Attenuation of Abnormal Scarring Using Spherical Nucleic Acids Targeting Transforming Growth Factor Beta 1.

Abnormal scarring is a consequence of dysregulation in the wound healing process, with limited options for effective and noninvasive therapies. Given the ability of spherical nucleic acids (SNAs) to penetrate skin and regulate gene expression within, we investigated whether gold-core SNAs (AuSNAs) and liposome-core SNAs (LSNAs) bearing antisense oligonucleotides targeting transforming growth factor beta 1 (TGF-ß1) can function as a topical therapy for scarring. Importantly, both SNA constructs appreciably downregulated TGF-ß1 protein expression in primary hypertrophic and keloid scar fibroblasts in vitro. In vivo, topically applied AuSNAs and LSNAs downregulated TGF-ß1 protein expression levels and improved scar histology as determined by the scar elevation index. These data underscore the potential of SNAs as a localized, self-manageable treatment for skin-related diseases and disorders that are driven by increased gene expression.

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.

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.

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.

Simplifying Skin Disease Diagnosis with Topical Nanotechnology.

A new study published in the journal Nature Biomedical Engineering1 documents a novel diagnostic technology that exploits topically applied nanotechnology to detect skin tissue biomarkers for diagnosis. This concept is demonstrated by noninvasively imaging connective tissue growth factor (CTGF) mRNA in abnormal scar cells, whole tissue, and animal models. In this commentary, we highlight the main findings and discuss their implications. Successful implementation in the clinic could give rise to self-applied, biopsy-free diagnostic technology and significantly reduce healthcare burden. Crucially, noninvasive visualization of disease biomarkers, mobile device signal acquisition, and Internet-enabled transmission could significantly transform the diagnosis of skin disease and other superficial tissues.

Real-Time Imaging of Dynamic Cell Reprogramming with Nanosensors.

Cellular reprogramming, the process by which somatic cells regain pluripotency, is relevant in many disease modeling, therapeutic, and drug discovery applications. Molecular evaluation of reprogramming (e.g., polymerase chain reaction, immunostaining) is typically disruptive, and only provides snapshots of phenotypic traits. Gene reporter constructs facilitate live-cell evaluation but is labor intensive and may risk insertional mutagenesis during viral transfection. Herein, the utilization of a non-integrative nanosensor is demonstrated to visualize key reprogramming events in situ within live cells. Principally based on sustained intracellular release of encapsulated molecular probes, nanosensors successfully monitored mesenchymal-epithelial transition, pluripotency acquisition, and transdifferentiation events. Tracking the dynamic expression of four pivotal biomarkers (i.e., THY1, E-CADHERIN, OCT4, and GATA4 mRNA), nanosensor signal showed great agreement with polymerase chain reaction and gene reporter imaging (R2 > 0.9). Overall, such facile, versatile nanosensor enables real-time monitoring of low-frequency reprogramming events, thereby useful for high-throughput assessment, optimization, and biomarker-specific cell enrichment.

Near-Infrared Fluorescent Molecular Probe for Sensitive Imaging of Keloid.

Early detection of skin diseases is imperative for their effective treatment. However, fluorescence molecular probes that allow this are rare. The first activatable near-infrared (NIR) fluorescent molecular probe is reported for sensitive imaging of keloid cells, skin cells from abnormal scar fibrous lesions. As keloid cells have high expression levels of fibroblast activation protein-alpha (FAPα), the probe (FNP1) is designed to have a caged NIR dye and a FAPα-cleavable peptide substrate linked by a self-immolative segment. FNP1 can quickly and specifically turn on its fluorescence at 710 nm by 45-fold in the presence of FAPα, allowing it to effectively recognize keloid cells from normal skin cells. Integration of FNP1 with a simple microneedle-assisted topical application enables sensitive detection of keloid cells in metabolically-active human skin tissue with a theoretical limit of detection down to 20 000 cells.

Abnormal scar identification with spherical-nucleic-acid technology.

The accurate diagnosis of scar type and severity relies on histopathology of biopsied tissue, which is invasive and time-consuming, causes discomfort and may exacerbate scarring. Here, we show that imaging nanoprobes for the live-cell detection of intracellular messenger RNA (mRNA) (also known as NanoFlares) enable measurements of the expression of connective tissue growth factor (CTGF) as a visual indicator of hypertrophic scars and keloids. During cell culture, NanoFlares enabled the distinction of hypertrophic and keloidal fibroblasts from normal fibroblasts, and the detection of changes in CTGF expression resulting from the regulatory effects of transforming growth factor-ß (TGF-ß) agonists and TGF-ß antagonists. We also applied the NanoFlares topically to the skin of live mice and rabbits, and to ex vivo human skin models. Transepidermal penetration of the NanoFlares enabled the visual and spectroscopic quantification of underlying abnormal fibroblasts on the basis of CTGF mRNA expression. Our proof-of-concept studies of topically applied NanoFlare technology as a means of biopsy-free scar diagnosis may eventually inform therapeutic decisions on the basis of the mRNA-expression patterns of skin disorders.

Anti-Scarring Drug Screening with Near-Infrared Molecular Probes Targeting Fibroblast Activation Protein-α.

Abnormal scarring is a hyper-proliferative wound healing disorder, which causes itch, pain, psychological distress, and even limiting limb mobility. Unfortunately, no satisfactory treatment exists to date. Drug screening identifies suitable drugs or drug combinations that inhibit abnormal scarring from a large selection of candidates. However, current techniques are often laborious and technically complex. Herein, we adopt a near-infrared fluorescence probe (FNP1) for fibroblast activation protein (FAP)α, a biomarker overexpressed in skin fibroblasts derived from abnormal scars, to rapidly assess (<30 min) drug candidates for the treatment. FNP1 has high sensitivity, even detecting FAPα up-regulation with as little as 0.016 ng/mL TGFß1, 125-fold lower than typical culture concentrations. The identified drug candidates (RepSox and Thiazovivin) show similar potent anti-scarring activity as the existing anti-scarring compounds and further suppress the expression of other scar biomarkers including connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), and collagen type 1 (COL1A1).

Microneedle physical contact as a therapeutic for abnormal scars.

BACKGROUND: Abnormal (keloid and hypertrophic) scars are a significant affliction with no satisfactory single modality therapy to-date. Available options are often ineffective, painful, potentially hazardous, and require healthcare personnel involvement. Herein a self-administered microneedle device based on drug-free physical contact for inhibiting abnormal scars is reported. Its therapeutic activity through microneedle contact eliminates hazards associated with toxic anti-scarring drugs while self-treatment enables administration flexibility. METHODS: The microneedle patch was fabricated with FDA-approved liquid crystalline polymer under good manufacturing practice. It was first tested to ascertain its ability to inhibit (keloid) fibroblast proliferation. Later the microneedle patch was examined on the rabbit ear hypertrophic scar model to explore its potential in inhibiting the generation of abnormal scars post-injury. Finally, the microneedle patch was applied to the caudal region of a hypertrophic scar located on a female patient's dorsum to verify clinical efficacy. RESULTS: On untreated control cultures, barely any non-viable fibroblasts could be seen. After 12-h treatment with the microneedle patch, the non-viable proportion increased to 83.8 ± 11.96%. In rabbit ear hypertrophic scar model, 100% of the control wounds without the presence of patches on rabbit ears generated regions of raised dermis originating from the wound site (3/3), whereas microneedle treatment prevented dermis tissue thickening in 83.33% of the wounds (15/18). In the clinical test, the microneedle patch was well tolerated by the patient. Compared to the untreated region, microneedle treatment decreased the number of infiltrated inflammatory cells, with less disrupted dermis tissue architecture and more flattened appearance. CONCLUSIONS: A self-administered, drug-free microneedle patch appears highly promising in reducing abnormal scarring as observed from in vitro, in vivo and clinical experiments. Larger cohort clinical studies need to be performed to validate its efficacy on abnormal scars.

Peptide delivery with poly(ethylene glycol) diacrylate microneedles through swelling effect.

Transdermal delivery of therapeutic biomolecules (including peptides) can avoid enzymatic digestion that occurs in the oral route. (Polyethylene glycol) diacrylate (PEGDA)-based microneedles, with good biocompatibility, are easily fabricated through photo-polymerization with a precisely controlled structure. It has successfully been used for the transdermal delivery of small molecule drugs such as 5-fluorouracil. However, the delivery of peptide-based therapeutics using this platform is seldom reported. This is because of the potential damage to the peptide during the photo-polymerization process of PEGDA. Herein, we introduce a method to load PEGDA microneedles with peptides without compromising peptide potency. Using gap junction inhibitor (Gap 26) as an example, the peptide was loaded into PEGDA microneedles through the swelling effect of PEGDA in the aqueous solution. The peptide-loaded microneedles were applied to a keloid scar model and exhibited inhibition expression of collagen I, a predominant marker of keloid scar, demonstrating its potential therapeutic effects.

Noninvasive Monitoring of Three-Dimensional Chondrogenic Constructs Using Molecular Beacon Nanosensors.

Chondrogenic differentiation of human mesenchymal stem cells (MSCs) in three-dimensional hydrogel holds promise as a method for repairing injured articular cartilage. Given MSC plasticity (its potential to mature into alternative lineages), nondestructive monitoring is critical for the optimization of chondrogenic differentiation conditions and the evaluation of the final product. However, conventional validation/assessments of the differentiation process (i.e., quantitative reverse transcription polymerase chain reaction [qRT-PCR] and histology) are end-point assays requiring disruption of the sample. This report introduces molecular beacon (MB)-based nanosensors to achieve noninvasive monitoring of chondrogenic differentiation. These nanosensors consist of biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) encapsulating MBs to detect Type II Collagen (Col2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs that serve as lineage-specific and housekeeping biomarkers, respectively. The sustainable release of MBs from MB-NPs allows longitudinal monitoring of MSCs undergoing chondrogenic differentiation over a period of 28 days. Dual-colored MB loading ensures accurate assessment of Col2 mRNA expression level, where potential heterogeneity in nanosensor uptake and retention by MSCs are taken into account. When normalized nanosensor signal was compared against qRT-PCR result, a tight correlation was observed (R2 = 0.9301). Finally, nanosensor usage was compatible with MSC potency with minimal influence on chondrogenic, adipogenic, and osteogenic differentiation.

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (>90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving >95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (10(6) cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

Nanosensors for Continuous and Noninvasive Monitoring of Mesenchymal Stem Cell Osteogenic Differentiation.

Assessing mesenchymal stem cell (MSC) differentiation status is crucial to verify therapeutic efficacy and optimize treatment procedures. Currently, this involves destructive methods including antibody-based protein detection and polymerase chain reaction gene analysis, or laborious and technically challenging genetic reporters. Development of noninvasive methods for real-time differentiation status assessment can greatly benefit MSC-based therapies. This report introduces a nanoparticle-based sensing platform that encapsulates two molecular beacon (MB) probes within the same biodegradable polymeric nanoparticles. One MB targets housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference, while another detects alkaline phosphatase (ALP), a functional biomarker. Following internalization, MBs are gradually released as the nanoparticle degrades. GAPDH MBs provide a stable reference signal throughout the monitoring period (18 days) regardless of differentiation induction. Meanwhile, ALP mRNA undergoes well-defined dynamics with peak expression observed during early stages of osteogenic differentiation. By normalizing ALP-MB signal with GAPDH-MB, changes in ALP expression can be monitored, to noninvasively validate osteogenic differentiation. As proof-of-concept, a dual-colored nanosensor is applied to validate MSC osteogenesis on 2D culture and polycaprolactone films containing osteo-inductive tricalcium phospate.

Interference-free Micro/nanoparticle Cell Engineering by Use of High-Throughput Microfluidic Separation.

Engineering cells with active-ingredient-loaded micro/nanoparticles is becoming increasingly popular for imaging and therapeutic applications. A critical yet inadequately addressed issue during its implementation concerns the significant number of particles that remain unbound following the engineering process, which inadvertently generate signals and impart transformative effects onto neighboring nontarget cells. Here we demonstrate that those unbound micro/nanoparticles remaining in solution can be efficiently separated from the particle-labeled cells by implementing a fast, continuous, and high-throughput Dean flow fractionation (DFF) microfluidic device. As proof-of-concept, we applied the DFF microfluidic device for buffer exchange to sort labeled suspension cells (THP-1) from unbound fluorescent dye and dye-loaded micro/nanoparticles. Compared to conventional centrifugation, the depletion efficiency of free dyes or particles was improved 20-fold and the mislabeling of nontarget bystander cells by free particles was minimized. The microfluidic device was adapted to further accommodate heterogeneous-sized mesenchymal stem cells (MSCs). Complete removal of unbound nanoparticles using DFF led to the usage of engineered MSCs without exerting off-target transformative effects on the functional properties of neighboring endothelial cells. Apart from its effectiveness in removing free particles, this strategy is also efficient and scalable. It could continuously process cell solutions with concentrations up to 10(7) cells·mL(-1) (cell densities commonly encountered during cell therapy) without observable loss of performance. Successful implementation of this technology is expected to pave the way for interference-free clinical application of micro/nanoparticle engineered cells.

Molecular beacon-loaded polymeric nanoparticles for non-invasive imaging of mRNA expression.

Assessment of intracellular mRNA expression is invaluable for understanding cellular signaling activities, identifying disease stages, and monitoring the gene expression pattern of therapeutic cells during their culture, expansion and/or differentiation process. Previous methods suffer from the need to disrupt the biological samples to perform polymerase chain reaction analysis which can be laborious, fragmented and destructive. Herein, we develop a mRNA nanosensor based on the sustained release of mRNA-specific molecular beacons (probes that fluoresce upon hybridization) from the biodegradable poly(d,l-lactide-co-glycolide) nanoparticles. Post cellular internalization, the particles gradually degrade and release the encapsulated probes which are initially weakly fluorescent. When the released probes meet and hybridize with target mRNA, they restore pre-quenched fluorescence. By virtue of quantifying the fluorescence intensity, we can estimate the cellular mRNA expression. As a case study, ß-actin mRNA expression in mesenchymal stem cells cultured on a 3D matrix was monitored and compared with those cultured on a 2D plate for one week. Critically, the observed expression profile shows a great correlation with the established quantitative polymerase chain reaction analysis.

Nanosensors for regenerative medicine.

Assessing biodistribution, fate, and function of implanted therapeutic cells in preclinical animal experiments is critical to realize safe, effective and efficient treatments for subsequent implementation within the clinic. Currently, tissue histology, the most prevalent analytical technique to meet this need, is limited by end-point analysis, high cost and long preparation time. Moreover, it is disadvantaged by an inability to monitor in real-time, qualitative interpretation and ethical issues arising from animal sacrifice. While genetic engineering techniques allow cells to express molecules with detectable signals (e.g., fluorescence, luminescence, T1 (spin-lattice)/T2 (spin-spin) contrast in magnetic resonance imaging, radionuclide), concerns arise regarding technical complexity, high-cost of genetic manipulation, as well as mutagenic cell dysfunction. Alternatively, cells can be labeled using nanoparticle-sensors-nanosensors that emit signals to identify cell location, status and function in a simple, cost-effective, and non-genetic manner. This review article provides the definition, classification, evolution, and applications of nanosensor technology and focuses on how they can be utilized in regenerative medicine. Several examples of direct applications include: (1) monitoring post-transplantation cell behavior, (2) revealing host response following foreign biomaterial implantation, and (3) optimization of cell bioprocess operating conditions. Incorporating nanosensors is expected to expedite the development of cell-based regenerative medicine therapeutics.

Stem cell bioprocessing: fundamentals and principles.

In recent years, the potential of stem cell research for tissue engineering-based therapies and regenerative medicine clinical applications has become well established. In 2006, Chung pioneered the first entire organ transplant using adult stem cells and a scaffold for clinical evaluation. With this a new milestone was achieved, with seven patients with myelomeningocele receiving stem cell-derived bladder transplants resulting in substantial improvements in their quality of life. While a bladder is a relatively simple organ, the breakthrough highlights the incredible benefits that can be gained from the cross-disciplinary nature of tissue engineering and regenerative medicine (TERM) that encompasses stem cell research and stem cell bioprocessing. Unquestionably, the development of bioprocess technologies for the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation and safety of the process and the product. The successful translation will require contributions from fundamental research (from developmental biology to the 'omics' technologies and advances in immunology) and from existing industrial practice (biologics), especially on automation, quality assurance and regulation. The timely development, integration and execution of various components will be critical-failures of the past (such as in the commercialization of skin equivalents) on marketing, pricing, production and advertising should not be repeated. This review aims to address the principles required for successful stem cell bioprocessing so that they can be applied deftly to clinical applications.