Symposium SM7 : Future Healthcare Needs through Biomaterials, Bioengineering and the Cellular Building Block

Laser light has become an indispensable tool in today’s biomedical research with applications ranging from spectroscopy and in vivo imaging to biosensing and surgery. However, direct generation of laser light by or within biological samples has so far required macroscopic external resonator structures or has relied on process of random lasing.

Here, we demonstrate that microscopic whispering gallery mode (WGM) resonators doped with a fluorescent dye can generate laser light within individual, freely migrating, live cells. By using confocal fluorescence microscopy we prove that the micron-sized resonators are fully internalized by a great number of cell types via the natural process of endocytosis. On pumping the internalized WGM resonators with nanojoule light pulses, green laser emission is generated inside the cells.

Characteristic features in the lasing spectrum of our cell laser provide each cell with a unique barcode-type tag. Importantly, the spectral information can be collected by applying only a single pump pulse, thus making this technique compatible with high throughput detection schemes. To demonstrate the applicability of the intracellular laser tags, we performed cell tracking experiments over extended time periods and analysed the corresponding changes in the laser characteristics. Finally, we describe an improved method to increase the uptake efficiency of WGM resonators by non-phagocytic cells.

M. Schubert, A. Steude, P. Liehm, N. M. Kronenberg, M. Karl, E. C. Campbell, S. J. Powis, M. C. Gather, “Lasing within Live Cells Containing Intracellular Optical Microresonators for Barcode-Type Cell Tagging and Tracking”, Nano Letters 15, 5647 (2015)

Whether a cell chooses to divide, migrate or die is the result of a chemical computation typically orchestrated by protein interactions. In the cell signaling field we seek to observe these interactions, both spatially and temporally, for deeper insight into the molecular basis of cellular behavior. The discovery and application of fluorescent proteins - biologically produced labels that are covalently linked to the protein of interest - has revolutionized this endeavor. Previously undetectable proteins can now be made visible on the optical microscope with sub-second temporal resolutions and spatial resolutions that rival scanning electron microscopy. Despite their ubiquitous use, today’s fluorescent proteins are unable to address numerous critical problems in cell signaling and new, functional materials are needed to fill that void.

I will introduce a number of these roadblocks, including: the challenge of imaging cellular secretions, the question of how forces on cells affect gene expression, and the need for co-localizing nanoprobes with the cellular membrane. Interdisciplinary approaches to solving these problems, such as nanoplasmonic biosensing, magnetic particle based force actuation, functional nanopillars, and 3D lithographically patterned scaffolds will then be overviewed as examples of how non-biological materials are being adapted to provide new insights into cell signaling.

Numerous methods such as electroporation, sonoporation, and optoporation are utilized for introducing exogenous materials into cells en masse by physical membrane poration. However, these techniques are often limited by low throughputs or risk of cell damage, which raise safety concerns for their use in therapeutic applications. We recently reported a new method for ultrahigh throughput (UHT) cellular manipulation via mechanical membrane poration, i.e. UHT mechanoporation. This concept relies upon a microelectromechanical systems (MEMS) functional core composed of cell capture sites with monolithically integrated, sub-micrometer scale solid penetrators. Negative flow through aspiration vias at the bottom of the capture sites pulls cells onto the penetrators thus causing membrane poration. Cells are then released by reversing flow through the aspiration vias. The transient nature of membrane disruption enables transfection via diffusion-driven influx of exogenous molecules from the surrounding suspension, while massive parallelization provides for UHT operation (e.g. 10k capture sites in the current device).

Though our original studies validated concept feasibility, low poration efficiencies were observed (~8%). Herein, we describe our recent work on increasing the efficiency of our MEMS-based UHT mechanoporation devices. The implementation of high-resolution fluorescence imaging during device operation and precise flow rate control in the aspiration circuit has provided a means for improved characterization of our UHT devices. Through our efforts, we have been able to increase our poration efficiencies by optimizing several device parameters. We have found that improved washing steps, specific flow rates for capture (40 µl/min) and penetration (30 µl/min), and the addition of an immobilization step during our wash cycle significantly improved the proportion of porated cells in the collected population. These improved parameters thereby increased our total device efficiency nearly 5 fold (~40%). Collectively, these results have demonstrated a need for a more detailed study of device functionalities, and have helped identify directions for our ongoing optimization efforts.

Good resolution, cost effectiveness and fast batch processing makes photolithography a widely used tool in the micro-electronics industry. Since its conception, photolithography has come a long way and is not limited to the electronics sector anymore. In the last few decades it has also been explored for bio-engineering approaches like protein patterning. Patterning of proteins on a surface finds applications in proteomics, nanostructures, drug delivery and sensing. It not only enables a better understanding of the function of patterned protein, but also to study its interaction with subsequent layers of bio-molecules/cells. However, due to the fragile nature of proteins, the approaches developed so far are indirect i.e., they involve extensive surface modifications prior to patterning proteins and are protein specific. Silanization, gold coating, self-assembled monolayers etc. are a few of the surface modifications being practiced. Thus, there arises a need to develop methods to directly pattern these biomolecules in a specific orientation and without involving extensive surface chemistries.
We present a versatile and direct approach to pattern proteins using neutravidin via photolithography and subsequent lift off of the photoresist. Functionality of patterned neutravidin is confirmed by showing binding of biotinylated polystyrene beads and biotinylated antibodies. We also show that the default process steps lead to a stronger interaction between neutravidin and glass surface thereby circumventing the need of additional surface modification to provide stronger adhesion to proteins. The developed process can also be used with electron beam lithography to give feature sizes in nanometer regime and can be used to pattern biotinylated proteins with high specificity even if they are not compatible with lithography processes.

It is well known that neurons in vitro generate tension along their neurites. However, in vivo, contractility of neurons and its role in neuronal functions remain elusive. Here we use embryonic drosophila motor neurons to address this question. We dissect the embryo after 16 hrs of embryogenesis, and measure tension in single axons of motor neurons. We find that axons of motor neurons generate tension within 2 hrs of synaptogenesis. Axons actively maintain this tension, i.e., if the tension is released mechanically, axons shorten and develop the tension. If tension is increased by stretching the axons, they relax by growing until a steady tension is reached. In search of the origin of tension, we applied a series of drugs on embryonic drosophila. We concluded that axons maintain the tension by employing acto-myosin machinery. Most importantly, we find that axonal tension is essential or clustering neurotransmitter vesicles at the presynaptic terminal. If tension is released by dissecting the axon at the middle, vesicle clustering disappears. Clustering is restored by resupplying tension on the axon by a micropipette. We propose a hypothesis on the link between axonal tension and vesicle clustering. The study suggests that neuronal tension might be necessary for memory and learning in animals.

Unlike traditional materials, living cells actively generate forces at the molecular scale that change their structure and mechanical properties. This nonequilibrium activity is essential for cellular function, and drives processes such as cell division. Single molecule studies have uncovered the detailed force kinetics of isolated motor proteins in-vitro, however their behavior in-vivo has been elusive due to the complex environment inside the cell. Here, we quantify active force generation in living oocytes using in-vivo optical trapping and laser interferometry of endogenous vesicles. We integrate an experimental and theoretical framework to connect mesoscopic measurements of nonequilibrium properties to the underlying molecular-scale force kinetics. Our results show that force generation by myosin-V drives the cytoplasmic-skeleton out-of-equilibrium (at frequencies below 300 Hz) and actively softens the environment. In vivo myosin-V activity generates a force of F ~ 0.4 pN, with a power-stroke of length △x ~ 20 nm and duration τ ~ 300 µs, that drives vesicle motion at v_ν ~ 320 nm/s. This framework is widely applicable to quantify nonequilibrium properties of living cells and other soft active materials.

Overall, autologous bone grafting continues to be the gold standard for the restoration of bone defects while other practices include metallic meshes and plates. These practices are not always suitable particularly when performing reconstructive surgery in the maxillofacial region as the defects tend to be complex in terms of size and shape. These bone defect usually occur due to trauma, infection or a result of oncologic surgeries and therefore the patient requires large amount of bone grafting material [1].

There is a need for alternative methods such as is artificial bone scaffolds with regenerative medicine approaches in order to enable original tissue regeneration. In order to stimulate tissue regeneration scaffolding materials are required to have certain properties such as biocompatibility, adequate mechanical properties and internal and surface topographical features in order to provide specific biological signals to promote cell attachment and proliferation. Ideally, it would also need to be biodegradable and provide sufficient support for both the particular defect area and cellular ingrowth but degrade over time as new bone tissue is formed [2].

This work analyses the mechanical and chemical properties of Hydroxyapatite-poly(ethylene glycol) dimethacrylate (PEGDMA) and Hydroxyapatite-poly(ethylene glycol) diacrylate (PEGDA) based composites used as artificial bone scaffold material with internal structures optimized using finite element analysis (FEA) and manufactured using commercially available additive manufacturing techniques in order to develop a product that can be introduced directly into the patient. The technique allows implants to be custom made, having the right dimensions, and the right mechanical properties to have much greater control of the internal structure of the implant and the material itself.

Testing of the ceramic-hydrogel composite include mechanical testing in compression, tension, bending, impact and hardness while chemical analysis include Fourier Transform Infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC). Morphology will be analyzed using Scanning Electron Microscopy (SEM) and Laser Scanning Confocal Microscopy.

1. Götz, C., Warnke, P. H., & Kolk, A. (2015). Current and future options of regeneration methods and reconstructive surgery of the facial skeleton. Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology, 120(3), 315–23.

2. Cama, G. (2014). 1 - Calcium phosphate cements for bone regeneration. In P. Dubruel & S. Van Vlierberghe (Eds.), Biomaterials for Bone Regeneration (pp. 3–25). Woodhead Publishing.

Recent progress of cell engineering and regenerative medicine has facilitated in vitro cellular assay. The cells have interaction with flexible extracellular matrix that influences on cell proliferation and differentiation. Especially, the cellular response to mechanical stimulation is one of the most important physiological functions. To apply mechanical stimulation such as stretching, the elastic cell-culture substrates that can withstand physical deformation have been required. Here, we proposed the stretchable hydrogel-based mechanical stimulation assay sheet.¹
Stretchable double network (DN) hydrogel composed of poly(2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt) (PNaAMPS) and poly(dimethylacrylamide) (PDMAAm) was photopolymerized as previously reported.² Then, the cell-adhesive PNaAMPS was additionally photopolymerized at the surface region of the DN hydrogel (named as surface-modified double network (smDN) hydrogel) while maintaining stretchability of the original DN hydrogel.
Photopolymerization technique enables the patterning of the surface modified region on the DN hydrogel. The keratinocytes adhered only on the linear modified region of the smDN hydrogel surface. Then, the smDN hydrogel was applied to the mechanical stimulation assay of the keratinocytes stained with fluorescent Ca²⁺ indicator. The cells cultured on the smDN hydrogel showed typical Ca²⁺ response when the smDN hydrogel was stretched.

Theranostic materials that can simultaneously detect and treat diseases hold great promise for treatment of debilitating diseases like cancer. The small size of nanoparticles (NP) allows for preferential accumulation in tumors due to enhanced permeability and retention effect. Our group has developed polymer dynamic organic theranostic spheres (PolyDOTS) which are near-infrared (NIR) fluorescent and generate hyperthermia upon 800 nm laser irradiation. The formulation comprises of a NIR heat generating, semiconducting polymer poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b’]dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe) and a NIR fluorescent polymer poly[(9,9-dihexylfluorene)-co-2,1,3-benzothiadiazole-co-4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole] (PFBTDBT10). The therapeutic formulation was optimized to achieve the highest thermal and fluorescence quantum efficiency. The PolyDOTS are spherical with hydrodynamic diameter of 120 nm and zeta potential of -16.8 mV. The NP showed excellent colloidal stability in acidic (pH 4), basic (pH 10), high salt buffer (10X PBS) as well as serum (10% FBS) conditions for over a month. The in vitro efficacy of photothermal ablation (PTA) therapy with the PolyDOTS was evaluated in non-tumorigenic mouse epithelial cells (Balb/C.CL7) as well as two types of mouse breast cancer cell lines, 4T1 and E0771 using various laser parameters and NP concentrations. An in vivo model utilized luciferase transfected murine breast cancer cells (4T1) injected into the mammary fat pad of Balb/C mice. A 100 µL NP solution at 0.1mg/mL concentration in PBS was injected into the tail-vein. The fluorescence imaging was conducted at 0, 6, and 24 h to determine optimum time for NP accumulation in a tumor. The tumors were then treated with NIR laser (K-laser 800 nm, 2W/2min) 24 h after NP injection and a response to PTA was monitored by measuring tumor size twice a week over forty days. In addition to NP + laser treatment group, NP + no laser, PBS + laser and PBS + no laser served as control groups. Our results indicated significant decrease in tumor size as well as significant increase in overall survival in NP + laser group compared to control groups. Due to preferential tumor accumulation, PolyDOTS can be of great use to highlight solid tumors, tumor margins and micro-metastasis during surgery.

We present experimental and theoretical results of the adhesion and entry of magnetite nanoparticles (MNP) and MNP/cancer drug (paclitaxel and prodigiosin) complexes into MDA-MB-231 breast cancer cells. The adhesion between luteinizing hormone releasing hormone (LHRH) and breast cancer cells is studied using atomic force microscopy (AFM) technique. The results present that the adhesion force between LHRH coated AFM tips and MDA-MB-231 breast cancer cells is about twice as much as that between bare AFM tips and breast caner cells; the adhesion force between LHRH-MNP coated AFM tips is also approximately twice as much as that between MNP coated AFM tips and breast cancer cells. The increased adhesion from LHRH coated tips elucidate that LHRH can be used for breast cancer targeting. Similarly, the adhesion between the constituents of MNP/cancer drug nanoparticle complexes is elucidated via atomic force microscopy. Finally, the receptor-mediated entry of nanoparticles into breast cancer cells is investigated using thermodynamics and kinetics models. The predictions are shown to be in good agreement with both in-vitro and in-vivo experimental observations of nanoparticle entry into cells.

A perceived disadvantage of nanoparticle-based therapeutics is the inability of the nanoconstructs to escape from vesicles and thus induce a biological response. This talk will describe how this challenge can be overcome by both targeting lysosomes and exploiting lysosomal degradation of the biomarkers by aptamer-loaded gold nanostars. Gold nanostar particles are advantageous as nano-carriers because they are anisotropic in shape and facile carriers of bio-functional ligands. As a model system, we focused on targeting HER2 as the well-known biomarker and transmembrane protein in breast cancer cells and designed gold nanostar constructs based on anti-HER2 aptamers grafted to gold nanostars (HApt-AuNS). We found that HApt-AuNS showed improved delivery and anti-cancer effects in targeted cancer cells over free HApt. In order to correlate progression in the endocytosis process with cellular response, we monitored the sub-cellular localization of HER2-HApt-AuNS complexes using confocal fluorescence microscopy and differential interference contrast microscopy. At the same time points, we measured accumulation of HER2-HApt-AuNS complexes in lysosomes, lysosomal activity, and lysosomal degradation of HER2 and discovered that all were positively correlated.

Ovarian cancer is one of the most challenging malignancies to diagnose and treat, although it is relatively less prevalent, as the 7^th most common form of cancer in women. The survival rates for women diagnosed with this disease has barely changed since the advent of platinum-based therapies in the past 30 years, with a median 5-year survival < 30% in patients diagnosed with late-stage (Stage III or IV) metastatic ovarian cancer. The standard clinical approach to disease management involves a first step of tumor removal surgery, followed by a second step of combination chemotherapy. There has been growing evidence that the amount of residual disease remaining after surgery is an important prognostic variable in predicting long-term survival of the patients. Therefore, microscopic residual tumor detection during surgery represents a critical challenge in the successful treatment of this disease. Current gold-standard clinical imaging modalities, such as contrast-enhanced computed tomography (CT), offer poor sensitivity < 10%, when the tumor size < 1 cm. In this work, we present a first approach utilizing a combination of near-infrared (NIR) fluorescent, targeted nanomolecular probes, along with a custom-designed real-time intraoperative system for image-guided surgical debulking, resulting in enhanced survival. We achieve this using single-walled carbon nanotubes (SWNTs) as NIR-fluorescent contrast agents, functionalized and aqueous-dispersed using M13 bacteriophage, along with a targeting peptide against secreted protein, acidic and rich in cysteine (SPARC), which is overexpressed in highly-invasive ovarian cancers. We demonstrate that our SWNT-based probe is very highly sensitive, with millimeter- to sub-mm scale resolution, at ~ 93 ± 4% true positive rate, with moderately good specificity ~ 67 ± 6%. In an orthotopic ovarian cancer mouse model, using our image-guided surgery we report an enhancement in median survival by > 40%, compared to the control group receiving visible eye-only (non-guided) surgery. These results provide hope for the deployment of fluorescence imaging in the clinical setting using SWNT-based probes, with the potential for making a positive societal impact by extending the lifespan of patients affected by late-stage metastatic cancers.

Bladder cancer is the costliest cancer to treat because about 70% of patients with superficial disease will develop tumor recurrence, thus requiring long-term follow-up and repeat interventions. The current standard therapy for non-muscle invasive bladder cancer is surgical transurethral resection followed by intravesical immunotherapy or chemotherapy. However, the success of intravesical chemotherapy is limited due to the bladder permeability barrier and periodic voiding of urine. We have formulated dual drug-loaded mucoadhesive nanocapsules to promote interaction with the urothelium in order to provide sustained release of the drug locally and increase drug residence time in the bladder. These nanocapsules were prepared from chitosan and methacrylic acid, and have an average size of ~100-200 nm and zeta potential of +15 mV. Experiments with ex vivo porcine bladder confirmed that these nanocapsules attach strongly on the luminal surface of the bladder. The vesicular structure of the nanocapsules allows doxorubicin (Dox) and peptide-modified cisplatin (Pt-ALy) to be co-loaded to ~ 50% of the initial nanocapsule weight. These dual drug-loaded nanocapsules were readily taken in by UMUC3 bladder cancer cells, and the in vitro killing efficacy is much higher (with 5 to 16 times lower IC₅₀) than either Dox- or Pt-ALy-loaded nanocapsules. Analysis using the Chou and Talalay method showed that these two drugs work in synergy when co-delivered in the nanocapsules. Thus, these mucoadhesive nanocapsules capable of simultaneous delivery of a "two-in-one" synergistic drug combination to bladder cancer cells provide a promising avenue for increasing therapeutic efficacy in intravesical chemotherapy.

L-cysteine capped Mn doped ZnS quantum dots (L-cys-ZnS:Mn QDs) is prepared via a wet chemical approach. The QDs display a prominent orange emission band peaking at ~598 nm, when exposed to 325-nm light. Since their room-temperature phosphorescence is efficiently quenched by dopamine (DA), they have been employed as phosphorescence probe for detecting DA. The linear working range and limit of detection of L-cys-ZnS:Mn QDs are ~2.5–37.5 and ~0.72, respectively. The possible quenching mechanism has been discussed in detail. The QDs probe is highly selective to DA over other common ions, amino acids, glucose and bovine serum album. Finally, they have been successfully applied for detection of DA in human urine samples with recoveries as high as 98–104%. Our work provides a simple and convenient phosphorescence method to determine DA in real samples.

Nanoparticles prepared from whey protein isolate can be used for encapsulation and sustained delivery of β-carotene and zinc ion. The loaded whey protein nanoparticles were prepared by pH cycle method at aggregation pH 6 and 22 h ageing time in the presence of calcium ion. Morphology, particle size, stability and releasing profile at neutral and acidic conditions of the loaded whey protein nanoparticles were evaluated. The results illustrated that whey protein nanoparticles were able to encapsulate β-carotene molecules in around 400 nm spherical compact structure that can protect sensitive β-carotene and limits its susceptibility to auto-oxidation reactions when compared to control sample. Moreover, Whey protein nanoparticles showed higher release profile at neutral condition compared to acidic condition even in the presence of proteo-lytic enzymes. These findings confirmed that whey protein nanoparticles can be used to encapsulate, protect and enhance the release profile of sensitive biological active ingredients and micronutrients to be used for food as well as pharmaceutical applications.

Macrophages (MΦ) are functionally heterogeneous cells with different phenotypes representing distinct sublineages. In general, the heterogeneity of MΦ can be described with M1 or M2 classifications. Classically activated, M1, MΦs, are known to be pro-inflammatory and cytotoxic as part of the type 1 T helper (Th1) response.¹ They are activated by microbial stimuli, such as lipopolysaccharide (LPS). Alternatively activated, M2 MΦs can be induced by interleukin (IL)-4. Tumor-associated MΦs (TAMs) are of the M2 pathway and promote tumor growth through the release of angiogenic molecules.² Polymeric systems can potentially be used to reprogram TAMs to produce pro-inflammatory molecules or to deliver anti-cancer therapeutics to tumors.
Understanding how material parameters influence particle internalization for different macrophage phenotypes is important for targeted delivery to specific cell populations. We have investigated the effects of a number of material characteristics on particle internalization for pro-inflammatory, pro-angiogenic, and naïve macrophages using biopolymers (~600nm), functionalized with 13 different molecules. Viability assays showed that these materials had >80% cytocompatibility. Markers of the Th1 response – tumor necrosis factor-α (TNF-α) – and the Th2 response – IL-10 – were monitored and used to determine the ability of the materials to alter MΦ phenotype. In this work, we demonstrate that both initial cell phenotype and material functionality are important for inducing shifts in polarization and for the phagocytic capacity of MΦs.
Determining the in vivo effects of these materials is also important for long term applications of these biopolymers. SKH1-E mice were injected with 10 w/v% of functionalized particles (n=5). Prosense 680, a fluorescence probe was used to assess cathepsin activity, an indicator of inflammation, on days 1, 3, and 7. Tissue sections (n=2) and tissue samples (n=3) were collected for each material. H & E staining, immunohistochemistry (IHC) for CD206, and IHC for F480/CD40 was performed on the tissue sections. The production of tumor necrosis factor α, IL-10, and reactive nitrogen intermediates were examined. Arginase activity, a measure of M2 MΦs, was also studied.
Here, we show how different biomaterials affect both M1 and M2 MΦs resulting in different cytokines and phagocytosis levels. The ability to control polarization or to target delivery to specific MΦ phenotypes has the potential to vertically impact cancer therapy and tissue engineering.

References:
1. Gordon, S. Alternative activation of macrophages. Nat. Rev. Immunol. 3, 23–35 (2003).
2. Schmid, M. C. & Varner, J. A. Myeloid cells in the tumor microenvironment: modulation of tumor angiogenesis and tumor inflammation. J. Oncol. 2010, 201026 (2010).

Dengue fever is a tropical and sub-tropical area disease spread by mosquitoes. Severe dengue fever leads to critical state of shock that can cause within 12 to 24 hours. We report on the design of a sensitive dengue biosensor based on impedimetric detection of nucleic acids. Electrochemical impedance spectroscopy (EIS) can accurately detect an analyte by measuring a change in surface resistance properties upon analyte-electrode material binding events. We fabricated a composite of 3-Aminopropyltriethoxysilane (APTES) functionalized graphene oxide (APTES-GO) wrapped on SiO2 particles (SiO2@APTES-GO) as electrode material and further used it to detect dengue DNA and RNA. The resulted biosensors was able to detect 1 femto-molar dengue DNA and RNA using a different nucleic acid primer for each target. The superior performance of SiO2@APTES-GO as electrode material is due to an enhanced specific surface available for detection derived from the three-dimensional structure, the electrical properties of grapheme oxide, and the ability for rapid hybridization of grapheme oxide-based material.

In recent years, lipid-polymer hybrid nanoparticles have gain attention as an efficient drug delivery device to treat various diseases, including cardiovascular disease, tuberculosis, and cancer. Functionalized lipid polymer nanoparticles are ideal drug delivery devices as it can elute desired concentrations of drug at targeted areas of the body. Nanoprecipitation and self assembly of lipid polymer particles is a common method to synthesize drug encapsulated nanoparticles in a low cost manner. However, the multi-step process of this synthesis method causes difficulty in consistently producing uniformly sized nanoparticles. Here we developed a microfluidic device that utilizes a three channel pathway and mixer channel to produce uniformly sized lipid polymer nanoparticles in a controlled manner. Dynamic light scattering results of the microfluidic synthesized nanoparticles show uniformity in size within the range of 100 to 150 nm in diameter. The time required to produce 5 mL of lipid polymer nanoparticles with 1mg/mL concentration has decreased by 10 folds using the microfluidic device. The microfluidics device can be customized to synthesize nanoparticles of different size, different encapsulated drug, and different surface functionalization. The production of higher quality nanoparticles in an efficient manner using our microfluidics device can expedite the research and development process of drug delivering lipid polymer nanoparticles.

Middle East respiratory syndrome (MERS) is a viral respiratory disease caused by a coronavirus. About 3 to 4 out of 10 MERS patients have died. A novel application of rolling circle amplification(RCA) as a versatile biosensor for MERS RNA pathogen detection is introduced. Following WHO indication, a simple and robust microfluidic array chip assay has been developed based on RCA of adaptor templates upon the hybridization of MERS pathogens. Long single strands of RCA products can be visualized by SYBR gold staining. When a biological sample containing MERS pathogen is applied, RCA of the DNA template can be achieved and can be visualized by under a UV lamp. In contrast, if there is no MERS pathogen in sample, no fluorescence signal can be seen. This assay shows high sensitivity and potent ability to detect various disease according to design of DNA template. Furthermore, this assay doesn’t need reverse transcription step and all process is at room temperature. It makes special equipment such as thermal cycler unnecessary contrast to PCR method. It is likely that the development of this facile and sensitive MERS pathogen detection assay may relieve excessive workloads.

Exosomes are nanovesicles secreted by cells that contain the molecular components of that cell. Exosomes are specific to their parent cells and are secreted into biological fluids such as urine and blood; making them potential biomarkers for an alternative non-invasive diagnostic test for prostate cancer. Raman spectroscopy detects the intensity of inelastically scattered light at various frequencies. A Raman spectrum provides detailed chemical and structural information that can be used to identify the molecular components of a sample. However, the ability of Raman spectroscopy to detect prostate cancer biomarkers in exosomes is still unclear. Here we show that Raman spectroscopy can be used to distinguish PC3 metastatic prostate cell line derived exosomes from non-PC3 macrophage derived exosomes. PC3 exosomes are expected to have a significant portion of CDCP1, CD9, CD81, and HSP 90 Heat Shock protein biomarkers. PC3 and non-PC3 derived exosomes were characterized using Raman spectroscopy at wavelengths of 532 nm and 1064 nm. Transmission electron microscopy (TEM) and Dynamic light scattering (DLS) were also performed to analyze the physical characteristics of both PC3 and non-PC3 exosomes. PC3 exosomes were characterized by three high intensity shifts corresponding to CDCP1, HSP90 and CD81. The spectrum associated with PC3 consisted of Raman shifts of 2932, 1655, and 1440 cm-1, with the simultaneous absence of peaks at 1089 and 552 cm-1. The non-PC3 exosomes were differentiated by peaks at 1089 and 522 cm-1. The TEM micrographs demonstrated that the exosome structure varies from pseudospherical to quadrilateral. The hydrodynamic diameter of exosomes was found to be 118nm using DLS. The previously identified prostate cancer biomarkers: CDCP1 protein, HSP90 Heat Shock protein, and CD81 protein; were confirmed to be good biomarkers for use in the characterization of PC3 derived exosomes. The use of exosomes as prostate cancer specific biomarkers could provide an alternative to the existing Prostate Specific Antigen (PSA) test for the screening and surveillance of prostate cancer. Since exosomes are secreted into the urinary tract system, exosomes in urine have potential as a noninvasive screening biomarker for future studies.

In cancer treatment strategies, hydrophobic chemotherapeutic compounds, such as podophyllotoxin (PODO) and its analogues are largely ignored despite their potency due to the difficulty of incorporating hydrophobic molecules into drug delivery systems. This solubility hurdle therefore necessitates development of novel delivery strategies to utilize these highly potent compounds in clinical applications. In an effort to develop such a strategy, this study focuses on conjugating PODO to phospholipid tails to promote their incorporation into liposome (lipid bilayer) and microbubble (lipid monolayer) phospholipid carriers. Using a steglich esterification reaction PODO was conjugated with phospholipid tails with a ~66% (PODO) yield, generating 2T-PODO prodrug. Particle size analysis of 2T-PODO prodrug loaded liposomes indicates 20 mol% prodrug loaded liposomes maintain a stable size distribution at least one week post generation, indicating formulation stability. Differential scanning calorimetry data generated for lipid films showed a decrease in phase transition as the concentration was increased, indicating successful incorporation of the drug conjugates. Using an MTT assay, the in-vitro toxicity (IC50) of 2T-PODO against HeLa (human cervical cancer) was 11±0.54uM. Preliminary ultrasound experiments show local delivery of prodrug loaded MBs to a specific target area. Further ultrasound and synthesis experiments are in progress to validate and explore these findings.

Hydrogel composites are found to be excellent materials for antibacterial applications. Diatomite was used as raw material for synthesizing a novel diatomite-poly (acrylic acid) (DT/PAA) hydrogel composite by graft polymerization in aqueous solution. The effect of diatomite on water absorbency was discussed and the highest water absorbency was obtained when the amount of diatomite in the feed was 25%. The diatomite-poly (acrylic acid) (DT/PAA) hydrogel composite was characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). The bioactive compound of Atractylodes lancea was loaded into the hydrogel composite. The influences of oil concentration, pH, cations and anions on equilibrium water absorbency of DT/PAA were investigated. The results showed the concentration of bioactive compound had strong effect on the swelling behaviors of DT/PAA.

New techniques for mitigating bacterial infections in tissue are needed in an effort to augment the delivery of antibiotics and support immune responses. Application of heat is one method which can be useful for either complete eradication of disease or as an adjuvant therapy. This report describes the development of hybrid polymer nanoparticles comprised of Poly(3-hexylthiophene-2,5-diyl) (P3HT) with Poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt−2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe) for photothermal ablation (PTA) of gram positive Streptococcus pyogenes (GAS) and Staphylococcus aureus (SA) bacteria. PCPDTBSe/P3HT nanoparticles were coated with the biopolymer, chitosan, to develop a positively charged nanoparticle that can bind to the negatively charged cell wall. The nanoparticles were characterized by FTIR, UV-Vis spectroscopy, dynamic light scattering and transmission electron microscopy to characterize their chemical, optical and size properties. FTIR confirms that the nanoparticles consist of a homogenous polymer blend with a size of approximately 130nm. Heating curves of increasing concentrations of the nanoparticle demonstrated the most variation in temperature occurred between 0µg/mL and 50µg/mL, making this the optimum range to see to differences in bacterial cell kill. The minimum inhibitory concentration (MIC) of the nanoparticles was measured to determine the appropriate working concentration (50µg/mL). PTA was performed, using a 5W, 800nm infrared laser for one minute to bring the PCPDTBSe /P3HT nanoparticles to temperatures in excess of 70^oC. The log reduction of S. aureus was quantified using fluorescence assays and colony forming unit measurement. Microscopic observation of SA and GAS, stained green, incubated with nanoparticles which exhibit red fluorescence, showed the nanoparticles attached to the bacterium. PTA with chitosan coated PCPDTBSe/P3HT nanoparticles was able to drastically reduce the bacterial load in both species. The use of chitosan coated PCPDTBSe/P3HT nanoparticles in PTA therapy is a promising alternative to pharmacotherapeutic treatments of infection.

With applications ranging from food packaging to wound dressings to local drug delivery, polymeric films and coatings have been used in industry and investigated in the lab for decades. Processing techniques on well-chosen materials can yield mechanically stable films with a wide range of diffusion, degradation, and surface energy characteristics that are flexible enough to conform to most contours. Petroleum-based polymers such as the widely used and various PLGA species often require harsh chemicals to process which are difficult or impossible to completely remove, which is undesirable for applications in the food and medical industries. Furthermore, recent cultural shifts in environmental attitudes toward green, renewable materials may cause both consumers and investors to shy away from petroleum-based products. In light of these factors, both industrial and academic researchers have been looking to ‘natural’ polymers as possible alternative materials. Here we present a fabrication method for producing free-standing agarose films.

Agarose is the polysaccharide derived from certain forms of red algae whose gelatin agar is used ubiquitously in microbiology laboratories. Our process begins by boiling agarose in water and allowing the solution to cool in a beaker until a gel disk forms. The gel disk is then placed under vacuum at room temperature until the gel is completely dehydrated. The diameter and thickness of the resulting film may be tuned by allowing the solution to gel in a larger beaker and adjusting the volume of the gel disk at the outset. A 3-mm-thick gel disk results in a film approximately 100 µm thick, while the diameter does not change during dehydration. The mechanical properties may be tuned by adding polyethylene glycol (PEG) to the original solution. Increasing the amount of PEG yields a film that is more flexible with more elongation under stress. Cargo may be added to the films by placing a small amount of aqueous solution containing the desired cargo to the original gel before dehydration and allowing the gel to soak up the cargo in a sponge-like manner. The release rate of the cargo is tunable by the introduction of the polysaccharide chitosan to the gel. We demonstrate this ability by altering the release rate of fluorescein into water from minutes to hours. Additionally, we show the possibility of using the films as a wound dressing by producing antibiotic-loaded films that inhibit bacterial growth. Furthermore, multi-stage release is possible by producing multi-layered films, which is demonstrated through an experiment in which a quick burst of fluorescein is followed by slow, sustained release.

The endogenous electric field plays an important role in accomplishing various functions including communication with brain and with different parts of the physiological system, wound healing, and cellular functions. Moreover, the endogenous electric field can be modified using external electric field to induce changes in cell functionality. Given that the advantage of dynamic flow of media on cell growth in tissue engineering, the objective of the study is to elucidate the effect of media flow (dynamic condition) on osteoblast functions at pulsed DC (direct current) electric field of strength of 0.5-1 V/cm and compared with the static condition (no flow of media and in the presence of electric field). It is underscored that the biological functionality is favorably altered in the presence of electrical field in dynamic condition with consequent effect on cell proliferation, growth, and expression level of prominent proteins, actin and vinculin.




Key words: Electric field; Dynamic culture; Tissue engineering; Bone growth

Biomaterials found in vertebrate tissue and skeletal systems are composites formed by combining collagens with a variety of other proteins and polysaccharides associated with the collagens. Here, we fabricated a bio-inspired scaffold mimicking natural extracellular matrix (ECM) for bone regeneration including gelatin which is denatured type I collagen and polysaccharide such as agarose, alginate or chitosan which provides structural integrity to form stable structures. In addition, inspired by the composition of adhesive proteins in mussels, we used oxidized diphenol self-polymerization for cross-linking of mixed materials. Tyramine functionalized gelatin, polysaccharide (PS) and silicatein-like protein (SLP) were mixed together and were cross-linked between oxidized tyramines that are conjugated to gelatin and/or modified surface tyrosine of SLP by horse radish peroxidase in the presence of hydrogen peroxide. Bio-silicification of hydrogel was mediated by hydrogel-immobilized SLC. Cell viability, mineral deposition and alkaline phosphatase activity were assessed. The cross-linked and mineralized composite maintained the high cell viability and increased synthesis of mineral deposition compared to gelatin alone. The increased stiffness and adhesiveness of scaffold and osteogenic biosilica enhanced both osteogenic cell viability and mineralization. Hence, this composite can be applied to not only bone tissue but also other biomedical applications including cartilage.

The advent of induced pluripotent stem cells (iPSCs) has significant potential as patient-specific disease models. However, even state-of-the-art methods still produce a heterogeneous population of mixed cell types as well as functional maturity. This heterogeneity and potential tumorgenicity severely limits iPSC utility. However, the sequence of expression and epi-genetic factors that cause a cell to evolve along a particular developmental pathway (lineage) are not well understood. Improved understanding of the factors that drive lineage progression at a single-cell level would allow design and feedback to guide cells along the desired pathway. While numerous mRNA analyses of single cells and cell populations at different development time points have been performed, these are fundamentally impaired because the analysis is destructive. Without the ability to watch the same cell over time, it is never possible to determine the temporal sequence of expression and protein activity that lead to the final phenotype.
Here we describe a new technology for direct, non-destructive cytosolic cell access to sample from the same cell or population of cells over an extended time period. Inorganic “nanostraws” were developed for direct delivery of reagents into the cell over the past several years. Based on the fact that these nanostraws enable material to directly diffuse into the cell cytoplasm, we reasoned that material from the cell should also be able to diffuse out when the membrane is temporarily permeated with a mild electric field. Our data for the CHO cell line shows that intracellular contents from single cells to thousands of cells can be non-destructively sampled and analyzed multiple times over the course of one week, a feat which has not been previously possible. This advance will enable new studies of how cells temporally evolve within fully interconnected cell monolayers in response to different therapies and differentiation conditions.

Nanopore ion transistor is an electrofluidic element conceived to manipulate the transport of molecular species through nanopores using electric fields. In this work, we report a newly discovered diode circuit element existing in the electrolyte-oxide interface, and its importance for electrofluidic gating in ion transistors. We built sub-20 nm nanopore ion transistor devices and characterized ionic transport of KCl electrolytes. Simultaneous monitoring of electric currents of source (Is), drain (Id) and gate probes (Ig), allowed us to characterize both ionic and interfacial transports, as a function of gate voltage (Vg). Ionic transport through the nanopore was modulated such that negative (-) gate voltage bias induced an increase of ionic current, representing p-type transport, suggesting that the majority carrier contributing to ionic transport is positively charged potassium ion (K+) which screens the surface charges of pore wall. Interestingly, a characterization of Ig showed the presence of a diode circuit element in the electrolyte-oxide interface: The electric field created by negative (-) gate voltage bias reaches the electrolyte more effectively than that created by a positive (+) gate voltage bias, thus leading more efficient electrical-control over ionic transport. This diode functionality in electrolyte-oxide interface results in the unipolar transport of ion transistors. Based on the analysis of secondary ion mass spectrometry (SIMS) of the gate oxide layer, we suggest that the diode functionality is attributed to the diffusion of permeable potassium ions into the gate oxide, driven by negative (-) gate voltage biases. Our interpretation of the electrolyte-oxide interfacial effect clarifies electrofluidic gating behavior in ion transistors.

Portable, robust detection systems capable of real-time identification of hazardous chemical and biological agents are needed for diagnostic, environmental monitoring, and security applications. Living cell–based sensors have proven effective as sensitive, specific, near real-time detectors; however, living cell-based sensors require frequent cell replenishment due to cell sensitivity to the ex-vivo environment, limiting sensor stability. In an effort to stabilize cells for sensor development, researchers have encapsulated various microbial cells in silica matrices accessed via the sol-gel process generating bio-nano interphases which protect cells from harsh ex-vivo conditions. While robust, traditional encapsulation strategies focus on encapsulation of planktonic cell phenotypes in either bulk silica gels or single cell silica coatings and are not easily tuned. In an effort to develop an encapsulation strategy with facile tunability of the encapsulating silica sol that allows for encapsulation of planktonic and/or natural multi-cellular communities, (i.e. yeast flocs), we explored the use of a novel Sol-Generating Chemical Vapor into Liquid (SG-CViL) process. In SG-CViL, the high vapor pressure of tetramethyl orthosilicate (TMOS) is utilized to deliver silica into an aqueous medium, creating a silica sol. By varying SG-CViL deposition time, temperature, and pH, we demonstrate tunable nanometer scale silica deposition in a liposome model system using dynamic light scattering. Employing biocompatible SG-CViL parameters, we encapsulated both non-flocculated (i.e. planktonic cells) and flocculated (i.e. multi-cellular flocs) Saccharomyces cerevisiae phenotypes in silica. Fluorescence microscopy shows Saccharomyces cerevisiae maintain their pre-encapsulation phenotype (i.e. non-flocculated vs. flocculated) post silica encapsulation and are approximately 45% viable after 21 days of storage under non-ideal storage conditions. Ultimately, these data demonstrate SG-CViL is a process whereby SG-CViL generated cell-inorganic interfaces can be tuned at both a material level (inorganic component) and a phenotypic level (cell component), leading to development of biosensing components with diverse structural and functional characteristics.

Human sweat contains plethora of information that can be utilized for successful diagnostics of several disease conditions. Cortisol is an established biomarker for estimation of stress levels and glucose is well known biomarker for diabetic conditions. Cortisol is a glucocorticoid hormone that also regulates carbohydrate metabolism. It enhances the expression of enzymes involved in glucogenesis to maintain the glucose levels in plasma. It would be beneficial to simultaneously monitor the sweat levels of these biomarkers for improved health management of diabetic patients. The rising interest in wearable sensors is coherent with growing popularity of personal health management than centralized hospitalized patient care. This is mainly with an intention of reduced health-care costs that can be availed using personal health monitoring. We demonstrate the development of a non-invasive, combinatorial and non-faradiac electrochemical biosensor employing flexible bioelectronics for simultaneous detection of cortisol and glucose from synthetic sweat. Pulsed laser deposition method was used to deposit zinc oxide thin films on flexible substrates. Electrodes were patterned and fabricated to form a zinc oxide device on flexible substrates. ZnO surface was functionalized with Dithiobis succinimidyl propionate (DSP) linker. The α-cortisol and glucose oxidase antibodies were used on two different sensors deposited on same substrate to setup the biosensor immunoassay. The concentration of cortisol was tested in the range of 1 ng/mL to 100 ng/mL and 10 µg/mL to 1mg/mL for glucose. The concentrations for glucose and cortisol were spiked in synthetic sweat simultaneously in 3 logarithmic steps. These ranges correspond to the clinically relevant range of biomarkers observed in human sweat. Electrochemical impedance spectroscopy was used to measure the impedance changes observed due to presence of varying biomarker concentrations. In order to analyze the combinatorial detection of glucose and cortisol, EIS measurements obtained due to the presence of both biomarkers were captured from both cortisol antibody functionalized and glucose oxidase antibody functionalized sensors. The individual calibration response of independent detection of glucose and cortisol were first fixed in synthetic sweat using EIS. These calibrations of individual biomarker were referred to as the perfect match. Calibration of combinatorial sensing of glucose and cortisol was achieved by comparing the combinatorial impedance changes to the individual perfect match ranges for corresponding biomarker concentrations. It was verified whether after combining the two biomarkers together in synthetic sweat, the change in impedance was within 15% variation of individual dynamic range for each biomarker. This biosensor utilized low sample volumes (less than 10μL) for ultra-sensitive and label free detection of biomolecules, making it suitable for wearable diagnostics.

Nanoparticles (NPs) are increasingly utilized to locate and optimize cell-based therapeutics. Existing NP agents (e.g. Iron oxide, quantum dots etc.) have been extensively used to reveal cell biodistribution following therapeutic administration. Nonetheless, their utilization is still far from perfect since the state-of-art consists of NPs that emit passive signals. These are limited by their failure to provide information regarding therapeutic cell status.

Currently, incorporating reporter genes through genomic integration is the most popular method for non-invasive detection of cellular behavior. However, establishing such system in therapeutic cells is highly inefficient, laborious, and accompanied by risk of introducing random mutations. Furthermore, detecting a novel molecular target requires a lengthy procedure to identify and create suitable clones. Developing a convenient, safe and integration-free technology to non-invasively examine molecular attributes of live cells is a clear priority.

Here we have developed a versatile nanosensor platform to probe various cellular functions such as cell differentiation. This platform consists of FDA-approved poly(lactic-co-glycolic acid) NPs which packages and achieves sustained intracellular delivery of oligonucleotide-based sensor probes (aptamers). These aptamers have high recognition specificity to its complementary target sequence, based on preferential hybridization. Initially, aptamers retain a hairpin structure with minimal fluorescence. Upon successful hybridization however, it forms a linear structure which restores its pre-quenched fluorescence. Thus, aptamers can be designed to non-invasively report the expression level of any target mRNA within live cells.

The performance of nanosensors was first validated using ubiquitously expressed β-actin as a target. As compared to the signal from bolus delivery (using Streptolysin-O toxin to reversibly induce pores) which declined significantly after 24hr, nanosensor signal was retained for ≥8 days. Then, this platform was used to track the osteogenic differentiation of mesenchymal stem cells (MSCs) by encapsulating two kinds of aptamers that target alkaline phosphatase (ALP) mRNA, an early osteogenic biomarker and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, a reference biomarker respectively. By monitoring the change in signal ratio between ALP and GAPDH aptamers, we successfully visualized early to late-stage osteogenic differentiation of MSCs. Crucially, observed signal correlated well with quantitative polymerase chain reaction.

A non-invasive, integration-free, nanosensor platform to probe intracellular gene expression in live cells is described herewith. It is a highly versatile platform, yet is highly specific and compatible for multiplexed monitoring of cells and other complex culture systems. We anticipate this nanosensor platform to play a vital role in cell tracking especially for optimizing cell-based therapeutics.

With the growing drive to interface biology with electronics, electrochemical (EC) techniques have emerged as a natural choice for bridging this divide. We have previously demonstrated such an approach in a lab-on-a-chip targeting clozapine (CLZ), the optimal antipsychotic for patients suffering from treatment-resistant schizophrenia. In this work, we investigate the impact of serum protein binding on the accuracy of in vitro EC testing of CLZ. For this mental health drug, we find that only the unbound drug is sensed by EC methods. This aids understanding the therapeutic availability of drug in patient’s blood, which is critical for managing symptoms and ensuring treatment efficacy. Therapeutic drug availability may be impacted by the presence of other molecules in circulation, and these interactions with the drug have yet to be understood thoroughly.
Like many therapeutics, CLZ becomes ineffective when forming a bound complex with native blood proteins, such as serum albumin (SA) or alpha-1 acid-glycoprotein (AAG). However, little is known about the amount of protein binding or the EC activity of this complex, as current detection methods cannot easily distinguish free versus bound species. Here, we use differential pulse voltammetry (DPV) in combination with ultrafiltration (UF) to isolate and determine the difference in EC activity (peak current) of the free drug compared to when it is bound in mixtures with bovine SA or human AAG. With UF, a membrane allows unbound CLZ to traverse the membrane under centrifugation while the free protein and bound complex remain above.
Results show almost no difference in EC activity between filtered and unfiltered solutions at therapeutic CLZ concentrations, suggesting that bound complexes do not significantly contribute to EC reactions in mixtures of CLZ with proteins. Ultraviolet-visible spectroscopy and mass spectrometry verified the ability of the filters to eliminate protein and bound drug during filtration. The experimental results showed that CLZ binds at a concentration-independent proportion of 30.5% to AAG and 79.4% to SA, showing that this combined UF-EC method can be used to estimate the protein-bound CLZ in blood that is unavailable for patient treatment. The impact of free protein on EC signals was also investigated, with a <10% and 20-30% reduction in signal observed for AAG and SA, respectively.
These measurements represent initial steps toward improving sensing specificity with electrochemical techniques, and offer insight as to strategic targets for either eliminating noise or amplifying the resulting signal. Ultimately, the above method can be used to optimize the concentration of free, and therefore therapeutically active CLZ in blood and further study the optimized dosage of a broad spectrum of mental health drugs.

Genetically engineered probiotics are rapidly gaining attention as highly programmable drug delivery vehicles for localized production of therapeutic molecules in situ in the gastrointestinal tract. Most of the research in this area has focused on developing methods to secrete the therapeutics (i.e. payload delivery) into the gut lumen. Here we describe progress toward an alternative approach, wherein a probiotic bacterial strain is programmed to produce a self-assembled material designed to mediate interactions with the gut epithelium and display immobilized therapeutic proteins in high density. We demonstrate the ability to engineer E. coli curli fibers to display trefoil factors – a family of anti-inflammatory cytokines – while the biological functionality of the cytokines is maintained. Overall, our results suggests that curli-containing biofilms could serve as a versatile platform for therapeutic display and delivery to the gut epithelium.

Attempts to apply artificial micro-/nanomotors for diverse biomedical applications have inspired a variety of strategies for designing motors with diverse propulsion mechanisms and functions. However, existing artificial motors are made exclusively of synthetic materials, which are subject to serious immune attack and clearance upon entering the bloodstream. Herein we present a platform by using red blood cell as camouflage coating for micro-/nanomotors. As the first example, a red blood cell membrane-camouflaged nanowire that can serve as new generation of biomimetic motor sponge is described. The biomimetic motor sponge is constructed by the fusion of biocompatible gold nanowire motors and RBC nanovesicles. The motor sponge possesses a high coverage of RBC vesicles, which remain totally functional due to its exclusively oriented extracellular functional portion on the surfaces of motor sponge. These biomimetic motors display efficient acoustical propulsion, including controlled movement in undiluted whole blood. The RBC vesicles on the motor sponge remain highly stable during the propulsion process, conferring thus the ability to absorb membrane-damaging toxins and allowing the motor sponge to be used as efficient toxin decoys. The efficient propulsion of the motor sponges under an ultrasound field results in accelerated neutralization of the membrane-damaging toxins. Such motor sponges connect artificial nanomotors with biological entities and hold great promise for treating a variety of injuries and diseases caused by membrane-damaging toxins.

As another example, an elegant approach is used to directly turn natural red blood cells (RBCs) into functional micromotors with the aid of ultrasound propulsion and magnetic guidance. Since the RBC motors preserve the biological and structural features of regular RBCs, these motors possess a wide range of antigenic, transport, and mechanical properties that common synthetic motors cannot achieve and thus hold considerable promise for a number of practical biomedical uses. The ability of the RBC micromotors to transport imaging and therapeutic agents at high speed and spatial precision through a complex microchannel network is also demonstrated. Such ability to load and transport diagnostic imaging agents and therapeutic drugs within a single cell-based motor, in addition to a lower toxicity observed once the drug is encapsulated within the multicargo RBC motor, opens the door to the development of theranostic micromotors that may simultaneously treat and monitor diseases.

Titanium and its alloys with open cellular structures such as mesh and foam are characterized by low Young’s modulus similar to that of human bone and provide the space required for bone tissue in-growth. However, to ensure long-term endurance and the ability to withstand abrupt impact fracture, the artificial cellular materials are expected to possess adequate strength in conjunction with high energy absorption capability. Thus, for the porous implant to attain long-term mechanical and biological stability, it is expected possess high porosity-high strength combination with long-term endurance and high energy absorption capability. By optimizing the unit cell structure, shape, distribution of pore size, pore morphology, and relative density, the aforementioned properties can be attained in a single material. Ti–6Al–4V meshes with different elements (cubic, G7, and rhombic dodecahedron) in Materialise software were fabricated by additive manufacturing using the electron beam melting (EBM) method, and the effects of cell shape on the mechanical properties of these samples were studied. The results showed that these cellular structures with porosities of 88–58% had compressive strength and elastic modulus in the range 10–300 MPa and 0.5–15 GPa, respectively. The compressive strength and deformation behavior of these meshes were determined by the coupling of the buckling and bending deformation of struts. Meshes that were dominated by buckling deformation showed relatively high collapse strength and were prone to exhibit brittle characteristics in their stress–strain curves, whereas, meshes dominated by bending deformation, showed ductile behavior. Furthermore, graded/gradient structures are fabricated to withstand varying mechanical stress at specific regions, which mimics the natural bone architecture. The study provides a foundation for obtaining ultimate functionalities in 3D-cellular mesh structures with interconnected porous architecture.

Drug-resistant bacteria are an emerging clinical heath threat, and currently cause millions of serious infections annually in the US alone. Some resistant microbial pathogens with current or potential clinical relevance include Burkholderia pseudomallei, Francisella tularensis, Bacillus anthracis, Yersinia pestis, and MRSA among many others. With Burkholderia pseudomallei (Bp) infections, pneumonic and septic forms of melioidosis can occur with as few as 10 aerosolized bacteria, and result in highly variable incubation periods (years), which require prolonged antibiotic therapy for effective treatment (months). In this work, targeted nanoparticles were used to kill Burkholderia pseudomallei and Burkholderia thailandensis by overcoming a natural resistance mechanism of these microbes.

The nanoparticle drug carrier developed for treatment of Burkholderia was comprised of mesoporous silica nanoparticles (MSNPs), which are enveloped by a fused lipid bilayer to form a therapeutic nanoparticle system referred to as a ‘protocell’. In this work, MSNPs were solute-loaded with antibiotics by equilibrium partitioning, and then fused with targeted liposomes to produce a biocompatible therapeutic nanoconstruct. To fully characterize this nanoparticle system, we employed a host of analysis techniques for both the MSNPs and protocells, including dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning mobility particle sizing (SMPS), optical particle sizing (OPS), nanoparticle tracking analysis (NTA), zeta potential analysis (Zeta), and surface area and porosity characterization (ASAP).

Protocells modified with targeting moieties conjugated to the lipid bilayer showed a high degree of specificity to the corresponding targeted cell lines in vitro. Resistant Bp was efficiently killed in vitro using targeted, antibiotic-loaded protocells by overcoming an efflux pumping resistance mechanism. In vivo, protocells modified with peptide ‘zipcodes’ showed highly specific uptake in targeted lung tissues following IV administration, as confirmed by ICP-MS. Additionally, in vivo studies with targeted protocells against Burkholderia resulted in significantly improved survival of rodents as compared to control groups.