructs may be retained within the reservoir 112. Materials suitable for use as impermeable backing layer 114 may include materials such as polyesters, polyethylene, polypropylene and other synthetic polymers. The material is generally heat or otherwise sealable to the backing layer to provide a barrier to transverse flow of reservoir contents.
Reservoir 112, defined by the space or gap between the impermeable backing layer 14 and the microneedle array 16, provides a storage structure in which to retain the suspension of siRNA constructs to be administered. The reservoir may be formed from a variety of materials that are compatible with an agent to be contained therein. By way of example, natural and synthetic polymers, metals, ceramics, semiconductor materials, and composites thereof may form the reservoir.
In one embodiment, the reservoir may be attached to the substrate upon which the microneedles are located. According to another embodiment, the reservoir may be separate and removably connectable to the microneedle array or in fluid communication with the microneedle array, for instance via appropriate tubing, leur locks, etc.
The device may include one or a plurality of reservoirs for storing agents to be delivered. For instance, the device may include a single reservoir that stores a single or multiple siRNA construct-containing formulation, or the device may include multiple reservoirs, each of which stores one or more agents for delivery to all or a portion of the array of microneedles. Multiple reservoirs may each store a different material that may be combined for delivery. For instance, a first reservoir may contain an siRNA construct and a second reservoir may contain a vehicle, e.g., saline, or a second siRNA construct. The different agents may be mixed prior to delivery. Mixing may be triggered by any means, including, for example, mechanical disruption (i.e. puncturing, degradation, or breaking), changing the porosity, or electrochemical degradation of the walls or membranes separating the chambers. Multiple reservoirs may contain different active agents for delivery that may be delivered in conjunction with one another or sequentially.
In one embodiment, the reservoir may be in fluid communication with one or more microneedles of the transdermal device, and the microneedles may define a structure (e.g., a central or lateral bore) to allow transport of delivered agents beneath the barrier layer.
In alternative embodiments, a device may include a microneedle assembly and a reservoir assembly with flow prevention between the two prior to use. For instance, a device may include a release member positioned adjacent to both a reservoir and a microneedle array. The release member may be separated from the device prior to use such that during use the reservoir and the microneedle array are in fluid communication with one another. Separation may be accomplished through the partial or complete detachment of the release member. For example, referring to
The rate control membrane may help slow down the flow rate of the drug compound upon its release. Specifically, fluidic drug compounds passing from the drug reservoir to the microneedle assembly via microfluidic channels may experience a drop in pressure that results in a reduction in flow rate. If this difference is too great, some backpressure may be created that may impede the flow of the compound and potentially overcome the capillary pressure of the fluid through the microfluidic channels. Thus, the use of the rate control membrane may ameliorate this difference in pressure and allow the drug compound to be introduced into the microneedle at a more controlled flow rate. The particular materials, thickness, etc. of the rate control membrane may vary based on multiple factors, such as the viscosity of the drug compound, the desired delivery time, etc.
The rate control membrane may be fabricated from permeable, semi-permeable or microporous materials that are known in the art to control the rate of drug compounds and having permeability to the permeation enhancer lower than that of drug reservoir. For example, the material used to form the rate control membrane may have an average pore size of from about 50 nanometers to about 5 micrometers, in some embodiments from about 100 nanometers to about 2 micrometers, and in some embodiments, from about 300 nanometers to about 1 micrometer (e.g., about 600 nanometers). Suitable membrane materials include, for instance, fibrous webs (e.g., woven or nonwoven), apertured films, foams, sponges, etc., which are formed from polymers such as polyethylene, polypropylene, polyvinyl acetate, ethylene n-butyl acetate and ethylene vinyl acetate copolymers. Such membrane materials are also described in more detail in U.S. Pat. Nos. 3,797,494, 4,031,894, 4,201,211, 4,379,454, 4,436,741, 4,588,580, 4,615,699, 4,661,105, 4,681,584, 4,698,062, 4,725,272, 4,832,953, 4,908,027, 5,004,610, 5,310,559, 5,342,623, 5,344,656, 5,364,630, and 6,375,978, which are incorporated in their entirety herein by reference for all relevant purposes. A particularly suitable membrane material is available from Lohmann Therapie-Systeme.
Referring to
Regardless of the particular configuration employed, the patch 300 also contains a release member 310 that is positioned between the drug delivery assembly 370 and the microneedle assembly 380. While the release member 310 may optionally be bonded to the adjacent support 312 and/or rate control membrane 308, it is typically desired that it is only lightly bonded, if at all, so that the release member 310 may be easily withdrawn from the patch 300. If desired, the release member 310 may also contain a tab portion 371 (
Referring to
In the embodiments shown in
Referring to
In this embodiment, the support 212 and the rate control membrane 208 are initially positioned horizontally adjacent to each other, and a release member 210 extends over the support 212 and the rate control member 208. In this particular embodiment, it is generally desired that the release member 210 is releasably attached to the support 212 and the rate control membrane 208 with an adhesive (e.g., pressure-sensitive adhesive). In its “inactive” configuration as shown in
The device may deliver an agent at a rate so as to be therapeutically useful. In accord with this goal, the transdermal device may include a housing with microelectronics and other micro-machined structures to control the rate of delivery either according to a preprogrammed schedule or through active interface with the patient, a healthcare professional, or a biosensor. The device may include a material having a predetermined degradation rate, so as to control release of an siRNA construct contained within the device. The delivery rate may be controlled by manipulating a variety of factors, including the characteristics of the formulation to be delivered (e.g., viscosity, electric charge, and/or chemical composition); the dimensions of the device (e.g., outer diameter and the volume of any openings); the number of microneedles on a transdermal patch; the number of individual devices in a carrier matrix; the application of a driving force (e.g., a concentration gradient, a voltage gradient, a pressure gradient); the use of a valve; and so forth.
Transportation of agents through the device may be controlled or monitored using, for example, various combinations of valves, pumps, sensors, actuators, and microprocessors. These components may be produced using standard manufacturing or microfabrication techniques. Actuators that may be useful with a device may include micropumps, microvalves, and positioners. For instance, a microprocessor may be programmed to control a pump or valve, thereby controlling the rate of delivery.
Flow of an agent through the device may occur based on diffusion or capillary action, or may be induced using conventional mechanical pumps or nonmechanical driving forces, such as electroosmosis or electrophoresis, or convection. For example, in electroosmosis, electrodes are positioned on a biological surface (e.g., the skin surface), a microneedle, and/or a substrate adjacent a microneedle, to create a convective flow which carries oppositely charged ionic species and/or neutral molecules toward or into the delivery site.
Flow of an agent may be manipulated by selection of the material forming the microneedle surface. For example, one or more large grooves adjacent the microneedle surface of the device may be used to direct the passage of the siRNA construct. Alternatively, the materials forming the nanostructured surface may be manipulated to either promote or inhibit transport of material along the surface, such as by controlling hydrophilicity or hydrophobicity.
The flow of an agent may be regulated using valves or gates as is known in the art. Valves may be repeatedly opened and closed, or they may be single-use valves. For example, a breakable barrier or one-way gate may be installed in the device between a reservoir and the patterned surface. When ready to use, the barrier may be broken or gate opened to permit flow through to the microneedle surface. Other valves or gates used in the device may be activated thermally, electrochemically, mechanically, or magnetically to selectively initiate, modulate, or stop the flow of molecules through the device. In one embodiment, flow is controlled by using a rate-limiting membrane as a “valve.”
In general, any agent delivery control system, including reservoirs, flow control systems, sensing systems, and so forth as are known in the art may be incorporated with devices. By way of example, U.S. Pat. Nos. 7,250,037, 7,315,758, 7,429,258, 7,582,069, and 7,611,481 describe reservoir and control systems as may be incorporated in devices.
During use, the presence of the nanostructured surface of the microneedles within the skin may affect formation and maintenance of cell/cell junctions including tight junctions and desmosomes. As previously mentioned, tight junctions have been found in the stratum granulosum and opening of the tight junctions may provide a paracellular route for improved delivery of siRNA constructs.
The present disclosure may be further understood with reference to the Examples provided below.
Several different molds were prepared using photolithography techniques similar to those employed in the design and manufacture of electrical circuits. Individual process steps are generally known in the art and have been described
Initially, silicon substrates were prepared by cleaning with acetone, methanol, and isopropyl alcohol, and then coated with a 258 nanometer (nm) layer of silicon dioxide according to a chemical vapor deposition process.
A pattern was then formed on each substrate via an electron beam lithography patterning process as is known in the art using a JEOL JBX-9300FS EBL system. The processing conditions were as follows:
Beam current=11 nA
Acceleration voltage=100 kV
Shot pitch=14 nm
Dose=260 μC/cm2
Resist=ZEP520A, ˜330 nm thickness
Developer=n-amyl acetate
Development=2 min. immersion, followed by 30 sec. isopropyl alcohol rinse.
A silicon dioxide etch was then carried out with an STS Advanced Oxide Etch (AOE). Etch time was 50 seconds utilizing 55 standard cubic centimeters per minute (sccm) He, 22 sccm CF4, 20 sccm C4F8 at 4 mTorr, 400 W coil, 200 W RIE and a DC Bias of 404-411 V.
Following, a silicon etch was carried out with an STS silicon oxide etch (SOE). Etch time was 2 minutes utilizing 20 sccm Cl2 and 5 sccm Ar at 5 mTorr, 600 W coil, 50 W RIE and a DC Bias of 96-102 V. The silicon etch depth was 500 nm.
A buffered oxide etchant (BOE) was used for remaining oxide removal that included a three minute BOE immersion followed by a deionized water rinse.
An Obducat NIL-Eitre®6 nanoimprinter was used to form nanopatterns on a variety of polymer substrates. External water was used as coolant. The UV module utilized a single pulsed lamp at a wave length of between 200 and 1000 nm at 1.8 W/cm2. A UV filter of 250-400 nm was used. The exposure area was 6 inches with a maximum temperature of 200° C. and 80 Bar. The nanoimprinter included a semi-automatic separation unit and automatic controlled demolding.
To facilitate the release of the nanoimprinted films from the molds, the molds were treated with Trideca-(1,1,2,2-tetrahydro)-octytrichlorosilane (F13-TCS). To treat a mold, the silicon mold was first cleaned with a wash of acetone, methanol, and isopropyl alcohol and dried with a nitrogen gas. A Petri dish was placed on a hot plate in a nitrogen atmosphere and 1-5 ml of the F13-TCS was added to the Petri dish. A silicon mold was placed in the Petri dish and covered for 10-15 minutes to allow the F13-TCS vapor to wet out the silicon mold prior to removal of the mold.
Five different polymers as given in Table 1, below, were utilized to form various nanotopography designs.
Several different nanotopography patterns were formed, schematic representations of which are illustrated in
The pattern illustrated in
The nanoimprinting process conditions used with polypropylene films are provided below in Table 2.
Films were formed as described above in Example 1 including various different patterns and formed of either polystyrene (PS) or polypropylene (PP). The underlying substrate varied in thickness. Patterns utilized were either DN2, DN3, or DN4 formed utilizing formation processes as described in Example 1. The pattern molds were varied with regard to hole depth and feature spacing to form a variety of differently-sized features having the designated patterns. Sample no. 8 (designated BB1) was formed by use of a 0.6 μm millipore polycarbonate filter as a mold. A 25 μm polypropylene film was laid over the top of the filter and was then heated to melt such that the polypropylene could flow into the pores of the filter. The mold was then cooled and the polycarbonate mold dissolved by use of a methylene chloride solvent.
SEMs of the formed films are illustrated in
1Pattern Features as shown on the figures.
2Cross sectional dimension values were derived from the mold and equated as an approximation of the maximum dimension of the structure, although it should be understood that the actual dimension of any given individual structure may vary slightly as may be seen in the figures.
3Feature heights are provided as the average of several individually determined feature heights.
For each sample AFM was utilized to characterize the film. Characterizations included formation of scanning electron micrograph (SEM), determination of surface roughness, determination of maximum measured feature height, and determination of fractal dimension.
The atomic force microscopy (AFM) probe utilized was a series 16 silicon probe and cantilever available from μMasch. The cantilever had a resonant frequency of 170 kHz, a spring constant of 40 N/m, a length of 230±5 μm, a width of 40±3 μm, and a thickness of 7.0±0.5 μm. The probe tip was an n-type phosphorous-doped silicon probe, with a typical probe tip radius of 10 nm, a full tip cone angle of 40°, a total tip height of 20-25 μm, and a bulk resistivity 0.01-0.05 ohm-cm.
The surface roughness value given in Table 3 is the arithmetical mean height of the surface areal roughness parameter as defined in the ISO 25178 series.
The Fractal Dimension was calculated for the different angles by analyzing the Fourier amplitude spectrum; for different angles the amplitude Fourier profile was extracted and the logarithm of the frequency and amplitude coordinates calculated. The fractal dimension, D, for each direction is then calculated as
D=(6+s)/2,
where s is the (negative) slope of the log-log curves. The reported fractal dimension is the average for all directions.
The fractal dimension may also be evaluated from 2D Fourier spectra by application of the Log Log function. If the surface is fractal the Log Log graph should be highly linear, with at negative slope (see, e.g., Fractal Surfaces, John C. Russ, Springer-Verlag New York, LLC, July, 2008).
HaCaT human skin epithelial cells were grown in DMEM, 10% FBS, 1% penicillin/streptomycin at 37° C., 5% CO2 for 24 hours at a concentration of 25,000 cell/cm2 in 6 well plates. Plates either had polypropylene nanopatterned films formed as described above in Example 1 and designate DN1, DN2 (Sample 4 of Table 3), DN3 or untreated surface at the bottom of the well. Nanopatterned films were adhered in place with cyanoacrylate.
Cells were detached from the surfaces with 1 mL of trypsin per well for 10 minutes, quenched with 1 mL growth medium (same as above), then transferred to a microfuge tube and pelleted at 1200 rpm for 7 minutes.
RNA was isolated from pelleted cells using the RNeasy miniprep kit from Qiagen using the manufacturer's protocol. Briefly, cells were lysed, mixed with ethanol and spun down in a column. Lysates were then washed 3 times, treated with DNase and eluted in 40 μl volumes.
cDNA was created from the RNA isolated using the RT first strand kit from SA Biosciences. Briefly, RNA was treated with DNase again at 42° C. for 5 minutes. Random primers and reverse transcriptase enzyme was then added and incubated at 42° C. for 15 minutes, then incubated at 95° C. for 5 minutes to stop reaction.
qPCR was then performed on the cDNA samples using RT profiler custom PCR array from SA Biosciences with primers for IL1-β, IL6, IL8, IL10, IL1R1, TNFα, TGFβ-1, PDGFA, GAPDH, HDGC, RTC and PPC. Briefly, cDNA was mixed with SYBR green and water, and then added to a PCR plate pre-fixed with the correct sense and antisense primer pair for the gene of interest. The plate was then run on an ABI StepOnePlus PCR machine heated to 95° C. for 10 minutes, then for 45 cycles of: 15 seconds at 95° C. and 1 minute at 60° C.
Delta delta CT analysis was performed using GAPDH as the internal control. HDGC, RTC and PPC levels were used as additional internal controls for activity and genomic DNA contamination.
One-way ANOVA and Tukey's 2-point tests were then used to determine statistical significance in the differences between surfaces.
Table 4, below, presents the protein expressions obtained as the fold change in expression on nanoimprinted structures produced on polypropylene films versus expression on an unstructured film.
HaCaT human skin epithelial cells were grown in DMEM, 10% FBS, 1% penicillin/streptomycin at 37° C., 5% CO2 for 24 hours at a concentration of 25,000 cell/cm2 in 6 well plates. Plates either had a polypropylene film formed as described above in Example 1 with designation DN1, DN2 (Sample 4 of Table 3), DN3 or an untreated surface at the bottom of the well. Films were adhered in place with cyanoacrylate.
Media was collected from each well and analyzed for cytokine production with a Milliplex Map Kit from Millipore. Beads to detect IL1-β, IL1RA, IL6, IL8, IL10, PDGF-AA, PGGF-AB/BB and TNF-α were used. Readings were done on a BioRad BioPlex machine. Briefly, media was placed into microplate wells with filters. Primary beads were added and incubated at room temperature for 1 hour with shaking. The plates were then washed and incubated with detection antibodies for 30 minutes at room temperature with shaking. Strepavidin-phycoertythrin was then added and incubated at room temperature for an additional 30 minutes. Plates were then washed, beads were resuspended in assay buffer and median fluorescent intensity was analyzed on the BioPlex.
The permeability effects of films patterned as described herein were determined on a monolayer of Caco-2 cells (human epithelial colorectal adenocarcinoma cells).
Films formed as described above in Example 1 were utilized including films formed with patterns designated as DN2, DN3, and DN4. A fourth film, designated as BB1 (described in Example 2, above) was also used. The protocol was run with multiple examples of each film type.
The general protocol followed for each film was as follows:
Materials
Cell culture inserts 0.4 μm pore size HDPET membrane (BD Falcon)
24 well plate (BD Falcon)
Caco-2 media
Nanostructured membranes as described above
IgG-FITC (Sigma Aldrich)
BSA-FITC (Sigma Aldrich)
Minimum Essential Medium no phenol red (Invitrogen)
TEER voltmeter
Warmed PBS
Black 96-well plate
Aluminum foil
Protocol
Films utilized and results obtained are summarized in Table 5, below.
Moduli were determined according to standard methods as are known in the art as described by Schubert, et al. (Sliding induced adhesion of stiff polymer microfiber arrays: 2. Microscale behaviour, Journal Royal Society, Interface, Jan. 22, 2008. 10.1098/rsif.2007.1309)
The contact angles were measured by placing a drop of water on the surface according to standard practice. (See, e.g., Woodward, First Ten Angstroms, Portsmouth, Va.).
The BSA signal was read on a fluorometer and the IgG signal was read on a spectrophotometer.
An array of microneedles including a nanopatterned surface was formed. Initially, an array of microneedles as illustrated in
Microneedles were formed according to a typical micromachining process on a silicon based wafer. The wafers were layered with resist and/or oxide layers followed by selective etching (oxide etching, DRIE etching, iso etching), resist stripping, oxide stripping, and lithography techniques (e.g., iso lithography, hole lithography, slit lithography) according to standard methods to form the array of microneedles.
Following formation of the microneedle array, a 5 μm polypropylene film including a DN2 pattern formed thereon as described above in Example 1, the characteristics of which are described at sample 2 in Table 3, was laid over the microneedle array. The wafer/film structure was held on a heated vacuum box (3 in. H2O vacuum) at elevated temperature (130° C.) for a period of one hour to gently pull the film over the surface of the microneedles while maintaining the nanopatterned surface of the film.
Methods as described in Example 5 were utilized to determine the permeability effects of films patterned as described herein on a monolayer of Caco-2 cells when considering permeability of a cell layer to siRNA.
siRNA utilized was the BLOCK-iT™ Fluorescent Oligo available from Invitrogen. The siRNA is a fluorescein-labeled dsRNA oligomer. The protocol was the same as described in Example 5. The structured film used included a DN2 pattern on a polypropylene film (sample 4 of Table 3) as well as an unpatterned film (PPUI) and a layer of cells with no film (cells). Results of permeability over time are shown in
While the subject matter has been described in detail with respect to the specific embodiments thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing, may readily conceive of alterations to, variations of, and equivalents to these embodiments. Accordingly, the scope of the present disclosure should be assessed as that of the appended claims and any equivalents thereto.
This application is a continuation of U.S. application Ser. No. 15/378,109 having a filing date of Dec. 14, 2016, which is a continuation of U.S. application Ser. No. 13/641,502 having a filing date of Feb. 22, 2013, which claims priority to International Patent Application No. PCT/IB2011/051862 having a filing date of Apr. 27, 2011, U.S. Provisional Patent Application Ser. No. 61/328,723 having a filing date of Apr. 28, 2010, and to U.S. Provisional Patent Application Ser. No. 61/416,057 having a filing date of Nov. 22, 2010, all of which are incorporated herein in their entirety by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 3797494 | Zaffaroni | Mar 1974 | A |
| 3964482 | Gerstel et al. | Jun 1976 | A |
| 4031894 | Urquhart et al. | Jun 1977 | A |
| 4051840 | Kantrowitz et al. | Oct 1977 | A |
| 4201211 | Chandrasekaran et al. | May 1980 | A |
| 4379454 | Campbell et al. | Apr 1983 | A |
| 4436741 | Urquhart et al. | Mar 1984 | A |
| 4588580 | Gale et al. | May 1986 | A |
| 4615699 | Gale et al. | Oct 1986 | A |
| 4661105 | Gale | Apr 1987 | A |
| 4681584 | Gale et al. | Jul 1987 | A |
| 4698062 | Gale et al. | Oct 1987 | A |
| 4725272 | Gale | Feb 1988 | A |
| 4832953 | Campbell et al. | May 1989 | A |
| 4880633 | Loper et al. | Nov 1989 | A |
| 4908027 | Enscore et al. | Mar 1990 | A |
| 5004610 | Osborne et al. | Apr 1991 | A |
| 5310559 | Shah et al. | May 1994 | A |
| 5328470 | Nabel | Jul 1994 | A |
| 5342623 | Enscore et al. | Aug 1994 | A |
| 5344656 | Enscore et al. | Sep 1994 | A |
| 5364630 | Osborne et al. | Nov 1994 | A |
| 6132755 | Eicher et al. | Oct 2000 | A |
| 6312612 | Sherman et al. | Nov 2001 | B1 |
| 6313612 | Honda et al. | Nov 2001 | B1 |
| 6334856 | Allen et al. | Jan 2002 | B1 |
| 6375978 | Kleiner et al. | Apr 2002 | B1 |
| 6471993 | Shastri et al. | Oct 2002 | B1 |
| 6569143 | Alchas et al. | May 2003 | B2 |
| 6591124 | Sherman et al. | Jul 2003 | B2 |
| 6611707 | Prausnitz et al. | Aug 2003 | B1 |
| 6656147 | Gertsek et al. | Dec 2003 | B1 |
| 6663820 | Arias et al. | Dec 2003 | B2 |
| 6767341 | Cho | Jul 2004 | B2 |
| 6881203 | Delmore et al. | Apr 2005 | B2 |
| 6908453 | Fleming et al. | Jun 2005 | B2 |
| 6926953 | Nealey et al. | Aug 2005 | B2 |
| 6979347 | Wu et al. | Dec 2005 | B1 |
| 6980855 | Cho | Dec 2005 | B2 |
| 6995336 | Hunt et al. | Feb 2006 | B2 |
| 7048723 | Frazier et al. | May 2006 | B1 |
| 7066922 | Angel et al. | Jun 2006 | B2 |
| 7108681 | Gartstein et al. | Sep 2006 | B2 |
| 7115108 | Wilkinson et al. | Oct 2006 | B2 |
| 7129554 | Lieber et al. | Oct 2006 | B2 |
| 7131987 | Sherman et al. | Nov 2006 | B2 |
| 7185663 | Koch et al. | Mar 2007 | B2 |
| 7189435 | Tuominen et al. | Mar 2007 | B2 |
| 7226439 | Prausnitz et al. | Jun 2007 | B2 |
| 7250037 | Shermer et al. | Jul 2007 | B2 |
| 7285113 | Yeshurun | Oct 2007 | B2 |
| 7315758 | Kwiatkowski et al. | Jan 2008 | B2 |
| 7332339 | Canham | Feb 2008 | B2 |
| 7364568 | Angel et al. | Apr 2008 | B2 |
| 7374864 | Guo | May 2008 | B2 |
| 7410476 | Wilkinson et al. | Aug 2008 | B2 |
| 7416541 | Yuzhakov et al. | Aug 2008 | B2 |
| 7429258 | Angel et al. | Sep 2008 | B2 |
| 7449200 | Sung et al. | Nov 2008 | B2 |
| 7473244 | Frazier et al. | Jan 2009 | B2 |
| 7531120 | Van Rijn et al. | May 2009 | B2 |
| 7537590 | Santini, Jr. et al. | May 2009 | B2 |
| 7544770 | Haynie | Jun 2009 | B2 |
| 7556615 | Pettis et al. | Jul 2009 | B2 |
| 7563451 | Lin et al. | Jul 2009 | B2 |
| 7572405 | Sherman et al. | Aug 2009 | B2 |
| 7578954 | Gartstein | Aug 2009 | B2 |
| 7582069 | Laurent et al. | Sep 2009 | B2 |
| 7588552 | Yeshurun | Sep 2009 | B2 |
| 7611481 | Cleary et al. | Nov 2009 | B2 |
| 7627938 | Kim et al. | Dec 2009 | B2 |
| 7651475 | Angel et al. | Jan 2010 | B2 |
| 7658728 | Yuzhakov | Feb 2010 | B2 |
| 7753888 | Mukerjee et al. | Jul 2010 | B2 |
| 7785301 | Yuzhakov | Aug 2010 | B2 |
| 7803574 | Desai et al. | Sep 2010 | B2 |
| 7828827 | Gartstein et al. | Nov 2010 | B2 |
| 7846488 | Johnson | Dec 2010 | B2 |
| 7901387 | Stemme et al. | Mar 2011 | B2 |
| 7914480 | Cleary et al. | Mar 2011 | B2 |
| 7914813 | Adachi et al. | Mar 2011 | B2 |
| 7918814 | Prausnitz et al. | Apr 2011 | B2 |
| 7972616 | Dubrow et al. | Jul 2011 | B2 |
| 7981346 | Griss et al. | Jul 2011 | B2 |
| 7997274 | Baska | Aug 2011 | B2 |
| 8052633 | Kendall | Nov 2011 | B2 |
| 8057842 | Choi et al. | Nov 2011 | B2 |
| 8088321 | Ferguson et al. | Jan 2012 | B2 |
| 8097456 | Borenstein et al. | Jan 2012 | B2 |
| 8118753 | Cho et al. | Feb 2012 | B2 |
| 8137697 | Sung | Mar 2012 | B1 |
| 8137736 | Zhu et al. | Mar 2012 | B2 |
| 8162901 | Gonnelli et al. | Apr 2012 | B2 |
| 8238995 | Chandrasekaran et al. | Aug 2012 | B2 |
| 8366677 | Kaspar et al. | Feb 2013 | B2 |
| 8389205 | Duerig et al. | Mar 2013 | B2 |
| 8419708 | Tokumoto et al. | Apr 2013 | B2 |
| 8506530 | Laermer et al. | Aug 2013 | B2 |
| 8574615 | Tenney et al. | Nov 2013 | B2 |
| 8690838 | Ozawa et al. | Apr 2014 | B2 |
| 8696637 | Ross | Apr 2014 | B2 |
| 8915957 | Arney et al. | Dec 2014 | B2 |
| 8944804 | Robeson et al. | Feb 2015 | B2 |
| 9028409 | Yodfat et al. | May 2015 | B2 |
| 20020082543 | Park et al. | Jun 2002 | A1 |
| 20020133129 | Arias et al. | Sep 2002 | A1 |
| 20020183688 | Lastovich et al. | Dec 2002 | A1 |
| 20040028875 | Van Rijn et al. | Feb 2004 | A1 |
| 20040063100 | Wang | Apr 2004 | A1 |
| 20040087992 | Gartstein et al. | May 2004 | A1 |
| 20040106904 | Gonnelli et al. | Jun 2004 | A1 |
| 20050049625 | Shaya et al. | Mar 2005 | A1 |
| 20050112135 | Cormier et al. | May 2005 | A1 |
| 20050118388 | Kingsford | Jun 2005 | A1 |
| 20050119723 | Peacock | Jun 2005 | A1 |
| 20050124967 | Kaestner et al. | Jun 2005 | A1 |
| 20050137531 | Prausnitz et al. | Jun 2005 | A1 |
| 20050143713 | Delmore et al. | Jun 2005 | A1 |
| 20050178760 | Chang et al. | Aug 2005 | A1 |
| 20050187521 | Fleming et al. | Aug 2005 | A1 |
| 20050203613 | Arney et al. | Sep 2005 | A1 |
| 20060024358 | Santini, Jr. et al. | Feb 2006 | A1 |
| 20060025848 | Weber et al. | Feb 2006 | A1 |
| 20060030538 | Hendriks | Feb 2006 | A1 |
| 20060051404 | Yeshurun et al. | Mar 2006 | A1 |
| 20060204738 | Dubrow et al. | Sep 2006 | A1 |
| 20060264893 | Sage et al. | Nov 2006 | A1 |
| 20070066934 | Etheredge, III et al. | Mar 2007 | A1 |
| 20070078376 | Smith | Apr 2007 | A1 |
| 20070081977 | Horstmann | Apr 2007 | A1 |
| 20070088248 | Glenn et al. | Apr 2007 | A1 |
| 20070110810 | Smith et al. | May 2007 | A1 |
| 20070112309 | Zucker | May 2007 | A1 |
| 20070112548 | Dickerson et al. | May 2007 | A1 |
| 20070191761 | Boone et al. | Aug 2007 | A1 |
| 20070249552 | Khalili et al. | Oct 2007 | A1 |
| 20070276318 | Henley | Nov 2007 | A1 |
| 20070282247 | Desai et al. | Dec 2007 | A1 |
| 20080026464 | Borenstein et al. | Jan 2008 | A1 |
| 20080088066 | Ferguson et al. | Apr 2008 | A1 |
| 20080091226 | Yeshurun et al. | Apr 2008 | A1 |
| 20080097352 | Beck et al. | Apr 2008 | A1 |
| 20080108958 | Carter et al. | May 2008 | A1 |
| 20080195035 | Frederickson et al. | Aug 2008 | A1 |
| 20080200883 | Tomono | Aug 2008 | A1 |
| 20080208076 | Cho et al. | Aug 2008 | A1 |
| 20080217180 | Doye et al. | Sep 2008 | A1 |
| 20080221408 | Hoarau et al. | Sep 2008 | A1 |
| 20080262416 | Duan et al. | Oct 2008 | A1 |
| 20080269666 | Wang et al. | Oct 2008 | A1 |
| 20080269685 | Singh et al. | Oct 2008 | A1 |
| 20080305989 | Wen | Dec 2008 | A1 |
| 20080311172 | Schapira et al. | Dec 2008 | A1 |
| 20080312610 | Binks | Dec 2008 | A1 |
| 20090012494 | Yeshurun et al. | Jan 2009 | A1 |
| 20090043279 | Kaspar et al. | Feb 2009 | A1 |
| 20090069788 | Yeshurun et al. | Mar 2009 | A1 |
| 20090093776 | Yue et al. | Apr 2009 | A1 |
| 20090093871 | Rea et al. | Apr 2009 | A1 |
| 20090093879 | Wawro et al. | Apr 2009 | A1 |
| 20090099427 | Jina et al. | Apr 2009 | A1 |
| 20090099502 | Tokumoto et al. | Apr 2009 | A1 |
| 20090099537 | DeVoe et al. | Apr 2009 | A1 |
| 20090118662 | Schnall | May 2009 | A1 |
| 20090118672 | Gonnelli et al. | May 2009 | A1 |
| 20090137926 | Srinivasan et al. | May 2009 | A1 |
| 20090177273 | Piveteau et al. | Jul 2009 | A1 |
| 20090187167 | Sexton et al. | Jul 2009 | A1 |
| 20090198189 | Simons et al. | Aug 2009 | A1 |
| 20090232870 | Srivastava et al. | Sep 2009 | A1 |
| 20090234301 | Tomono | Sep 2009 | A1 |
| 20100004733 | Atanasoska et al. | Jan 2010 | A1 |
| 20100021464 | Archambeau et al. | Jan 2010 | A1 |
| 20100028604 | Bhushan et al. | Feb 2010 | A1 |
| 20100076035 | Carter et al. | Mar 2010 | A1 |
| 20100121307 | Lockard et al. | May 2010 | A1 |
| 20100168506 | Moon et al. | Jul 2010 | A1 |
| 20100215580 | Hanes et al. | Aug 2010 | A1 |
| 20100256568 | Frederickson et al. | Oct 2010 | A1 |
| 20100274203 | Lee et al. | Oct 2010 | A1 |
| 20110021996 | Lee et al. | Jan 2011 | A1 |
| 20110046557 | Lee et al. | Feb 2011 | A1 |
| 20110059150 | Kendall et al. | Mar 2011 | A1 |
| 20110144591 | Ross et al. | Jun 2011 | A1 |
| 20110160069 | Corrie et al. | Jun 2011 | A1 |
| 20110270221 | Ross | Nov 2011 | A1 |
| 20110276003 | Luttge et al. | Nov 2011 | A1 |
| 20120089117 | Junginger et al. | Apr 2012 | A1 |
| 20120109065 | Backes | May 2012 | A1 |
| 20120114734 | Desai et al. | May 2012 | A1 |
| 20120128932 | Veith et al. | May 2012 | A1 |
| 20130144217 | Ross | Jun 2013 | A1 |
| 20130144257 | Ross | Jun 2013 | A1 |
| 20130150822 | Ross | Jun 2013 | A1 |
| 20130165861 | Ross | Jun 2013 | A1 |
| 20130211310 | Bommarito | Aug 2013 | A1 |
| 20130331792 | Karp et al. | Dec 2013 | A1 |
| 20140112921 | Ross | Apr 2014 | A1 |
| 20150329362 | Aria et al. | Nov 2015 | A1 |
| 20170157381 | Ross | Jun 2017 | A1 |
| Number | Date | Country |
|---|---|---|
| 2100850 | Sep 2009 | EP |
| WO 9945860 | Sep 1999 | WO |
| WO 2000074764 | Dec 2000 | WO |
| WO 0175164 | Oct 2001 | WO |
| WO 0230506 | Apr 2002 | WO |
| WO 0232480 | Apr 2002 | WO |
| WO 03020359 | Mar 2003 | WO |
| WO 03024508 | Mar 2003 | WO |
| WO 03092785 | Nov 2003 | WO |
| WO 2005049128 | Jun 2005 | WO |
| WO 2006062974 | Jun 2006 | WO |
| WO 2007012114 | Feb 2007 | WO |
| WO 2007070004 | Jun 2007 | WO |
| WO 2007081876 | Jul 2007 | WO |
| WO 2008081876 | Jul 2007 | WO |
| WO 2007112309 | Oct 2007 | WO |
| WO 2008003564 | Jan 2008 | WO |
| WO 2008024141 | Feb 2008 | WO |
| WO 2009079589 | Jun 2009 | WO |
| WO 2009113856 | Sep 2009 | WO |
| WO 2010070628 | Jun 2010 | WO |
| WO 2010087971 | Aug 2010 | WO |
| WO 2010126640 | Nov 2010 | WO |
| WO 2011116388 | Sep 2011 | WO |
| WO 2011135531 | Nov 2011 | WO |
| WO 2012006677 | Jan 2012 | WO |
| WO 2012046149 | Apr 2012 | WO |
| WO 2013061208 | May 2013 | WO |
| WO 2013061209 | May 2013 | WO |
| Entry |
|---|
| Abstract of Japanese Patent—JP2001238964, Sep. 4, 2001, 1 page. |
| Abstract of Japanese Patent—JP2008511382, Apr. 17, 2008, 2 pages. |
| Abstract of Japanese Patent—JP2008237673, Oct. 9, 2008, 1 page. |
| Abstract of Japanese Patent—JP2009207733, Sep. 17, 2009, 1 page. |
| Abstract of Japanese Patent—JPH08337521, Dec. 24, 1996, 2 pages. |
| Abstract of WIPO Patent—WO 2006/075689, Jul. 20, 2006, 1 page. |
| Ahmed et al; “Bis(diacetylene)s I: Langmuir-Blodgett films”, Thin Solid Films 187; 141-153; 1990. |
| Ainslie et al., “Microfabricated implants for applications in therapeutic delivery, tissue engineering, and biosensing,” Royal Society of Chemistry, 8. (2008): 1864-1878. |
| Ainslie et al.. “Microfabricated Devices for Enhanced Bioadhesive Drug Delivery: Attachment to and Small-Molecule Release Through a Cell Monolayer Under Flow” Small, (2009). |
| Al-Qallaf et al., “Optimizing Microneedle Arrays to Increase Skin Permeability for Transdermal Drug Delivery”, Interdisciplinary Transport Phenomena V: Ann. N.Y. Acad. Sci., 2009, pp. 1-12. |
| Bekarde, lil Gercek, “Biomimetic Apatite-coated PCL Scaffolds: Effect of Surface Nanotopography on Cellular Functions.” Journal of Bioactive and Compatible Polymers. 24.6 (2009): 507-524. |
| Berry et al., “The interaction of human bone marrow cells with nanotopographical features in three dimensional constructs.” Journal of Biomedical Materials Research Part A. 79A.2 :2006): 431-439. |
| Biehl et al., “Proliferation of Mouse Embryonic Stem Cell Progeny and the Spontaneous Contractile Activity of Cardiomyocytes Are Affected by Microtopography.” Developmental Dynamics . 238. (2009): 1964-1973. |
| Biggs et al., “Interactions with Nanoscale Topography: Adhesion quantification and signal transduction in cells of osteogenic and multipotent lineage,” Journal of Biomedical Materials Research Part A, 2008, pp. 195-208. |
| Brunauer et al., “Adsorption of Gases in Multimolecular Layers.” Journal of the American Chemical Society. 60. (1932): 309-319. |
| Chandler, David L.. “PhysOrg.com.” Harnessing nanopatterns: Tiny textures can produce big differences, N.p., Sep. 24, 2009. Web. Dec. 1, 2009. <http://www.physorg.com/news173004362.html>. |
| Chen et al., “Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo”, 1994, Proc. Natl. Sci USA, vol. 91: 3054-3057. |
| Choi et al. “Cell interaction with three-dimensional sharp-tip nanotopography.” Biomaterials. 28.9 (2007): 1672-1679. |
| Chun et al., “The role of polymer nanosurcace roughness and the submicron pores in improving bladder urothelial cell density and inhibiting calcium oxalate stone formation.” Nanotechnology. 20.8 (2009): 85104. |
| Citi, S., “Protein Kinase Inhibitors Prevent Junction Dissociation Induced by Low Extracellular Calcium in MDCK Epithelial Cells”, J Cell Biology 1992 117(1) 169-178. |
| Cohn, Abby. “Ding Delivery, Nanoscale.” Innovations. 3.4 (2009). |
| Curtis et al., “Cell signaling arising from nanotopography: implications for nanomedicaldevices.” Nanomedicine. 1.1 (2006): 67-72. |
| Dalby, Matthew J. “Nanostructured surfaces: cell engineering and cell biology.” Nanomedicine. 4.3 (2009): 247-248. |
| Dalby et al., “Attempted endocytosis of nano-environment produced by colloidal lithography by human fibrobhists ” Experimental Cell Research. 295. (2004); 387-394. |
| Dalby et al., “Nano-Topography Induces Mechanotransduction in Human Fibroblasts,” European Cells and Materials . 6.2 (2003): 31. |
| Dalby et al., “Increasing Fibroblast Response to Materials Using Nanotopography: Morphological and Genetic measurements or Cell Response to 13-nm-High Polymer Demixed Islands.” Experimental Cell Research. 276.1 (2002): 1-9. |
| Elbashir et al., “RNA interference is mediated by 21- and 22-nucleotide RNAs”, Genes & Development, 2000, 15:188-200. |
| Fischer et al., “Biomimetic Nanowire Coatings for Next Generation Adhesive Drug Delivery Systems.” Nano Letters. 9.2 (2009): 716-720. |
| Geiger & Ayalon, “Cadherins”, Annu Rev Cell Biol, 1992, 8:307-32. |
| Gumbiner & Simons, “A Functional Assay for Proteins Involved in Establishing an Epithelial Occluding Barrier: Identification at an Uvomorulin-like Polypetide”, J. Cell Biology, 1986, 102 457-468. |
| Hart et al., “Filapodial Sensing of Nanotopography in Osteoprogenitor Cells,” European Cells and Materials . 10.2 (2005): 65. |
| He, et al., “The anatase phase of nanotopography titania plays an important role on osteoblast cell morphology and proliferation”, Journal of Mater. Sci. Mater Med (2008), 19;3465-3472. |
| Hirano et al.; “Calcium-dependent Cell-Cell Adhesion Molecules (Cadherins): Subclass Specificities and Possible Involvement of Actin Bundles”, J. Cell Biology, 1987, 105(6): 2501-2510. |
| Hu et al., “Surface Energy Induced Patterning of Polymer Nanostructures for Cancer Diagnosis and Therapy.” IEEE Nano 2007 Conference Paper. (2007). |
| Kaushal et al, Influence of Piperline on Transcutaneous Permeation of Repaglinide in Rats and on Tight Junction Proteins in HaCaT Cells: Unveiling the Mechanisms for Enhances Permeation; Sci. Pharm. 2009; 77; 877-897. |
| Kitner, C., “Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain”, Cell, 1992, 69(2):225-36. |
| Korhonen et al., “Nitric oxide production and signaling in inflammation”, Curr Drug Targets Inflamm Allergy 4(4) 471-9, 2005. |
| Kumar et al. “Transdermal Drug Delivery System: An Overview.” International Journal of Pharmaceutical Sciences Review and Research. 3.2 (2010); 49-54. |
| Langeler et al., “Norepinephrine and iloprost improve barrier function of human endothelial cellmonolayers: role of cAMP”, Am J Physio Cell Physiol, 1991, vol. 260(5), C1052-C1059. |
| Lim et al., “Human foetal osteoblastic cell response to polymer-demixed nanotopographic interfaces.” Journal of the Royal Society Interface. 2.2 (2005): 97-108. |
| Madara, JL, “Regulation of the movement of solutes across tight junctions”; Annu Rev Physiol, 1998, 60:143-59. |
| Mahdavi et al., “A biodegradable and biocompatible gecko-inspired tissue adhesive.” PNAS. 105.7 (2008): 2307-2312. |
| Martinez-Palomo et al., “Experimental Modulation of Occluding Junctions in a Cultured Transporting Epithelium”, J. Cell Biology, 1980, 87: 736-746. |
| Meirelles et al., “The effect of chemical and nanotopographical modifications on the early stages of osseointegration” International Journal of Oral and Maxillofacial Implants. 23.4 (2008): 641-647. |
| Mendelsohn et al. “Inorganic Nanoporous Membranes for Immunoisolated Cell-Based Drug Delivery.” Therapeutic Applications of Cell Microencapsulation, 2010. |
| Nagafuchi & Takechi, “Cell binding function of E-cadherin is regulated by the cytoplasmic domain”, EMBO Journal, 1988, 7(12): 3679-3684. |
| Ng et al., “Study of substrate topographical effects on epithelial cell behavior using etched alpha-particle tracks on PADC films.” Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms. 266.14 (2008): 3247-3256. |
| Ojakian, GK, “Tumor promotor-induced changes in the permeability of epithelial cell tight junctions”, Cell, 1981, 23(1): 95-403. |
| Orr et al., “Submicrometer and Nanoscale Inorganic Particles Exploit the Actin Machinery to Be Propelled along Microvilli-likestructures into Alveolar Cells.” American Chemical Society NANO. 1.5 (2007): 463-475. |
| Ozawa et al., “The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independnet proteins structurally related in different species” EMBO Journal, 1989, 8(6): 1711-1717. |
| Ozawa et al., “Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule”, Dev Biology, 1990, 87: 4246-4250. |
| Park et al., “Towards the silicon nanowire-based sensor for intracellular biochemical detection.” Biosensors and Bioelectronics, vol. 22, No. 9-10, Apr. 1, 2007, 6 pages. |
| Peng et al.. “Long-Term Small Molecule and Protein Elution from TiO2 Nanotubes.” Nano Letters 9.5 (2009): 1932-1936. |
| Peng et al., “The effect of TiO2 nanotubes on endothelial function and smooth muscle proliferation.” Journal of Biomaterials. 30. (2009): 1268-1272. |
| Rubin et al. “A Cell Culture Model of the Blood-Brain Barrier”, J. Cell Biology, 1991, 115:1725-1735. |
| Robin, LL, “Endothelial cells: adhesion and tight junctions”, Curr Opin Cell Biol, 1992, 4(5):830-3. |
| Rutten et al., “Electrical resistance and macro-molecular permeability of brain endothelial monolayer cultures”, Brain Res, 1987, 425(2):301-10. |
| Sapra et al., “Transdermal Delivery of Carvedilol Containing Glycyrrhizin and Chitosan as Permeation Enhancers:Biochemical, Biophysical, Microscopic and Pharmacodynamic Evaluation.” Drug Delivery . 15.7 (2008); 443-454. |
| Sapra et al.. “Transdermal delivery of carvedilol in rats: probing the percutaneous permeation enhancement mechanism of soybean extract-chitosan mixture.” Drug Delivery . 35.10 (2009): 1230-1241. |
| Sapra et al., “Effect of Asparagus racemous Extract on Transdermal Delivery of Carvedilol: A Mechanistic Study.” American Association of Pharmaceutical Scientists PharmSciTech. 10.1 (2009): 199. |
| Schubert et al, “Sliding-induced adhesion of stiff polymer microfibre arrays. II. Microscale behavior”, J Royal Society; Jan. 2008, 5, 845-853. |
| Stappert et al., “A short core region of E-cadherin is essential for certain binding and is highly phosphorylated”, 1993, Cell Adhes Commun. 2(4):319-27. |
| Stelzner et al., “Role of cyclic adenosine monophosphate in the induction of endothelial barrier properties”, J. Cell Physiol, 1989. 139(1):157-66. |
| Teo et al. “The effect of micro and nanotopography on endocytosis in drug and gene delivery systems”, Biomaterials, 32 (2011), 9866-9875. |
| Thakar et al., “Contractility-Dependent Modulation or Cell Proliferation and Adhesion by Microscale : Topographical Cues.” Small. 4.9 (2008): 1416-1424. |
| Valenta et al., “The use of polymers for dermal and transdermal delivery.” European Journal or Pharmaceutics and Biopharmaceutics. 58.2 (2004): 279-289. |
| Wang et al., “Nano patterned PDMS for periodontal ligament fibroblast culture.” Surface and Coatings Technology. 204.4 (2009): 525-530. |
| Wei et al., “Protein adsorption on materials surfaces with nano-topography.” Chinese Science Bulletin. 52.23 (2007): 3169-3173. |
| Wood, M.A. “Colloidal lithography and current fabrication techniques producing in-plane nanotopography for biological applications,” Journal of the Royal Society Interface. 4.12 (2007): 1-17. |
| Yao et al., “Nano-Surface Modification on Titanium Implants the Drug Delivery.” Materials Research Society . (2007). |
| Yim et al., “Nanopattern-induced changes in morphology and motility of smooth muscle cells.” Journal of Biomaterials. 58.1 (2005). |
| Supplementary European Search Report dated Nov. 25, 2013, 12 pages. |
| Number | Date | Country | |
|---|---|---|---|
| 20200345993 A1 | Nov 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61416057 | Nov 2010 | US | |
| 61328723 | Apr 2010 | US |
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| Parent | 15378109 | Dec 2016 | US |
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