Oxygenated Hydrogel Promotes Re-Epithelialization and Reduces Inflammation in a Perturbed Wound Model in Rat.

Chronic wounds can take months or even years to heal and require proper medical intervention. Normal wound healing processes require adequate oxygen supply. Accordingly, destroyed or inefficient vasculature leads to insufficient delivery to peripheral tissues and impair healing. Oxygen is critical for vital processes such as proliferation, collagen synthesis and antibacterial defense. Hyperbaric oxygen therapy (HBOT) is commonly used to accelerate healing however, this can be costly and requires specialized training and equipment. Efforts have turned to the development of topical oxygen delivery systems. Oxysolutions has developed oxygenated gels (P407, P407/P188, nanocellulose based gel (NCG)) with high levels of dissolved oxygen. This study aims to evaluate the efficacy of these newly developed oxygenated products by assessing their impact on healing rates in a rat perturbed wound model. Here, P407/P188 oxygenated gels demonstrated greater re-epithelialization distances compared to its controls at Day 3. In addition, all oxygenated gels had a higher proportion of wounds with complete wound closure. All three oxygenated gels also minimized further escalation in inflammation from Day 3 to Day 10. This highlights the potential of this newly-developed oxygenated gels as an alternative to existing oxygen therapies.

Battery-free and AI-enabled multiplexed sensor patches for wound monitoring.

Wound healing is a dynamic process with multiple phases. Rapid profiling and quantitative characterization of inflammation and infection remain challenging. We report a paper-like battery-free in situ AI-enabled multiplexed (PETAL) sensor for holistic wound assessment by leveraging deep learning algorithms. This sensor consists of a wax-printed paper panel with five colorimetric sensors for temperature, pH, trimethylamine, uric acid, and moisture. Sensor images captured by a mobile phone were analyzed by neural network-based machine learning algorithms to determine healing status. For ex situ detection via exudates collected from rat perturbed wounds and burn wounds, the PETAL sensor can classify healing versus nonhealing status with an accuracy as high as 97%. With the sensor patches attached on rat burn wound models, in situ monitoring of wound progression or severity is demonstrated. This PETAL sensor allows early warning of adverse events, which could trigger immediate clinical intervention to facilitate wound care management.

Carbon Dot-Doped Hydrogel Sensor Array for Multiplexed Colorimetric Detection of Wound Healing.

Effective wound care and treatment require a quick and comprehensive assessment of healing status. Here, we develop a carbon dot-doped hydrogel sensor array in polydimethylsiloxane (PDMS) for simultaneous colorimetric detections of five wound biomarkers and/or wound condition indicators (pH, glucose, urea, uric acid, and total protein), leading to the holistic assessment of inflammation and infection. A biogenic carbon dot synthesized using an amino acid and a polymer precursor is doped in an agarose hydrogel matrix for constructing enzymatic sensors (glucose, urea, and uric acid) and dye-based sensors (pH and total protein). The encapsulated enzymes in such a matrix exhibit improved enzyme kinetics and stability compared to those in pure hydrogels. Such a matrix also provides stable colorimetric responses for all five sensors. The sensor array exhibits high accuracy (recovery rates of 91.5-113.1%) and clinically relevant detection ranges for all five wound markers. The sensor array is established for simulated wound fluids and validated with rat wound fluids from perturbed wound models. Distinct color patterns are obtained that can clearly distinguish healing vs nonhealing wounds visually and quantitatively. This hydrogel sensor array shows great potential for on-site wound sensing due to its long-term stability, lightweight, and flexibility.

Targeting connexin 43 expression via scaffold mediated delivery of antisense oligodeoxynucleotide preserves neurons, enhances axonal extension, reduces astrocyte and microglial activation after spinal cord injury.

Injury to the central nervous system (CNS) provokes an inflammatory reaction and secondary damage that result in further tissue damage and destruction of neurons away from the injury site. Upon injury, expression of connexin 43 (Cx43), a gap junction protein, upregulates and is responsible for the spread and amplification of cell death signals through these gap junctions. In this study, we hypothesise that the downregulation of Cx43 by scaffold-mediated controlled delivery of antisense oligodeoxynucleotide (asODN), would minimise secondary injuries and cell death, and thereby support tissue regeneration after nerve injuries. Specifically, using spinal cord injury (SCI) as a proof-of-principle, we utilised a fibre-hydrogel scaffold for sustained delivery of Cx43asODN, while providing synergistic topographical cues to guide axonal ingrowth. Correspondingly, scaffolds loaded with Cx43asODN, in the presence of NT-3, suppressed Cx43 up-regulation after complete transection SCI in rats. These scaffolds facilitated the sustained release of Cx43asODN for up to 25 days. Importantly, asODN treatment preserved neurons around the injury site, promoted axonal extension, decreased glial scarring, and reduced microglial activation after SCI. Our results suggest that implantation of such scaffold-mediated asODN delivery platform could serve as an effective alternative SCI therapeutic approach.

Challenges faced in developing an ideal chronic wound model.

INTRODUCTION: Chronic wounds are a major drain on healthcare resources and can lead to substantial reductions in quality of life for those affected. Moreover, they often precede serious events such as limb amputations and premature death. In the long run, this burden is likely to escalate with an ageing population and lifestyle diseases such as obesity. Thus far, the identification of beneficial therapeutics against chronic wounds have been hindered by the lack of an ideal chronic wound animal model. Although animal models of delayed healing have been developed, none of these models fully recapitulate the complexity of the human chronic wound condition. Furthermore, most animals do not develop chronic wounds. Only the thoroughbred racehorse develops chronic ulcers. AREAS COVERED: In this review, the different characteristics of chronic wounds that highlight its complexity are described. In addition, currently available models reflecting different aspects of chronic wound pathology and their relevance to human chronic wounds are discussed. This article concludes by listing relevant features representative of an ideal chronic wound model. Additionally, alternative approaches for the development of chronic wound models are discussed. EXPERT OPINION: Delayed models of healing, including the streptozotocin diabetic model, skin flap model and magnet-induced IR models have emerged. While these models have been widely adopted for preclinical therapeutic testing, their relevance towards human chronic wounds remains debatable. In particular, current delayed healing models often fail to fully incorporate the key characteristics of chronic ulcers. Ultimately, more representative models are required to expedite the advancement of novel therapeutics to the clinic.

Bio-Mimicking Acellular Wet Electrospun Scaffolds Promote Accelerated Integration and Re-Epithelialization of Full-Thickness Dermal Wounds.

Scaffolds can promote the healing of burns and chronic skin wounds but to date have suffered from issues with achieving full skin integration. Here, we characterise the wound response by both tissue integration and re-epithelialization to a scaffold using wet electrospinning to fabricate 3D fibrous structures. Two scaffold materials were investigated: poly(ε-caprolactone) (PCL) and PCL + 20% rat tail type 1 collagen (PCL/Coll). We assessed re-epithelisation, inflammatory responses, angiogenesis and the formation of new extracellular matrix (ECM) within the scaffolds in rat acute wounds. The 3D PCL/Coll scaffolds impeded wound re-epithelisation, inducing a thickening of wound-edge epidermis as opposed to a thin tongue of migratory keratinocytes as seen when 3D PCL scaffolds were implanted in the wounds. A significant inflammatory response was observed with 3D PCL/Coll scaffolds but not with 3D PCL scaffolds. Enhanced fibroblast migration and angiogenesis into 3D PCL scaffolds was observed with a significant deposition of new ECM. We observed that this deposition of new ECM within the scaffold was key to enabling re-epithelialization over the scaffold. Such scaffolds provide a biocompatible environment for cell integration to lay down new ECM and encourage re-epithelisation over the implanted scaffold.

Delivery of Wnt inhibitor WIF1 via engineered polymeric microspheres promotes nerve regeneration after sciatic nerve crush.

Injuries within the peripheral nervous system (PNS) lead to sensory and motor deficits, as well as neuropathic pain, which strongly impair the life quality of patients. Although most current PNS injury treatment approaches focus on using growth factors/small molecules to stimulate the regrowth of the injured nerves, these methods neglect another important factor that strongly hinders axon regeneration-the presence of axonal inhibitory molecules. Therefore, this work sought to explore the potential of pathway inhibition in promoting sciatic nerve regeneration. Additionally, the therapeutic window for using pathway inhibitors was uncovered so as to achieve the desired regeneration outcomes. Specifically, we explored the role of Wnt signaling inhibition on PNS regeneration by delivering Wnt inhibitors, sFRP2 and WIF1, after sciatic nerve transection and sciatic nerve crush injuries. Our results demonstrate that WIF1 promoted nerve regeneration (p < 0.05) after sciatic nerve crush injury. More importantly, we revealed the therapeutic window for the treatment of Wnt inhibitors, which is 1 week post sciatic nerve crush when the non-canonical receptor tyrosine kinase (Ryk) is significantly upregulated.

A 3D Fiber-Hydrogel Based Non-Viral Gene Delivery Platform Reveals that microRNAs Promote Axon Regeneration and Enhance Functional Recovery Following Spinal Cord Injury.

Current treatment approaches toward spinal cord injuries (SCI) have mainly focused on overcoming the inhibitory microenvironment that surrounds lesion sites. Unfortunately, the mere modulation of the cell/tissue microenvironment is often insufficient to achieve desired functional recovery. Therefore, stimulating the intrinsic growth ability of injured neurons becomes crucial. MicroRNAs (miRs) play significant roles during axon regeneration by regulating local protein synthesis at growth cones. However, one challenge of using miRs to treat SCI is the lack of efficient delivery approaches. Here, a 3D fiber-hydrogel scaffold is introduced which can be directly implanted into a spinal cord transected rat. This 3D scaffold consists of aligned electrospun fibers which provide topographical cues to direct axon regeneration, and collagen matrix which enables a sustained delivery of miRs. Correspondingly, treatment with Axon miRs (i.e., a cocktail of miR-132/miR-222/miR-431) significantly enhances axon regeneration. Moreover, administration of Axon miRs along with anti-inflammatory drug, methylprednisolone, synergistically enhances functional recovery. Additionally, this combined treatment also decreases the expression of pro-inflammatory genes and enhance gene expressions related to extracellular matrix deposition. Finally, increased Axon miRs dosage with methylprednisolone, significantly promotes functional recovery and remyelination. Altogether, scaffold-mediated Axon miR treatment with methylprednisolone is a promising therapeutic approach for SCI.

Scaffold-Based Delivery of CRISPR/Cas9 Ribonucleoproteins for Genome Editing.

The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are delivered in the format of plasmids, mRNA, or ribonucleoprotein (RNP) complexes. In particular, non-viral approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local delivery and provide possible synergistic effects with other factors such as topographical cues that are co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be used to validate the efficiency of gene disruption.

Cell Membrane-Coated Electrospun Fibers Enhance Keratinocyte Growth through Cell-Type Specific Interactions.

Although cell membrane-coated fiber scaffolds can be useful for regenerative medicine by presenting both cell surface antigens and topographical cues, it remains unknown whether changes in cellular behavior on cell membrane-coated scaffolds are due to specific cell-cell interactions. In this work, the effects of scaffold fiber diameters and surface charges on the cell membrane coating efficiency were explored. Furthermore, fibroblast membrane-coated scaffolds improved the growth of human keratinocytes as compared to red blood cell membrane-coated and plain scaffolds. These results suggest the biofunctionality of cell membrane-coated scaffolds and the specific cell-cell interactions that are preserved to modulate cellular response.

A laser microdissection-based axotomy model incorporating the use of biomimicking fiber scaffolds reveals that microRNAs promote axon regeneration over long injury distances.

The regeneration of injured neurons over long injury distances remains suboptimal. In order to successfully stimulate nerve regrowth, potent biomolecules are necessary. Furthermore, reproducible and translatable methods to test the potency of candidate drugs for enhancing nerve regeneration over long axotomy distances are also needed. To address these issues, we report a novel laser microdissection-based axotomy model that involves the use of biomimicking aligned fiber substrates to precisely control neuronal axotomy distances. Correspondingly, we demonstrate that in the absence of therapeutics, dorsal root ganglion (DRG) explants (consisting of neurons) axotomized within short distances from the main cell somas regenerated significantly longer than axons that were injured more distally (p < 0.05). However, when treated with a cocktail of microRNAs (miR-132/miR-222/miR-431), robust neurite outgrowth was observed (p < 0.05). Specifically, microRNA treatment promoted neurite outgrowth by ∼2.2-fold as compared to untreated cells and this enhancement was more significant under the less conducive regeneration condition of a long axotomy distance (i.e. 1000 µm from the cell soma). Besides that, we demonstrated that the treatment of miR-132/miR-222/miR-431 led to a longer length of nerve regeneration as well as a bigger nerve extension area after sciatic nerve transection injury. These results indicate that distance effects on axonal regrowth may be overcome by the effects of microRNAs and that these microRNAs may serve as promising therapeutics for nerve injury treatment.

Localized delivery of CRISPR/dCas9 via layer-by-layer self-assembling peptide coating on nanofibers for neural tissue engineering.

The clustered regularly interspaced short palindromic repeat (CRISPR) systems have a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or interference (CRISPRi). However, the safety concerns associated with viral vectors and the possible off-target issues of systemic administration remain huge concerns to be safe delivery methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide (SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 systems. Specifically, an amphiphilic negatively charged SAP- is first coated onto PCL nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively charged SAP+-RGD are then absorbed via electrostatic interactions. The SAPcoated scaffolds facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF expression in mammalian cells, and the secreted GDNF subsequently promotes neurite outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide coated nanofibers can be a new route to establish a bioactive interface, which provides a simple and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine.

Biofunctional scaffolds with high packing density of aligned electrospun fibers support neural regeneration.

Neurons of the central nervous system do not regenerate spontaneously after injury. As such, biofunctional tissue scaffolds have been explored to provide a growth-promoting environment to enhance neural regeneration. In this regard, aligned electrospun fibers have proven invaluable for regeneration by offering guidance for axons to cross the injury site. However, a high fiber density could potentially limit axonal ingrowth into the scaffold. Here, we explore which fiber density provides the optimal environment for neurons to regenerate. By changing fiber electrospinning time, we generated scaffolds with different fiber densities and implanted these in a rat model of spinal cord injury (SCI). We found that neurons were able to grow efficiently into scaffolds with high fiber density, even if the gaps between fiber bundles were very small (<1 µm). Scaffolds with high fiber density showed good host-implant integration. Cell infiltration was not affected by fiber density. Efficient blood vessel ingrowth likely requires larger gaps between fibers or faster degrading fibers. We conclude that scaffolds with high fiber densities, and thus a large number of small gaps in between fiber bundles, provide the preferred environment for nerve regeneration after SCI.

Regenerative rehabilitation: exploring the synergistic effects of rehabilitation and implantation of a bio-functional scaffold in enhancing nerve regeneration.

Clinically, rehabilitation is one of the most common treatment options for traumatic injuries. Despite that, recovery remains suboptimal and recent breakthroughs in regenerative approaches may potentially improve clinical outcomes. To date, there have been numerous studies on the utilization of either rehabilitative or regenerative strategies for traumatic injury treatment. However, studies that document the combined effects of rehabilitation and regenerative tissue engineering options remain scarce. Here, in the context of traumatic nerve injury treatment, we use a rat spinal cord injury (SCI) model as a proof of concept to evaluate the synergistic effects of regenerative tissue engineering and rehabilitation. Specifically, we implanted a pro-regenerative hybrid fiber-hydrogel scaffold and subjected SCI rats to intensive rehabilitation. Of note, the rehabilitation session was augmented by a novel customized training device that imparts normal hindlimb gait movements to rats. Morphologically, more regenerated axons were observed when rats received rehabilitation (∼2.5 times and ∼2 times enhancement after 4 and 12 weeks of recovery, respectively, p < 0.05). Besides that, we also observed a higher percentage of anti-inflammatory cells (36.1 ± 12.9% in rehab rats vs. 3.31 ± 1.48% in non-rehab rats, p < 0.05) and perineuronal net formation in rehab rats at Week 4. Physically, rehab animals were also able to exert higher ankle flexion force (∼0.779 N vs. ∼0.495 N at Week 4 and ∼1.36 N vs. ∼0.647 N at Week 12 for rehab vs. non-rehab rats, p < 0.001) and performed better than non-rehab rats in the open field test. Taken together, we conclude that coupling rehabilitation with regenerative scaffold implantation strategies can further promote functional recovery after traumatic nerve injuries.

Biomimicking Fiber Scaffold as an Effective In Vitro and In Vivo MicroRNA Screening Platform for Directing Tissue Regeneration.

MicroRNAs effectively modulate protein expression and cellular response. Unfortunately, the lack of robust nonviral delivery platforms has limited the therapeutic application of microRNAs. Additionally, there is a shortage of drug-screening platforms that are directly translatable from in vitro to in vivo. Here, a fiber substrate that provides nonviral delivery of microRNAs for in vitro and in vivo microRNA screening is introduced. As a proof of concept, difficult-to-transfect primary neurons are targeted and the efficacy of this system is evaluated in a rat spinal cord injury model. With this platform, enhanced gene-silencing is achieved in neurons as compared to conventional bolus delivery (p < 0.05). Thereafter, four well-recognized microRNAs (miR-21, miR-222, miR-132, and miR-431) and their cocktails are screened systematically. Regardless of age and origin of the neurons, similar trends are observed. Next, this fiber substrate is translated into a 3D system for direct in vivo microRNA screening. Robust nerve ingrowth is observed as early as two weeks after scaffold implantation. Nerve regeneration in response to the microRNA cocktails is similar to in vitro experiments. Altogether, the potential of the fiber platform is demonstrated in providing effective microRNA screening and direct translation into in vivo applications.

Drug therapies and delivery mechanisms to treat perturbed skin wound healing.

Acute wound healing is an orderly process of four overlapping events: haemostasis, inflammation, proliferation and remodelling. A drug delivery system with a temporal control of release could promote each of these events sequentially. However, acute wound healing normally proceeds very well in healthy individuals and there is little need to promote it. In the elderly and diabetics however, healing is often slow and wounds can become chronic and we need to promote their healing. Targeting the events of acute wound healing would not be appropriate for a chronic wound, which have stalled in the proinflammatory phase. They also have many additional problems such as poor circulation, low oxygen, high levels of leukocytes, high reactive oxygen species, high levels of proteolytic enzymes, high levels of proinflammatory cytokines, bacterial infection and high pH. The future challenge will be to tackle each of these negative factors to create a wound environment conducive to healing.

Scaffold-mediated non-viral delivery platform for CRISPR/Cas9-based genome editing.

Genome editing, especially via the simple and versatile type II CRISPR/Cas9 system, offers an effective avenue to precisely control cell fate, an important aspect of tissue regeneration. Unfortunately, most CRISPR/Cas9 non-viral delivery strategies only utilise micro-/nano-particle delivery methods. While these approaches provide reasonable genomic editing efficiencies, their systemic delivery may lead to undesirable off-target effects. For in vivo applications, a more localized and sustained delivery approach may be useful, particularly in the context of tissue regeneration. Here, we developed a scaffold that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and non-viral manner. Specifically, using mussel-inspired bioadhesive coating, polyDOPA-melanin (pDOPA), we adsorbed Cas9:sgRNA lipofectamine complexes onto bio-mimicking fiber scaffolds. To evaluate the genome-editing efficiency of this platform, U2OS.EGFP cells were used as the model cell type. pDOPA coating was essential in allowing Cas9:sgRNA lipofectamine complexes to adhere onto the scaffolds with a higher loading efficiency, while laminin coating was necessary for maintaining cell viability and proliferation on the pDOPA-coated fibers for effective gene editing (21.5% editing efficiency, p < 0.001). Importantly, U2OS.EGFP cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds via reverse transfection. Overall, we demonstrate the efficacy of such fiber scaffolds in providing localized, sustained and non-viral delivery of Cas9:sgRNA complexes. Such genome editing scaffolds may find useful applications in tissue regeneration. STATEMENT OF SIGNIFICANCE: Currently, there is a lack of effective non-viral means to deliver CRISPR/Cas9 components for genome editing. Most existing approaches only utilize micro-/nano-particles by injection or systemic delivery, which may lead to undesirable off-target effects. Here, we report a platform that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and sustained manner. We used mussel-inspired bioadhesive coating to functionalize the bio-mimicking fiber scaffolds with Cas9:sgRNA lipofectamine complexes, to allow effective gene editing for the cells seeded on the scaffolds. Importantly, the cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds. Such genome editing scaffolds may find useful applications in tissue regeneration.

Scaffold-Mediated Sustained, Non-viral Delivery of miR-219/miR-338 Promotes CNS Remyelination.

The loss of oligodendrocytes (OLs) and subsequently myelin sheaths following injuries or pathologies in the CNS leads to debilitating functional deficits. Unfortunately, effective methods of remyelination remain limited. Here, we present a scaffolding system that enables sustained non-viral delivery of microRNAs (miRs) to direct OL differentiation, maturation, and myelination. We show that miR-219/miR-338 promoted primary rat OL differentiation and myelination in vitro. Using spinal cord injury as a proof-of-concept, we further demonstrate that miR-219/miR-338 could also be delivered non-virally in vivo using an aligned fiber-hydrogel scaffold to enhance remyelination after a hemi-incision injury at C5 level of Sprague-Dawley rats. Specifically, miR-219/miR-338 mimics were incorporated as complexes with the carrier, TransIT-TKO (TKO), together with neurotrophin-3 (NT-3) within hybrid scaffolds that comprised poly(caprolactone-co-ethyl ethylene phosphate) (PCLEEP)-aligned fibers and collagen hydrogel. After 1, 2, and 4 weeks post-treatment, animals that received NT-3 and miR-219/miR-338 treatment preserved a higher number of Olig2+ oligodendroglial lineage cells as compared with those treated with NT-3 and negative scrambled miRs (Neg miRs; p < 0.001). Additionally, miR-219/miR-338 increased the rate and extent of differentiation of OLs. At the host-implant interface, more compact myelin sheaths were observed when animals received miR-219/miR-338. Similarly within the scaffolds, miR-219/miR-338 samples contained significantly more myelin basic protein (MBP) signals (p < 0.01) and higher myelination index (p < 0.05) than Neg miR samples. These findings highlight the potential of this platform to promote remyelination within the CNS.

Localised non-viral delivery of nucleic acids for nerve regeneration in injured nervous systems.

Axons damaged by traumatic injuries are often unable to spontaneously regenerate in the adult central nervous system (CNS). Although the peripheral nervous system (PNS) has some regenerative capacity, its ability to regrow remains limited across large lesion gaps due to scar tissue formation. Nucleic acid therapy holds the potential of improving regeneration by enhancing the intrinsic growth ability of neurons and overcoming the inhibitory environment that prevents neurite outgrowth. Nucleic acids modulate gene expression by over-expression of neuronal growth factor or silencing growth-inhibitory molecules. Although in vitro outcomes appear promising, the lack of efficient non-viral nucleic acid delivery methods to the nervous system has limited the application of nucleic acid therapeutics to patients. Here, we review the recent development of efficient non-viral nucleic acid delivery platforms, as applied to the nervous system, including the transfection vectors and carriers used, as well as matrices and scaffolds that are currently used. Additionally, we will discuss possible improvements for localised nucleic acid delivery.