Monitoring Wound Healing with Topically Applied Optical NanoFlare mRNA Nanosensors.

An effective wound management strategy needs accurate assessment of wound status throughout the whole healing process. This can be achieved by examining molecular biomarkers including proteins, DNAs, and RNAs. However, existing methods for quantifying these biomarkers such as immunohistochemistry and quantitative polymerase chain reaction are usually laborious, resource-intensive, and disruptive. This article reports the development and utilization of mRNA nanosensors (i.e., NanoFlare) that are topically applied on cutaneous wounds to reveal the healing status through targeted and semi-quantitative examination of the mRNA biomarkers in skin cells. In 2D and 3D in vitro models, the efficacy and efficiency of these nanosensors are demonstrated in revealing the dynamic changes of mRNA biomarkers for different stages of wound development. In mouse models, this platform permits the tracking and identification of wound healing stages and a normal and diabetic wound healing process by wound healing index in real time.

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.

Transdermal delivery of Chinese herbal medicine extract using dissolvable microneedles for hypertrophic scar treatment.

Hypertrophic scars are unfavorable skin diseases characterized by excessive collagen deposition. Although systemic treatments exist in clinic to manage hypertrophic scars, they pose significant side effects and tend to lose efficacy over prolonged applications. Traditional Chinese medicine (TCM) offers as a promising candidate to treat pathological scars. A large number of TCMs have been studied to show anti-scarring effect, however, the natural barrier of the skin impedes their penetration, lowering its therapeutic efficacy. Herein, we reported the use of dissolvable hyaluronic acid (HA) microneedles (MNs) as a vehicle to aid the transdermal delivery of therapeutic agent, a model TCM called shikonin for the treatment of hypertrophic scars. Here, shikonin was mixed with HA to make MNs with adequate mechanical strength for skin penetration, making its dosage controllable during the fabrication process. The therapeutic effect of the shikonin HA MNs was studied in vitro using HSFs and then further verified with quantitative reverse transcriptase polymerase chain reaction. Our data suggest that the shikonin HA MNs significantly reduce the viability and proliferation of the HSFs and downregulate the fibrotic-related genes (i.e., TGFß1, FAP-α and COL1A1). Furthermore, we observed a localized therapeutic effect of the shikonin HA MNs that is beneficial for site-specific treatment.

Ocular Delivery of Predatory Bacteria with Cryomicroneedles Against Eye Infection.

The development of potent antibiotic alternatives with rapid bactericidal properties is of great importance in addressing the current antibiotic crisis. One representative example is the topical delivery of predatory bacteria to treat ocular bacterial infections. However, there is a lack of suitable methods for the delivery of predatory bacteria into ocular tissue. This work introduces cryomicroneedles (cryoMN) for the ocular delivery of predatory Bdellovibrio bacteriovorus (B. bacteriovorus) bacteria. The cryoMN patches are prepared by freezing B. bacteriovorus containing a cryoprotectant medium in a microneedle template. The viability of B. bacteriovorus in cryoMNs remains above 80% as found in long-term storage studies, and they successfully impede the growth of gram-negative bacteria in vitro or in a rodent eye infection model. The infection is significantly relieved by nearly six times through 2.5 days of treatment without substantial effects on the cornea thickness and morphology. This approach represents the safe and efficient delivery of new class of antimicrobial armamentarium to otherwise impermeable ocular surface and opens up new avenues for the treatment of ocular surface disorders.

Nephrotoxicity Assessment with Human Kidney Tubuloids using Spherical Nucleic Acid-Based mRNA Nanoflares.

Drug-induced nephrotoxicity represents an important cause of acute kidney injury with associated patient morbidity and mortality and is often responsible for termination of drug development, after extensive resource allocation. We have developed a human kidney tubuloid system that phenocopies, in 3D culture, kidney proximal tubules, a primary injury site of most nephrotoxicants. Traditional end point assays are often performed on 2D cultures of cells that have lost their differentiated phenotype. Herein, we pair a tubuloid system with Nanoflare (NF) mRNA nanosensors to achieve a facile, real-time assessment of drug nephrotoxicity. Using kidney injury molecule-1 (KIM-1) mRNA as a model injury biomarker, we verify NF specificity in engineered and adenovirus-transfected cells and confirm their efficacy to report tubular cell injury by aristolochic acid and cisplatin. The system also facilitates nephrotoxicity screening as demonstrated with 10 representative anticancer moieties. 5-Fluorouracil and paclitaxel induce acute tubular injury, as reflected by an NF signal increase.

Cryomicroneedles for transdermal cell delivery.

Cell therapies for the treatment of skin disorders could benefit from simple, safe and efficient technology for the transdermal delivery of therapeutic cells. Conventional cell delivery by hypodermic-needle injection is associated with poor patient compliance, requires trained personnel, generates waste and has non-negligible risks of injury and infection. Here, we report the design and proof-of-concept application of cryogenic microneedle patches for the transdermal delivery of living cells. The microneedles are fabricated by stepwise cryogenic micromoulding of cryogenic medium with pre-suspended cells, and can be easily inserted into porcine skin and dissolve after deployment of the cells. In mice, cells delivered by the cryomicroneedles retained their viability and proliferative capability. In mice with subcutaneous melanoma tumours, the delivery of ovalbumin-pulsed dendritic cells via the cryomicroneedles elicited higher antigen-specific immune responses and led to slower tumour growth than intravenous and subcutaneous injections of the cells. Biocompatible cryomicroneedles may facilitate minimally invasive cell delivery for a range of cell therapies.

Nanodelivery Systems for Topical Management of Skin Disorders.

Topical drug delivery has inherent advantages over other administration routes. However, the existence of stratum corneum limits the diffusion to small and lipophilic drugs. Fortunately, the advancement of nanotechnology brings along opportunities to address this challenge. Taking the unique features in size and surface chemistry, nanocarriers such as liposomes, polymeric nanoparticles, gold nanoparticles, and framework nucleic acids have been used to bring drugs across the skin barrier to epidermis and dermis layers. This article reviews the development of these formulations and focuses on their applications in the treatment of skin disorders such as acne, skin inflammation, skin infection, and wound healing. Existing hurdles and further developments are also discussed.

Temporal pressure enhanced topical drug delivery through micropore formation.

Transdermal drug delivery uses chemical, physical, or biochemical enhancers to cross the skin barrier. However, existing platforms require high doses of chemical enhancers or sophisticated equipment, use fragile biomolecules, or are limited to a certain type of drug. Here, we report an innovative methodology based on temporal pressure to enhance the penetration of all kinds of drugs, from small molecules to proteins and nanoparticles (up to 500 nm). The creation of micropores (~3 µm2) on the epidermal layer through a temporal pressure treatment results in the elevated expression of gap junctions, and reduced expression of occludin tight junctions. A 1 min treatment of 0.28-MPa allows nanoparticles (up to 500 nm) and macromolecules (up to 20 kDa) to reach a depth of 430-µm into the dermal layer. Using, as an example, the delivery of insulin through topical application after the pressure treatment yields up to 80% drop in blood glucose in diabetic mice.

A self-adhesive microneedle patch with drug loading capability through swelling effect.

Microneedles (MNs) offer a rapid method of transdermal drug delivery through penetration of the stratum corneum. However, commercial translation has been limited by fabrication techniques unique to each drug. Herein, a broadly applicable platform is explored by drug-loading via swelling effect of a hydrogel MN patch. A range of small molecule hydrophilic, hydrophobic, and biomacromolecule therapeutics demonstrate successful loading and burst release from hydrogel MNs fabricated from methacrylated hyaluronic acid (MeHA). The post-fabrication drug loading process allows MeHA MN patches with drug loadings of 10 µg cm-2. Additional post-fabrication processes are explored with dendrimer bioadhesives that increase work of adhesion, ensuring stable fixation on skin, and allow for additional drug loading strategies.

A Double-Layered Microneedle Platform Fabricated through Frozen Spray-Coating.

This work reports a frozen spray-coating method for the fabrication of double-layered microneedles (MNs). Taking swellable methacrylated hyaluronic acid (MeHA)-derived MNs as the model, both hydrophobic molecules (Nile red, Cy5) and hydrophilic ones (FITC, FITC-Dextran, Insulin) can be homogeneously coated without impacting the mechanical properties of the original MeHA MNs. The prepared double-layered MNs can execute multiple roles. It is demonstrated that insulin-coated MeHA double-layered MNs allow the effective delivery of the insulin into circulation of mice for controlling the blood glucose level while they also permit the extraction of skin interstitial fluid for the timely analysis of the biomarker (glucose).

Unraveling Framework Nucleic Acid-Skin Cell Interactions with a Co-Culture System.

Framework nucleic acid (FNA) is an emerging drug carrier platform, with its biodegradability and uniform, tunable structures. Recently, its applicability in transdermal drug delivery has been demonstrated, extending the range of applications that are predominantly based on intravenous injection. However, FNA's interaction and impact toward the skin cells are yet to be elucidated. This study employs an optically clear keratinocyte/fibroblast co-culture system to visualize the FNA-skin cell interactions. FNA's influence on these cells is evaluated through polymerase chain reaction analyses and metabolism assays. A size-dependent interaction and cellular internalization on both keratinocytes and fibroblasts is observed, with no adverse effects on cell viability and functions.

Reprogramming of macrophages with macrophage cell membrane-derived nanoghosts.

Macrophages can be polarized to M1 or M2 type with pro-inflammatory or anti-inflammatory properties. Nanoparticles have recently been found to be a promising platform to polarize macrophages to desired phenotypes. This article explores the usage of cell membrane-derived nanoparticles (nanoghosts) for reprogramming macrophages. The efficacy and efficiency of this technology are examined via cytokine analysis and immunostaining of the nanoghost-treated cells. We find that several cytokines/chemokines are highly expressed on nanoghosts. In addition, a 2D wound healing model is deployed to reveal their potential application in clinical settings.

Upconversion Nanoparticle Powered Microneedle Patches for Transdermal Delivery of siRNA.

Microneedles (MNs) permit the delivery of nucleic acids like small interfering RNA (siRNA) through the stratum corneum and subsequently into the skin tissue. However, skin penetration is only the first step in successful implementation of siRNA therapy. These delivered siRNAs need to be resistant to enzymatic degradation, enter target cells, and escape the endosome-lysosome degradation axis. To address this challenge, this article introduces a nanoparticle-embedding MN system that contains a dissolvable hyaluronic acid (HA) matrix and mesoporous silica-coated upconversion nanoparticles (UCNPs@mSiO2 ). The mesoporous silica (mSiO2 ) shell is used to load and protect siRNA while the upconversion nanoparticle (UCNP) core allows the tracking of MN skin penetration and NP diffusion through upconversion luminescence imaging or optical coherence tomography (OCT) imaging. Once inserted into the skin, the HA matrix dissolves and UCNPs@mSiO2 diffuse in the skin tissue before entering the cells for delivering the loaded genes. As a proof of concept, this system is used to deliver molecular beacons (MBs) and siRNA targeting transforming growth factor-beta type I receptor (TGF-ßRI) that is potentially used for abnormal scar treatment.

Transdermal delivery of small interfering RNAs with topically applied mesoporous silica nanoparticles for facile skin cancer treatment.

Small interfering RNA (siRNA) is a promising tool for the treatment of skin disorders including skin squamous cell carcinoma (SCC). This article develops a topical formulation for the transdermal delivery of siRNA. The formulation is built on mesoporous silica nanoparticles (MSNPs) with a loading capacity of 1.4 µg of oligonucleotide per mg of MSNPs. Cell experiments are employed to study the functionality of the formulation including the cellular uptake, the qualitative and quantitative detection of specific gene biomarkers. The clinical potential of this system is examined by topically delivering siRNA targeting TGFßR-1 (TGFßR-1) to the SCC in a mouse xenograft model. In comparison to the controls, MSNPs containing TGFßR-1 siRNA show a 2-fold suppression of TGFßR-1.

Framework Nucleic Acids: A Paradigm Shift in Transdermal Drug Delivery.

Transdermal drug delivery (TDD) provides a direct drug administration route bypassing gastrointestinal and liver metabolism. Until now, topical nanocarriers responsible for efficient TDD are predominantly polymeric or lipid based. The size-dependent skin penetration ability of framework nucleic acids (FNAs) has recently been reported, along with their efficacy in delivering doxorubicin for skin melanoma therapy. This commentary is to highlight the paradigm shift of nucleic acid delivery from being a cargo moiety to serving as a drug carrier instead. Further development directions to maximize the potential of FNAs for TDD are also discussed.

Framework nucleic acids as programmable carrier for transdermal drug delivery.

DNA nanostructures are promising drug carriers with their intrinsic biocompatibility, uniformity and versatility. However, rapid serum disintegration leads to low bioavailability at targeted sites following systemic administration, hindering their biomedical applications. Here we demonstrate transdermal delivery of framework nucleic acids (FNAs) through topical applications. By designing FNAs with distinct shapes and sizes, we interrogate their penetration on mice and human skin explant. Skin histology reveals size-dependent penetration, with FNAs ≤75 nm effectively reaching dermis layer. 17 nm-tetrahedral FNAs show greatest penetration to 350 µm from skin periphery. Importantly, structural integrity is maintained during the skin penetration. Employing a mouse melanoma model, topical application of doxorubicin-loaded FNAs accommodates ≥2-fold improvement in drug accumulation and tumor inhibition relative to topically-applied free doxorubicin, or doxorubicin loaded in liposomes and polymeric nanoparticles. Programmable penetration with minimal systemic biodistribution underlines FNA potential as localized transdermal drug delivery carriers.

Functional Imaging with Nucleic-Acid-Based Sensors: Technology, Application and Future Healthcare Prospects.

Timely monitoring and assessment of human health plays a crucial role in maintaining the wellbeing of our advancing society. In addition to medical tools and devices, suitable probe agents are crucial to assist such monitoring, either in passive or active ways (i.e., sensors) through inducible signals. In this review we highlight recent developments in activatable optical sensors based on nucleic acids. Sensing mechanisms and bio-applications of these nucleic acid sensors in ex vivo assays, intracellular or in vivo settings are described. In addition, we discuss the limitations of these sensors and how nanotechnology can complement/enhance sensor properties to promote translation into clinical applications.

A Concise Review of Gold Nanoparticles-Based Photo-Responsive Liposomes for Controlled Drug Delivery.

The focus of drug delivery is shifting toward smart drug carriers that release the cargo in response to a change in the microenvironment due to an internal or external trigger. As the most clinically successful nanosystem, liposomes naturally come under the spotlight of this trend. This review summarizes the latest development about the design and construction of photo-responsive liposomes with gold nanoparticles for the controlled drug release. Alongside, we overview the mechanism involved in this process and the representative applications.

Hairpin-structured probe conjugated nano-graphene oxide for the cellular detection of connective tissue growth factor mRNA.

Identification of abnormal scars at their early stage has attracted increasing attentions as the scars can only be assessed qualitatively and subjectively upon maturity, when no invasive procedure is involved. This report introduces a fluorescent probe that targets a potential abnormal scar biomarker (connective tissue growth factor (CTGF) mRNA) in skin fibroblasts. This probe is constructed of hairpin-structured probes (HPs) targeting CTGF mRNA and the nano-graphene oxide (nano-GO) base. The HPs are non-covalently absorbed on the surface of nano-GO, which pre-quenches the fluorescence of HPs. Close proximity of complementary CTGF mRNA would lead to preferential HP hybridization and dissociation from nano-GO, which restores the fluorescence signal from HPs. Utilizing this probe, we can distinguish abnormal fibroblasts derived from abnormal scars and assess the effectiveness of anti-scarring drugs like Repsox and transforming growth factor-beta type I receptor (TGF-ßRI) siRNA.

Oligonucleotide Molecular Sprinkler for Intracellular Detection and Spontaneous Regulation of mRNA for Theranostics of Scar Fibroblasts.

Early diagnosis and timely intervention are key for the successful treatment of skin diseases like abnormal scars. This study introduces a nucleic-acid-based probe (i.e., molecular sprinkler) for the diagnosis and spontaneous regulation of the abnormal expression of fibrosis-related mRNA in scar-derived skin fibroblasts. Using mRNA encoding connective tissue growth factor (CTGF) as the model gene, a probe with three oligonucleotides is constructed, including a recognition sequence complementary to the CTGF mRNA, a siRNA against transforming growth factor receptor I (TGFßRI) as the CTGF mRNA suppressor, and a connecting sequence. The probe can detect CTGF mRNA with a limit of 10 × 10-9 m and distinguishes scar fibroblasts from normal ones in both 2D and 3D environments. Two days after transfection, the siRNA released from the probe reduces the expression of TGFßRI and, consequently, decreases the cellular expression of CTGF mRNA (up to 70%). This dual-role probe presents opportunities to monitor the TGF- ß signaling pathway, screen for drugs that target the CTGF pathway, and determine the role of inhibition of the CTGF pathway in therapeutic efficacy.