In-situ vaccination using dual responsive organelle targeted nanoreactors.

The poor translation of nanomedicines from bench to bedside can be attributed to (i) lack of a delivery system with precise drug compositions with no batch-to-batch variations, (ii) off-target or undesirable release of payload, and (iii) lack of a method to monitor the fate of the specific drug of interest, which often has to be modified with a fluorescent tag or replaced with a model drug which can be tracked. To overcome these translation hurdles, we developed dual responsive organelle targeted nanoreactors (DRONEs) with precise drug composition, site specific payload release and which enable accurate in-vivo monitoring. DRONEs consist of a polyprodrug inner core composed of a dual responsive backbone containing a photosensitizer (Protoporphyrin IX) grafted with functionalized polyethylene glycol (PEG) outer shell to prolong blood circulation and a tumour homing pro-apoptotic peptide (CGKRKD[KLAKLAK]2) (THP). DRONEs can significantly reduce the tumour burden in an orthotopic glioblastoma model due to its BBB penetrating and tumour homing capabilities. DRONEs exhibit good safety profile and biocompatibility along with a reliable route of elimination. DRONEs showed great potential as an in-situ vaccine which can not only eliminate the tumour but also trigger an adaptive immune response which would provide long-term anti-tumoural immunity.

Bioengineered immunomodulatory organelle targeted nanozymes for photodynamic immunometabolic therapy.

Intelligent nanomedicines integrated with stimuli-responsive components enable on-demand customizable treatment options which would improve therapeutic outcome and reduce systemic toxicity. In this work, we explore the synergistic therapeutic potential of photodynamic therapy and immunometabolic modulation to achieve tumour regression and to trigger an adaptive immunity to prevent tumour recurrence. The therapeutic potential of the fabricated Bioengineered Immunomodulatory Organelle targeted Nanozymes (BIONs) was tested on 3D printed mini-brains which could effectively recapitulate the biologically relevant interactions between glioblastoma cells and macrophages. In the presence of glioblastoma organotypic brain slices, activated BIONs upregulated the cell surface expression of CD86, a costimulatory molecule and CD83, maturation marker, on monocyte derived dendritic cells, suggesting its ability to elicit a strong immune response. Furthermore, the antigen pulsed dendritic cells by chemotaxis and transendothelial migration readily relocate into the draining lymph node where they present the antigenic cargo to enable the proliferation of T lymphocytes. The stealth and tunable catalytic activity of BIONs prevent ROS mediated diseases such as acute kidney injury by providing environment dependent protection without compromising on its promising anti-cancer activity.

3D Printing Methyl Cellulose Hydrogel Wound Dressings with Parameter Exploration Via Computational Fluid Dynamics Simulation.

PURPOSE: To investigate and optimize the use of methyl cellulose in the fabrication of three-dimensional (3D) printed drug-loaded hydrogel wound dressings for the treatment of burns. METHOD: The effects of incorporating various salts on the properties of methyl cellulose, especially the gelation temperature was investigated for methyl cellulose to undergo gelation at skin temperature (i.e., 31.7°C). The optimized methyl cellulose and salt compositions were then loaded with various drugs beneficial for the treatment of burns. Printability and cumulative release profiles for selected drugs were then obtained, which were then fitted to common release kinetic models. Computational Fluid Dynamics (CFD) simulation was also explored to investigate the relationship between printing parameters and the hydrogel filament produced during extrusion. RESULTS: The printed hydrogels had moderate dimensional integrity, were found to be stable for up to 2 weeks and demonstrated good swelling properties. In vitro drug release studies of various drugs showed that the hydrogel was able to release various drugs within 6 h and release profiles were fitted to common in vitro drug release models, such as the Korsmeyer Peppas model and the Weibull model. While there were deviations from the actual printing process, CFD simulation was able to predict the shape of the printed structure and showed fair accuracy in determining the mass flow rate and line width of extruded hydrogels. CONCLUSIONS: Methyl cellulose hydrogels with optimized salt composition demonstrated suitable properties for a wound dressing application, revealing its potential to be used for in situ wound dressing applications.

Sewage sludge ash-based mortar as construction material: Mechanical studies, macrofouling, and marine toxicity.

Incinerated sewage sludge ash is tested here as a cement and aggregate substitute in mortar blocks. It can be used at various percentages to reduce the overall cost of production and promote ash recycling. The compressive strength of the cast blocks was tested at 28 days to determine the optimal combination of ball milled ash (replacing cement) and sewage sludge ash (replacing sand). This was compared with a control block made of cement and sand only. The cast blocks with the optimal ash formulation were tested for their flexural strength and other properties such as surface functional groups, constituent phases and porosity. The control and ash mortars exhibited similar properties. A potential application of these blocks is to use them as part of seawalls. These blocks were thus suspended in the sea for 6 months. Marine organism attachment was observed over time in both control and ash mortar blocks. There was no significant difference between the mortars after 6 months. The mortar blocks were also subjected to leaching tests (NEN-7345). The leachates did not exhibit toxicity to microalgae. In contrast, mild toxicity was observed in the sea urchin embryo development assay. Overall, the study suggests that sewage sludge ash is a potential material to be used for seawall construction as it has the desirable mechanical properties. However, there remain some residual marine toxicity concerns that need to be further addressed.

Toxicity effects of size fractions of incinerated sewage sludge bottom ash on human cell lines.

Sewage sludge bottom ash (SSBA) from the incineration plant used for the production of construction materials possibly possess heavy metals which might cause a negative impact on human health. Considering biosafety, we investigated the toxicity effects of 0.5-2 mm (aggregate substitute) and < 0.075 mm (cement substitute) in its solid and leachate form on human lung fibroblast cells (MRC-5) and human skin epidermal cells (HaCaT) on exposure through contact. MTS assay revealed the cellular responses of lung and skin cell lines to the leachates showing that the skin cells, which often interact with the external environment displayed better tolerance than the lung cells, whereas solid ash showed a concentration and size-dependent toxicity. Solid ash was found to downregulate the intracellular glutathione/superoxide dismutase activities and upregulate lactate dehydrogenase/lipid peroxidation activities thus inducing oxidative stress to the cell and subsequently resulting in the cell membrane leakage, destructive mitochondrial membrane potential (Δψm), apoptosis, and DNA damage, which is nearly 7-fold higher than the negative control. At a high concentration, DNA damage index of 1.09 and 1.29 was observed for the 0.5-2 mm sized ash leachate on skin cells and lung cells respectively, whereas for ash (<0.075 mm size) leachate, this fraction was 1.29 and 2.96, respectively. Overall, the ash leachate is found to be safer/biocompatible if they come in contact with humans as compared to SSBA in its solid form.

Fabricating scalable, personalized wound dressings with customizable drug loadings via 3D printing.

In recent times, 3D printing has been gaining traction as a fabrication platform for customizable drug dosages as a form of personalized medicine. While this has been recently demonstrated as oral dosages, there is potential to provide the same customizability and personalization as topical applications for wound healing. In this paper, the application of 3D printing to fabricate hydrogel wound dressings with customizable architectures and drug dosages was investigated. Chitosan methacrylate was synthesized and mixed with Lidocaine Hydrochloride and Levofloxacin respectively along with a photoinitiator before being used to print wound dressings of various designs. These designs were then investigated for their effect on drug release rates and profiles. Our results show the ability of 3D printing to customize drug dosages and drug release rates through co-loading different drugs at various positions and varying the thickness of drug-free layers over drug-loaded layers in the wound dressing respectively. Two scale-up approaches were also investigated for their effects on drug release rates from the wound dressing. The influence that each wound dressing design has on the release profile of drugs was also shown by fitting them with drug release kinetic models. This study thus shows the feasibility of utilizing 3D printing to fabricate wound dressings with customizable shapes, drug dosage and drug release rates that can be tuned according to the patient's requirements.

Convection enhanced delivery of light responsive antigen capturing oxygen generators for chemo-phototherapy triggered adaptive immunity.

In recent years, combination therapy has emerged as the cornerstone of clinical practice in treating glioblastoma multiforme. However, their ability to trigger and leverage the body's adaptive immunity has rarely been studied. Tumour heterogeneity, the presence of the blood-brain barrier, and an immunosuppressive tumor microenvironment play a crucial role in the 90% local tumor recurrence post-treatment. Herein, we report an improved combination therapy approach capable of stimulating an immune response that utilizes Light responsive antigen-capturing oxygen generators (LAGs). The engineered LAGs loaded with a non-genotoxic molecule, Nutlin-3a, and a photosensitizer, Protoporphyrin IX, can release the payload on-demand when exposed to light of a specific wavelength. The in-situ oxygen generation capability of LAGs enables tumor oxygenation enhancement, thereby alleviating the tumor hypoxia and enhancing the efficacy of chemo-photodynamic therapy. Furthermore, by modulating the surface properties of LAGs, we demonstrated that the tumor-derived protein antigens released can be captured and retained in-situ, which improves antigen uptake and presentation by the antigen-presenting cells. Dual drug-loaded LAGs (DD-LAGs) upregulated the expression of cell surface CD83 maturation and CD86 costimulatory markers on monocyte-derived-dendritic cells, suggesting intrinsic immune adjuvancy. In the presence of 3D printed hypoxic U87 spheroids (h-U87), DD-LAGs induced cancer cell death, upregulated IL-1ß, and downregulated IL-10 resulting in CD3+, helper CD4+, and cytotoxic CD8+ proliferation. Finally, we have investigated convection-enhanced delivery as a potential route of administration for DD-LAGs. Our work presents a novel strategy to induce tumor cell death both during and post-treatment, thereby reducing the possibility of recurrence.