A review of genetic engineering biotechnologies for enhanced chronic wound healing.

Traditional methods for addressing chronic wounds focus on correcting dysfunction by controlling extracellular elements. This review highlights technologies that take a different approach - enhancing chronic wound healing by genetic modification to wound beds. Featured cutaneous transduction/transfection methods include viral modalities (ie adenoviruses, adeno-associated viruses, retroviruses and lentiviruses) and conventional non-viral modalities (ie naked DNA injections, microseeding, liposomal reagents, particle bombardment and electroporation). Also explored are emerging technologies, focusing on the exciting capabilities of wound diagnostics such as pyrosequencing as well as site-specific nuclease editing tools such as CRISPR-Cas9 used to both transiently and permanently genetically modify resident wound bed cells. Additionally, new non-viral transfection methods (ie conjugated nanoparticles, multi-electrode arrays, and microfabricated needles and nanowires) are discussed that can potentially facilitate more efficient and safe transgene delivery to skin but also represent significant advances broadly to tissue regeneration research.

Transgene delivery via intracellular electroporetic nanoinjection.

Development of an effective cytoplasmic delivery technique has remained an elusive goal for decades despite the success of pronuclear microinjection. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In this work we report a cytoplasmic delivery method termed intracellular electroporetic nanoinjection (IEN). IEN is unique in that it manipulates transgenes using electrical forces. The microelectromechanical system (MEMS) uses electrostatic charge to physically pick up transgenes and place them in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronuclei using electrical pulses. Standard electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm for transgene delivery from the cytoplasm to the pronucleus. In this report we describe the principles which allow localized electroporation of the pronuclei including: the location of mouse pronuclei between 21 and 28 h post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on electric-field-driven movement of transgenes. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.

Carbon-infiltrated carbon nanotubes inhibit the development of Staphylococcus aureus biofilms.

Staphylococcus aureus forms biofilms that cause considerable morbidity and mortality in patients who receive implanted devices such as prosthetics or fixator pins. An ideal surface for such medical devices would inhibit biofilm growth. Recently, it was reported that surface modification of stainless steel materials with carbon-infiltrated carbon nanotubes (CICNT) inhibits the growth of S. aureus biofilms. The purpose of this study was to investigate this antimicrobial effect on titanium materials with CICNT coated surfaces in a variety of surface morphologies and across a broader spectrum of S. aureus isolates. Study samples of CICNT-coated titanium, and control samples of bare titanium, a common implant material, were exposed to S. aureus. Viable bacteria were removed from adhered biofilms and quantified as colony forming units. Scanning electron microscopy was used to qualitatively analyze biofilms both before and after removal of cells. The CICNT surface was found to have significantly fewer adherent bacteria than bare titanium control surfaces, both via colony forming unit and microscopic analyses. This effect was most pronounced on CICNT surfaces with an average nanotube diameter of 150 nm, showing a 2.5-fold reduction in adherent bacteria. Since S. aureus forms different biofilm structures by isolate and by growth conditions, we tested 7 total isolates and found a significant reduction in the biofilm load in six out of seven S. aureus isolates tested. To examine whether the anti-biofilm effect was due to the structure of the nanotubes, we generated an unstructured carbon surface. Significantly more bacteria adhered to a nonstructured carbon surface than to the 150 nm CICNT surface, suggesting that the topography of the nanotube structure itself has anti-biofilm properties. The CICNT surface possesses anti-biofilm properties that result in fewer adherent S. aureus bacteria. These anti-biofilm properties are consistent across multiple isolates of S. aureus and are affected by nanotube diameter. The experiments performed in this study suggest that this effect is due to the nanostructure of the CICNT surface.

Nanoinjection: pronuclear DNA delivery using a charged lance.

We present a non-fluidic pronuclear injection method using a silicon microchip "nanoinjector" composed of a microelectromechanical system with a solid, electrically conductive lance. Unlike microinjection which uses fluid delivery of DNA, nanoinjection electrically accumulates DNA on the lance, the DNA-coated lance is inserted into the pronucleus, and DNA is electrically released. We compared nanoinjection and microinjection side-by-side over the course of 4 days, injecting 1,013 eggs between the two groups. Nanoinjected zygotes had significantly higher rates of integration per injected embryo, with 6.2% integration for nanoinjected embryos compared to 1.6% integration for microinjected embryos. This advantage is explained by nanoinjected zygotes' significantly higher viability in two stages of development: zygote progress to two-cell stage, and progress from two-cell stage embryos to birth. We observed that 77.6% of nanoinjected zygotes proceeded to two-cell stage compared to 54.7% of microinjected zygotes. Of the healthy two-cell stage embryos, 52.4% from the nanoinjection group and 23.9% from the microinjected group developed into pups. Structural advantages of the nanoinjector are likely to contribute to the high viability observed. For instance, because charge is used to retain and release DNA, extracellular fluid is not injected into the pronucleus and the cross-sectional area of the nanoinjection lance (0.06 µm(2)) is smaller than that of a microinjection pipette tip (0.78 µm(2)). According to results from the comparative nanoinjection versus microinjection study, we conclude that nanoinjection is a viable method of pronuclear DNA transfer which presents viability advantages over microinjection.

Curvature-induced defects on carbon-infiltrated carbon nanotube forests.

A morphological study of the micro-scale defects induced by growing a carbon-infiltrated carbon nanotube (CICNT) forest on concave substrates was conducted. Two CICNT heights (roughly 60 µm and 400 µm) and 4 curvatures (1-4 mm ID) were studied in order to test the geometric limitations. Defects were categorized and quantified by scanning electron microscopy (SEM) of the tops and cross-sections. These deformities were categorized as increased roughness on the top surface, a corrugated (also called wavy or rippled) forest, a curved forest, an inside crevice where the forest separates, and increased forest density on the top surface. Roughness increased nearly 3-fold with the taller forest heights no matter the substrate curvature. Due to the geometric limitations of CICNT height and substrate curvature, all other microscale defects were significantly more present on samples with a small radius of curvature and a tall CICNT forest (p < 0.05). These buckling and warping types of defects were attributed to the increase in circumferential compression as the forest grows as well as the van der Waals interactions between the nanotubes. Because the fabrication process for CICNT involves growing a CNT forest and then infiltrating it with pyrolytic carbon, this work may be applicable to other CNT forests on concave substrates within these forest heights and substrate curvatures.

Structural biofilm resistance of carbon-infiltrated carbon nanotube coatings.

Periprosthetic joint infection (PJI) is a devastating complication of orthopedic implant surgeries, such as total knee and hip arthroplasties. Treatment requires additional surgeries because antibiotics have limited efficacy due to biofilm formation and resistant bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA). A non-pharmaceutical approach is needed, and examples of this are found in nature; dragonfly and cicada wings are antibacterial because of their nanopillar surface structure rather than their chemistry. Carbon-infiltrated carbon nanotube (CICNT) surfaces exhibit a similar nanopillar structure, and have been shown to facilitate osseointegration, and it is postulated that they might provide a structurally-derived resistance to bacterial proliferation and biofilm formation. The objective of this study was to test the biofilm resistance of CICNT coatings. Two types of CICNT were produced: a vertically aligned CNT forest on a silicon substrate using a layer of iron as the catalyst (CICNT-Si) and a random-oriented CNT forest on stainless steel (SS) substrate using the substrate as the catalyst (CICNT-SS). These were tested against SS and carbon controls. After 48 h in an MRSA biofilm reactor, samples demonstrated that both types of CICNT coatings significantly (p < 0.0001) reduced MRSA biofilm formation by 60%-80%. Morphologically, biofilm presence on both types of CICNT was also significantly reduced. Clinical Significance: Results suggest that a CICNT surface modification could be suitable and advantageous for medical devices susceptible to MRSA cell attachment and biofilm proliferation, particularly orthopedic implants.

The effect of injection speed and serial injection on propidium iodide entry into cultured HeLa and primary neonatal fibroblast cells using lance array nanoinjection.

BACKGROUND: Although site-directed genetic engineering has greatly improved in recent years, particularly with the implementation of CRISPR-Cas9, the ability to deliver these molecular constructs to a wide variety of cell types without adverse reaction is still a challenge. One non-viral transfection method designed to address this challenge is a MEMS based biotechnology described previously as lance array nanoinjection (LAN). LAN delivery of molecular loads is based upon the combinational use of electrical manipulation of loads of interest and physical penetration of target cell membranes. This work explores an original procedural element to nanoinjection by investigating the effects of the speed of injection and also the ability to serially inject the same sample. RESULTS: Initial LAN experimentation demonstrated that injecting at speeds of 0.08 mm/s resulted in 99.3 % of cultured HeLa 229 cells remaining adherent to the glass slide substrate used to stage the injection process. These results were then utilized to examine whether or not target cells could be injected multiple times (1, 2, and 3 times) since the injection process was not pulling the cells off of the glass slide. Using two different current control settings (1.5 and 3.0 mA) and two different cell types (HeLa 229 cells and primary neonatal fibroblasts [BJ(ATCC(®) CRL-2522™)], treatment samples were injected with propidium iodide (PI), a cell membrane impermeable nucleic acid dye, to assess the degree of molecular load delivery. Results from the serial injection work indicate that HeLa cells treated with 3.0 mA and injected twice (×2) had the greatest mean PI uptake of 60.47 % and that neonatal fibroblasts treated with the same protocol reached mean PI uptake rates of 20.97 %. CONCLUSIONS: Both experimental findings are particularly useful because it shows that greater molecular modification rates can be achieved by multiple, serial injections via a slower injection process.

CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection.

BACKGROUND: CRISPR-Cas9 genome editing and labeling has emerged as an important tool in biologic research, particularly in regards to potential transgenic and gene therapy applications. Delivery of CRISPR-Cas9 plasmids to target cells is typically done by non-viral methods (chemical, physical, and/or electrical), which are limited by low transfection efficiencies or with viral vectors, which are limited by safety and restricted volume size. In this work, a non-viral transfection technology, named lance array nanoinjection (LAN), utilizes a microfabricated silicon chip to physically and electrically deliver genetic material to large numbers of target cells. To demonstrate its utility, we used the CRISPR-Cas9 system to edit the genome of isogenic cells. Two variables related to the LAN process were tested which include the magnitude of current used during plasmid attraction to the silicon lance array (1.5, 4.5, or 6.0 mA) and the number of times cells were injected (one or three times). RESULTS: Results indicate that most successful genome editing occurred after injecting three times at a current control setting of 4.5 mA, reaching a median level of 93.77 % modification. Furthermore, we found that genome editing using LAN follows a non-linear injection-dose response, meaning samples injected three times had modification rates as high as nearly 12 times analogously treated single injected samples. CONCLUSIONS: These findings demonstrate the LAN's ability to deliver genetic material to cells and indicate that successful alteration of the genome is influenced by a serial injection method as well as the electrical current settings.

A self-reconfiguring metamorphic nanoinjector for injection into mouse zygotes.

This paper presents a surface-micromachined microelectromechanical system nanoinjector designed to inject DNA into mouse zygotes which are ≈90 µm in diameter. The proposed injection method requires that an electrically charged, DNA coated lance be inserted into the mouse zygote. The nanoinjector's principal design requirements are (1) it must penetrate the lance into the mouse zygote without tearing the cell membranes and (2) maintain electrical connectivity between the lance and a stationary bond pad. These requirements are satisfied through a two-phase, self-reconfiguring metamorphic mechanism. In the first motion subphase a change-point six-bar mechanism elevates the lance to ≈45 µm above the substrate. In the second motion subphase, a compliant folded-beam suspension allows the lance to translate in-plane at a constant height as it penetrates the cell membranes. The viability of embryos following nanoinjection is presented as a metric for quantifying how well the nanoinjector mechanism fulfills its design requirements of penetrating the zygote without causing membrane damage. Viability studies of nearly 3000 nanoinjections resulted in 71.9% of nanoinjected zygotes progressing to the two-cell stage compared to 79.6% of untreated embryos.