Gold nanobipyramids-based laser-activated sealants for effective skin sealing and repair.

Anisotropic gold nanostructures have gained increased attention for biomedical applications because of their remarkable optical properties. An emerging type of gold nanostructure-gold nanobipyramids (AuNBP)-has been shown to exhibit superior absorption properties compared to conventionally used gold nanoparticles, which makes them attractive for photothermal applications. We generated a high-shape-purity dispersion of AuNBP using a seed-mediated method and embedded them as photothermal conversion agents in a silk fibroin matrix to investigate their efficacy in photothermal sealing of incisional wounds in immunocompetent mice. These AuNBP-doped laser-activated sealants, or AuNBP-LASE were able to absorb near-infrared laser energy and convert it to heat, thereby inducing transient hyperthermia in the wound and the surrounding tissue. This photothermal conversion facilitated rapid sealing of the skin tissue by the AuNBP-LASE, which resulted in faster functional recovery of skin barrier function compared to nylon sutures at the early stages of repair. Further, the biomechanical properties of the healing skin closed with AuNBP-LASE those of intact skin more rapidly compared to incisions approximated with sutures. Histology studies indicated higher penetration of the LASE within the volume of the incision in skin tissue, lower scab formation, and a similar epidermal gap compared to conventional suturing. These results demonstrate that AuNBP-LASEs can be effective as wound approximation devices for photothermal sealing.

Evaluation of drug carrier hepatotoxicity using primary cell culture models.

This study aims to establish a primary rat hepatocyte culture model to evaluate dose-dependent hepatotoxic effects of drug carriers (lipopolymer nanoparticles; LPNs) temporal. Primary rat hepatocyte cell cultures were used to determine half-maximal Inhibition Concentrations (IC50) of the drug-carrier library. Drug-carrier library, at concentrations <50 µg/mL, is benign to primary rat hepatocytes as determined using albumin and urea secretions. Albumin, as a hepatic biomarker, exhibited a more sensitive and faster outcome, compared to urea, for the determination of the IC50 value of LPNs. Temporal measurements of hepatic biomarkers including urea and albumin, and rigorous physicochemical (hydrodynamic diameter, surface charge, etc.) characterization, should be combined to evaluate the hepatotoxicity of drug carrier libraries in screens.

Radiation-Responsive Amino Acid Nanosensor Gel (RANG) for Radiotherapy Monitoring and Trauma Care.

Accurate detection of doses is critical for the development of effective countermeasures and patient stratification strategies in cases of accidental exposure to ionizing radiation. Existing detection devices are limited by high fabrication costs, long processing times, need for sophisticated detection systems, and/or loss of readout signal over time, particularly in complex environments. Here, we describe fundamental studies on amino acid-facilitated templating of gold nanoparticles following exposure to ionizing radiation as a new colorimetric approach for radiation detection. Tryptophan demonstrated spontaneous nanoparticle formation, and parallel screening of a library of amino acids and related compounds led to the identification of lead candidates, including phenylalanine, which demonstrated an increase in absorbance at wavelengths typical of gold nanoparticles in the presence of ionizing radiation (X-rays). Evaluation of screening, i.e., absorbance data, in concert with chemical informatics modeling led to the elucidation of physicochemical properties, particularly polarizable regions and partial charges, that governed nanoparticle formation propensities upon exposure of amino acids to ionizing radiation. NMR spectroscopy revealed key roles of amino and carboxy moieties in determining the nanoparticle formation propensity of phenylalanine, a lead amino acid from the screen. These findings were employed for fabricating radiation-responsive amino acid nanosensor gels (RANGs) based on phenylalanine and tryptophan, and efficacy of RANGs was demonstrated for predicting clinical doses of ionizing radiation in anthropomorphic thorax phantoms and in live canine patients undergoing radiotherapy. The use of biocompatible templating ligands (amino acids), rapid response, simplicity of fabrication, efficacy, ease of operation and detection, and long-lasting readout indicate several advantages of the RANG over existing detection systems for monitoring radiation in clinical radiotherapy, radiological emergencies, and trauma care.

Inorganic Nanomaterials for Soft Tissue Repair and Regeneration.

Inorganic nanomaterials have witnessed significant advances in areas of medicine including cancer therapy, imaging, and drug delivery, but their use in soft tissue repair and regeneration is in its infancy. Metallic, ceramic, and carbon allotrope nanoparticles have shown promise in facilitating tissue repair and regeneration. Inorganic nanomaterials have been employed to improve stem cell engraftment in cellular therapy, material mechanical stability in tissue repair, electrical conductivity in nerve and cardiac regeneration, adhesion strength in tissue approximation, and antibacterial capacity in wound dressings. These nanomaterials have also been used to improve or replace common surgical materials and restore functionality to damaged tissue. We provide a comprehensive overview of inorganic nanomaterials in tissue repair and regeneration, and discuss their promise and limitations for eventual translation to the clinic.

Protein-facilitated gold nanoparticle formation as indicators of ionizing radiation.

The use of X-ray radiation in radiotherapy is a common treatment for many cancers. Despite several scientific advances, determination of radiation delivered to the patient remains a challenge due to the inherent limitations of existing dosimeters including fabrication and operation. Here, we describe a colorimetric nanosensor that exhibits unique changes in color as a function of therapeutically relevant radiation dose (3-15 Gy). The nanosensor is formulated using a gold salt and maltose-binding protein as a templating agent, which upon exposure to ionizing radiation is converted to gold nanoparticles. The formation of gold nanoparticles from colorless precursor salts renders a change in color that can be observed visually. The dose-dependent multicolored response was quantified through a simple ultraviolet-visible spectrophotometer and the peak shift associated with the different colored dispersions was used as a quantitative indicator of therapeutically relevant radiation doses. The ease of fabrication, visual color changes upon exposure to ionizing radiation, and quantitative read-out demonstrates the potential of protein-facilitated biomineralization approaches to promote the development of next-generation detectors for ionizing radiation.

Rapid Soft Tissue Approximation and Repair using Laser-activated Silk Nanosealants.

Tissue approximation and repair have been conventionally performed with sutures and staples, but these means are inherently traumatic. Tissue approximation using laser-responsive nanomaterials can lead to rapid tissue sealing and repair, and is an attractive alternative to existing clinical methods. Here, we demonstrate the use of laser-activated nanosealants (LANS) with gold nanorods (GNRs) embedded in silk fibroin polypeptide matrices. The adaptability of LANS for sealing soft tissues is demonstrated using two different modalities: insoluble thin films for internal, intestinal tissue repair, and semi-soluble pastes for external repair, shown by skin repair in live mice. Laser repaired intestinal tissue held over seven times more fluid pressure than sutured intestine and also prevented bacterial leakage. Skin incisions in mice closed using LANS' showed indication of increased mechanical strength and faster repair compared to suturing. Laser-activated silk-GNR nanosealants rapidly seal soft-tissue tears and show high promise for tissue approximation and repair in trauma and routine surgery.

Parallel fabrication of macroporous scaffolds.

Scaffolds generated from naturally occurring and synthetic polymers have been investigated in several applications because of their biocompatibility and tunable chemo-mechanical properties. Existing methods for generation of 3D polymeric scaffolds typically cannot be parallelized, suffer from low throughputs, and do not allow for quick and easy removal of the fragile structures that are formed. Current molds used in hydrogel and scaffold fabrication using solvent casting and porogen leaching are often single-use and do not facilitate 3D scaffold formation in parallel. Here, we describe a simple device and related approaches for the parallel fabrication of macroporous scaffolds. This approach was employed for the generation of macroporous and non-macroporous materials in parallel, in higher throughput and allowed for easy retrieval of these 3D scaffolds once formed. In addition, macroporous scaffolds with interconnected as well as non-interconnected pores were generated, and the versatility of this approach was employed for the generation of 3D scaffolds from diverse materials including an aminoglycoside-derived cationic hydrogel ("Amikagel"), poly(lactic-co-glycolic acid) or PLGA, and collagen. Macroporous scaffolds generated using the device were investigated for plasmid DNA binding and cell loading, indicating the use of this approach for developing materials for different applications in biotechnology. Our results demonstrate that the device-based approach is a simple technology for generating scaffolds in parallel, which can enhance the toolbox of current fabrication techniques.

Correction to: Bioreducible Poly(Amino Ethers) Based mTOR siRNA Delivery for Lung Cancer.

Under the heading "Methods-Synthesis of the Bioreducible Modified-PAE (mPAE)", on page 3, line 14-17, there is an error. The quantity unit of PAE and 2-iminothiolane hydrochloride needs to be corrected to mg instead of g.

Bioreducible Poly(Amino Ethers) Based mTOR siRNA Delivery for Lung Cancer.

PURPOSE: Lung cancer is one of the leading causes of deaths in the United States, but currently available therapies for lung cancer are associated with reduced efficacy and adverse side effects. Small interfering RNA (siRNA) can knock down the expression of specific genes and result in therapeutic efficacy in lung cancer. Recently, mTOR siRNA has been shown to induce apoptosis in NSCLC cell lines but its use is limited due to poor stability in biological conditions. METHODS: In this study, we modified an aminoglyocisde-derived cationic poly (amino-ether) by introducing a thiol group using Traut's reagent to generate a bio-reducible modified-poly (amino-ether) (mPAE). The mPAE polymer was used to encapsulate mTOR siRNA by nanoprecipitation method, resulting in the formation of stable and bio-reducible nanoparticles (NPs) which possessed an average diameter of 114 nm and a surface charge of approximately +27 mV. RESULTS: The mTOR siRNA showed increased release from the mTS-mPAE NPs in the presence of 10 mM glutathione (GSH). The polymeric mTS-mPAE-NPs were also capable of efficient gene knockdown (60 and 64%) in A549 and H460 lung cancer cells, respectively without significant cytotoxicity at 30 µg/ml concentrations. The NPs also showed time-dependent cellular uptake for up to 24 h as determined using flow cytometry. Delivery of the siRNA using these NPs also resulted in significant inhibition of A549 and H460 cell proliferation in vitro, respectively. CONCLUSIONS: The results demonstrate that the mPAE polymer based NPs show strong potential for siRNA delivery to lung cancer cells. It is anticipated that future modification can help improve the efficacy of nucleic acid delivery, leading to higher inhibition of lung cancer growth in vitro and in vivo.

Spatiotemporal modeling of laser tissue soldering using photothermal nanocomposites.

OBJECTIVE: Laser tissue soldering using photothermal solders is a technology that facilitates rapid sealing using heat-induced changes in the tissue and the solder material. The solder material is made of gold nanorods embedded in a protein matrix patch that can be placed over the tissue rupture site and heated with a laser. Although laser tissue soldering is an attractive approach for surgical repair, potential photothermal damage can limit the success of this approach. Development of predictive mathematical models of photothermal effects including cell death, can lead to more efficient approaches in laser-based tissue repair. METHODS: We describe an experimental and modeling investigation into photothermal solder patches for sealing porcine and mouse cadaver intestine sections using near-infrared laser irradiation. Spatiotemporal changes in temperature were determined at the surface as well as various depths below the patch. A mathematical model, based on the finite element method, predicts the spatiotemporal temperature distribution in the patch and surrounding tissue, as well as concomitant cell death in the tissue is described. RESULTS: For both the porcine and mouse intestine systems, the model predicts temperatures that are quantitatively similar to the experimental measurements with the model predictions of temperature increase often being within a just a few degrees of experimental measurements. CONCLUSION: This mathematical model can be employed to identify optimal conditions for minimizing healthy cell death while still achieving a strong seal of the ruptured tissue using laser soldering. Lasers Surg. Med. 50:143-152, 2018. © 2017 Wiley Periodicals, Inc.

The histone deacetylase inhibitor Entinostat enhances polymer-mediated transgene expression in cancer cell lines.

Eukaryotic cells maintain an immense amount of genetic information by tightly wrapping their DNA around positively charged histones. While this strategy allows human cells to maintain more than 25,000 genes, histone binding can also block gene expression. Consequently, cells express histone acetyl transferases (HATs) to acetylate histone lysines and release DNA for transcription. Conversely, histone deacetylases (HDACs) are employed for restoring the positive charge on the histones, thereby silencing gene expression by increasing histone-DNA binding. It has previously been shown that histones bind and silence viral DNA, while hyperacetylation of histones via HDAC inhibition restores viral gene expression. In this study, we demonstrate that treatment with Entinostat, an HDAC inhibitor, enhances transgene (luciferase) expression by up to 25-fold in human prostate and murine bladder cancer cell lines when used with cationic polymers for plasmid DNA delivery. Entinostat treatment altered cell cycle progression, resulting in a significant increase in the fraction of cells present in the G0/G1 phase at low micromolar concentrations. While this moderate G0/G1 arrest disappeared at higher concentrations, a modest increase in the fraction of apoptotic cells and a decrease in cell proliferation were observed, consistent with the known anticancer effects of the drug. DNase accessibility studies revealed no significant change in plasmid transcriptional availability with Entinostat treatment. However, quantitative PCR studies indicated that Entinostat treatment, at the optimal dose for enhancing transgene expression, led to an increase in the amount of plasmid present in the nucleus in two cancer cell lines. Taken together, our results show that Entinostat enhances polymer- mediated transgene expression and can be useful in applications related to transient protein expression in mammalian cells. Biotechnol. Bioeng. 2016;113: 1345-1356. © 2015 Wiley Periodicals, Inc.

Biotemplating plasmonic nanoparticles using intact microfluidic vasculature of leaves.

Leaves are an abundant natural resource, and consist of a sophisticated microfluidic network of veins that transport nutrients and water, thereby enabling photosynthesis. Here, we simultaneously exploit the microfluidics as well as chemistry of processed leaf vasculature (venation) in order to template the in situ generation of plasmonic metal (gold and silver) nanoparticles under ambient conditions. This biotemplating approach involves capillary flow of metal salts through skeleton leaf vasculature, and does not require additional reducing agents for plasmonic nanoparticle formation. Gold nanoparticles, 30-40 nm in diameter, and silver nanoparticles, approximately 9 nm in diameter, were formed within the intact leaf vasculature using this method. Absorption spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and electron diffraction analyses were employed to ascertain the formation of nanoparticles in the leaf veins. Fourier transform infrared (FT-IR) spectroscopy was employed in order to obtain insights into functional groups responsible for formation of the plasmonic nanoparticles within the leaves. Gold nanoparticles, templated within leaves, demonstrated excellent catalytic properties, thereby imparting catalytic and plasmonic properties to the leaf itself. Furthermore, nanoparticles can be recovered from the leaves as soluble dispersions by simply combusting the organic leaf matter. Taken together, this is a simple yet powerful biotemplating approach for the generation of plasmonic nanoparticles and formation of biotic-abiotic structures for diverse, low-cost applications in sensing, catalysis, and medicine.

Design, synthesis, and functionalization of nanomaterials for therapeutic drug delivery.

Nanomaterials have the potential to solve some of the toughest challenges facing modern medicine. Their unique optical, magnetic and chemical properties at the nanoscale make them different from their macroscale counterparts. Successful application of nanomaterials can revolutionize therapeutics, diagnostics and imaging in several biomedical applications. Self-assembled amphiphilic polymeric nanoparticles have been employed to carry poorly soluble chemotherapeutic drugs. Loading of anticancer chemotherapeutic drugs into self assembled polymeric nanoparticles have shown to increase their circulation time, tumor localization and therapeutic potential. This book chapter provides an introductory discussion to organic nanotechnologies for drug delivery. Promising advances in the field of nanomedicine will be discussed and an outlook to the future will be provided.

In situ light-activated materials for skin wound healing and repair: A narrative review.

Dermal wounds are a major global health burden made worse by common comorbidities such as diabetes and infection. Appropriate wound closure relies on a highly coordinated series of cellular events, ultimately bridging tissue gaps and regenerating normal physiological structures. Wound dressings are an important component of wound care management, providing a barrier against external insults while preserving the active reparative processes underway within the wound bed. The development of wound dressings with biomaterial constituents has become an attractive design strategy due to the varied functions intrinsic in biological polymers, such as cell instructiveness, growth factor binding, antimicrobial properties, and tissue integration. Using photosensitive agents to generate crosslinked or photopolymerized dressings in situ provides an opportunity to develop dressings rapidly within the wound bed, facilitating robust adhesion to the wound bed for greater barrier protection and adaptation to irregular wound shapes. Despite the popularity of this fabrication approach, relatively few experimental wound dressings have undergone preclinical translation into animal models, limiting the overall integrity of assessing their potential as effective wound dressings. Here, we provide an up-to-date narrative review of reported photoinitiator- and wavelength-guided design strategies for in situ light activation of biomaterial dressings that have been evaluated in preclinical wound healing models.

Inflammasome modulation with P2X7 inhibitor A438079-loaded dressings for diabetic wound healing.

The inflammasome is a multiprotein complex critical for the innate immune response to injury. Inflammasome activation initiates healthy wound healing, but comorbidities with poor healing, including diabetes, exhibit pathologic, sustained activation with delayed resolution that prevents healing progression. In prior work, we reported the allosteric P2X7 antagonist A438079 inhibits extracellular ATP-evoked NLRP3 signaling by preventing ion flux, mitochondrial reactive oxygen species generation, NLRP3 assembly, mature IL-1ß release, and pyroptosis. However, the short half-life in vivo limits clinical translation of this promising molecule. Here, we develop a controlled release scaffold to deliver A438079 as an inflammasome-modulating wound dressing for applications in poorly healing wounds. We fabricated and characterized tunable thickness, long-lasting silk fibroin dressings and evaluated A438079 loading and release kinetics. We characterized A438079-loaded silk dressings in vitro by measuring IL-1ß release and inflammasome assembly by perinuclear ASC speck formation. We further evaluated the performance of A438079-loaded silk dressings in a full-thickness model of wound healing in genetically diabetic mice and observed acceleration of wound closure by 10 days post-wounding with reduced levels of IL-1ß at the wound edge. This work provides a proof-of-principle for translating pharmacologic inhibition of ATP-induced inflammation in diabetic wounds and represents a novel approach to therapeutically targeting a dysregulated mechanism in diabetic wound impairment.

Bioactive nanomaterials kickstart early repair processes and potentiate temporally modulated healing of healthy and diabetic wounds.

Slow-healing and chronic wounds represent a major global economic and medical burden, and there is significant unmet need for novel therapies which act to both accelerate wound closure and enhance biomechanical recovery of the skin. Here, we report a new approach in which bioactives that augment early stages of wound healing can kickstart and engender effective wound closure in healthy and diabetic, obese animals, and set the stage for subsequent tissue repair processes. We demonstrate that a nanomaterial dressing made of silk fibroin and gold nanorods (GNR) stimulates a pro-neutrophilic, innate immune, and controlled inflammatory wound transcriptomic response. Further, Silk-GNR, lasered into the wound bed, in combination with exogeneous histamine, accelerates early-stage processes in tissue repair leading to effective wound closure. Silk-GNR and histamine enhanced biomechanical recovery of skin, increased transient neoangiogenesis, myofibroblast activation, epithelial-to-mesenchymal transition (EMT) of keratinocytes and a pro-resolving neutrophilic immune response, which are hitherto unknown activities for these bioactives. Predictive and temporally coordinated delivery of growth factor nanoparticles that modulate later stages of tissue repair further accelerated wound closure in healthy and diabetic, obese animals. Our approach of kickstarting healing by delivering the "right bioactive at the right time" stimulates a multifactorial, pro-reparative response by augmenting endogenous healing and immunoregulatory mechanisms and highlights new targets to promote tissue repair.

Skin repair and infection control in diabetic, obese mice using bioactive laser-activated sealants.

Conventional wound approximation devices, including sutures, staples, and glues, are widely used but risk of wound dehiscence, local infection, and scarring can be exacerbated in these approaches, including in diabetic and obese individuals. This study reports the efficacy and quality of tissue repair upon photothermal sealing of full-thickness incisional skin wounds using silk fibroin-based laser-activated sealants (LASEs) containing copper chloride salt (Cu-LASE) or silver nanoprisms (AgNPr-LASE), which absorb and convert near-infrared (NIR) laser energy to heat. LASE application results in rapid and effective skin sealing in healthy, immunodeficient, as well as diabetic and obese mice. Although lower recovery of epidermal structure and function was seen with AgNPr-LASE sealing, likely because of the hyperthermia induced by laser and presence of this material in the wound space, this approach resulted in higher enhancement in recovery of skin biomechanical strength compared to sutures and Cu-LASEs in diabetic, obese mice. Histological and immunohistochemical analyses revealed that AgNPr-LASEs resulted in significantly lower neutrophil migration to the wound compared to Cu-LASEs and sutures, indicating a more muted inflammatory response. Cu-LASEs resulted in local tissue toxicity likely because of effects of copper ions as manifested in the form of a significant epidermal gap and a 'depletion zone', which was a region devoid of viable cells proximal to the wound. Compared to sutures, LASE-mediated sealing, in later stages of healing, resulted in increased angiogenesis and diminished myofibroblast activation, which can be indicative of lower scarring. AgNPr-LASE loaded with vancomycin, an antibiotic drug, significantly lowered methicillin-resistant Staphylococcus aureus (MRSA) load in a pathogen challenge model in diabetic and obese mice and also reduced post-infection inflammation of tissue compared to antibacterial sutures. Taken together, these attributes indicate that AgNPr-LASE demonstrated a more balanced quality of tissue sealing and repair in diabetic and obese mice and can be used for combating local infections, that can result in poor healing in these individuals.

Poly(aminoether)-gold nanorod assemblies for shRNA plasmid-induced gene silencing.

Gold nanorods (GNRs) have emerged as promising nanomaterials for biosensing, imaging, photothermal hyperthermia treatments, and therapeutic delivery for several diseases. We generated poly(aminoether)-GNR nanoassemblies using a layer-by-layer deposition approach based on the 1,4C-1,4Bis polymer from a library recently synthesized in our laboratory. Subtoxic concentrations of 1,4C-1,4Bis-GNR nanoassemblies were employed to deliver expression vectors that express shRNA ("shRNA plasmid") against firefly luciferase gene to knock down expression of the protein constitutively expressed in prostate cancer cells. The role of hydrodynamic size and zeta potential in determining nanoassembly mediated luciferase silencing was investigated. Finally, the theranostic potential of 1,4C-1,4Bis-GNR nanoassemblies was demonstrated using live cell two-photon induced luminescence bioimaging. Our results indicate that poly(aminoether)-GNR nanoassemblies are a promising theranostic platform for delivery of therapeutic payloads capable of simultaneous gene silencing and bioimaging.

Generation of polypeptide-templated gold nanoparticles using ionizing radiation.

Ionizing radiation, including γ rays and X-rays, are high-energy electromagnetic radiation with diverse applications in nuclear energy, astrophysics, and medicine. In this work, we describe the use of ionizing radiation and cysteine-containing elastin-like polypeptides (C(n)ELPs, where n = 2 or 12 cysteines in the polypeptide sequence) for the generation of gold nanoparticles. In the presence of C(n)ELPs, ionizing radiation doses higher than 175 Gy resulted in the formation of maroon-colored gold nanoparticle dispersions, with maximal absorbance at 520 nm, from colorless metal salts. Visible color changes were not observed in any of the control systems, indicating that ionizing radiation, gold salt solution, and C(n)ELPs were all required for nanoparticle formation. The hydrodynamic diameters of nanoparticles, determined using dynamic light scattering, were in the range of 80-150 nm, while TEM imaging indicated the formation of gold cores 10-20 nm in diameter. Interestingly, C2ELPs formed 1-2 nm diameter gold nanoparticles in the absence of radiation. Our results describe a facile method of nanoparticle formation in which nanoparticle size can be tailored based on radiation dose and C(n)ELP type. Further improvements in these polypeptide-based systems can lead to colorimetric detection of ionizing radiation in a variety of applications.

Alpha-ketoglutaric acid based polymeric particles for cutaneous wound healing.

Metabolites are not only involved in energy pathways but can also act as signaling molecules. Herein, we demonstrate that polyesters of alpha-ketoglutararte (paKG) can be generated by reacting aKG with aliphatic diols of different lengths, which release aKG in a sustained manner. paKG polymer-based microparticles generated via emulsion-evaporation technique lead to faster keratinocyte wound closures in a scratch assay test. Moreover, paKG microparticles also led to faster wound healing responses in an excisional wound model in live mice. Overall, this study shows that paKG MPs that release aKG in a sustained manner can be used to develop regenerative therapeutic responses.