Showing papers by "Ennio Tasciotti published in 2018"

Design and Development of Biomimetic Nanovesicles Using a Microfluidic Approach.

Abstract: The advancement of nanotechnology toward more sophisticated bioinspired approaches has highlighted the gap between the advantages of biomimetic and biohybrid platforms and the availability of manufacturing processes to scale up their production. Though the advantages of transferring biological features from cells to synthetic nanoparticles for drug delivery purposes have recently been reported, a standardizable, batch-to-batch consistent, scalable, and high-throughput assembly method is required to further develop these platforms. Microfluidics has offered a robust tool for the controlled synthesis of nanoparticles in a versatile and reproducible approach. In this study, the incorporation of membrane proteins within the bilayer of biomimetic nanovesicles (leukosomes) using a microfluidic-based platform is demonstrated. The physical, pharmaceutical, and biological properties of microfluidic-formulated leukosomes (called NA-Leuko) are characterized. NA-Leuko show extended shelf life and retention of the biological functions of donor cells (i.e., macrophage avoidance and targeting of inflamed vasculature). The NA approach represents a universal, versatile, robust, and scalable tool, which is extensively used for the assembly of lipid nanoparticles and adapted here for the manufacturing of biomimetic nanovesicles.

Biomimetic nanoparticles with enhanced affinity towards activated endothelium as versatile tools for theranostic drug delivery.

Abstract: Activation of the vascular endothelium is characterized by increased expression of vascular adhesion molecules and chemokines. This activation occurs early in the progression of several diseases and triggers the recruitment of leukocytes. Inspired by the tropism of leukocytes, we investigated leukocyte-based biomimetic nanoparticles (i.e., leukosomes) as a novel theranostic platform for inflammatory diseases. Methods: Leukosomes were assembled by combining phospholipids and membrane proteins from leukocytes. For imaging applications, phospholipids modified with rhodamine and gadolinium were used. Leukosomes incubated with antibodies blocking lymphocyte function-associated antigen 1 (LFA-1) and CD45 were administered to explore their roles in targeting inflammation. In addition, relaxometric assessment of NPs was evaluated. Results: Liposomes and leukosomes were both spherical in shape with sizes ranging from 140-170 nm. Both NPs successfully integrated 8 and 13 µg of rhodamine and gadolinium, respectively, and demonstrated less than 4% variation in physicochemical features. Leukosomes demonstrated a 16-fold increase in breast tumor accumulation relative to liposomes. Furthermore, quantification of leukosomes in tumor vessels demonstrated a 4.5-fold increase in vessel lumens and a 14-fold increase in vessel walls. Investigating the targeting mechanism of action revealed that blockage of LFA-1 on leukosomes resulted in a 95% decrease in tumor accumulation. Whereas blockage of CD45 yielded a 60% decrease in targeting and significant increases in liver and spleen accumulation. In addition, when administered in mice with atherosclerotic plaques, leukosomes exhibited a 4-fold increase in the targeting of inflammatory vascular lesions. Lastly, relaxometric assessment of NPs demonstrated that the incorporation of membrane proteins into leukosomes did not impact the r1 and r2 relaxivities of the NPs, demonstrating 6 and 30 mM-1s-1, respectively. Conclusion: Our study demonstrates the ability of leukosomes to target activated vasculature and exhibit superior accumulation in tumors and vascular lesions. The versatility of the phospholipid backbone within leukosomes permits the incorporation of various contrast agents. Furthermore, leukosomes can potentially be loaded with therapeutics possessing diverse physical properties and thus warrant further investigation toward the development of powerful theranostic agents.

Biomimetic Tissue Engineering: Tuning the Immune and Inflammatory Response to Implantable Biomaterials.

Abstract: Regenerative medicine technologies rely heavily on the use of well-designed biomaterials for therapeutic applications. The success of implantable biomaterials hinges upon the ability of the chosen biomaterial to negotiate with the biological barriers in vivo. The most significant of these barriers is the immune system, which is composed of a highly coordinated organization of cells that induce an inflammatory response to the implanted biomaterial. Biomimetic platforms have emerged as novel strategies that aim to use the principle of biomimicry as a means of immunomodulation. This principle has manifested itself in the form of biomimetic scaffolds that imitate the composition and structure of biological cells and tissues. Recent work in this area has demonstrated the promising potential these technologies hold in overcoming the barrier of the immune system and, thereby, improve their overall therapeutic efficacy. In this review, a broad overview of the use of these strategies across several diseases and future avenues of research utilizing these platforms is provided.

Smart cancer therapy with DNA origami.

Abstract: VOLUME 36 NUMBER 3 MARCH 2018 nature biotechnology Ennio Tasciotti is at the Center for Biomimetic Medicine and Center for Musculoskeletal Regeneration, Houston Methodist Orthopedic and Sports Medicine, Houston Methodist Hospital, Houston Methodist Research Institute, Houston, Texas, USA. e-mail: etasciotti@houstonmethodist.org because of its pleiotropic effects. In principle, their strategy could be used to deliver a multitude of other therapeutics whose clinical use is precluded by unwanted side effects. Vascular occlusion may be especially valuable for the treatment of chemoresistant cancers, since the mechanism of action of the nanorobot is independent of the cells’ molecular background, and potential resistance mechanisms are unlikely to overlap with those induced by other therapeutics8. A clever aspect of the design is that the DNA aptamer targeting agent also serves as the actuator, reducing the number of building blocks and simplifying the assembly. Other types of nanodevices are being developed to contain an ever-increasing number of moving parts. By contrast, DNA origami can be used to engineer folds and self-contained hinges in the primary structure of the material to accomplish the same functions. Creating complex, dynamic, three-dimensional structures from simple oneor two-dimensional shapes represents a novel approach that could transform how we think about drug delivery. By fine-tuning the release kinetics through the conversion of the structure in response to a microenvironmental change, DNA nanorobots could inspire the generation of a new class of logic-gated therapeutics. If we define a robot as a device capable of carrying out a complex series of actions automatically, we can infer that the device presented by Li et al.3 does indeed qualify as a first-generation DNA nanorobot. In the authors’ present design, recognition and dissociation of the DNA fasteners by nucleolin is not intended to be a reversible process, making it impossible for the sheet to fold up a second time. It is conceivable that in future studies some of the structural and functional DNA strands could be made to reconfigure themselves at the end of every activation cycle, allowing the nanorobot to complete its task several times. Finally, one can envision combinations of the presence of short DNA sequences that act as ‘staples’ and ‘fasteners’ to stabilize the final conformation. Once mixed, scaffold, staple and fastener strands self-assemble in a single step. The process can be reversed by displacing the fasteners. In addition, chemical modification of nucleic acids with functional groups allows precise patterning of molecules that can act as sensors or actuators of the shape modification. DNA origami has been used to design a variety of complex shapes, devices, and logic switches6. Li et al.3 focused instead on building a device that could overcome the sequential challenges faced by any drug delivery system injected into the circulation. Protection of the payload, avoidance of healthy tissues, and in situ activation were all achieved by clever assembly of DNA building blocks that served as structural and functional domains of the device. The design, which resembles a nanoparticle developed by the Church lab7, consists of a rectangular DNA sheet formed from an M13 bacteriophage genome DNA strand, held together by multiple short ‘staple’ strands (Fig. 1). The sheet is rolled up into a hollow tube using DNA fasteners. Li et al.3 call their device a DNA ‘nanorobot’ because it is functionalized with molecules that enable it to deliver an active drug only in the presence of a specific tumor marker. The DNA fasteners contain a DNA aptamer that recognizes the tumor-vasculature marker nucleolin. The blood coagulation enzyme thrombin is bound to the inner surface of the tube via DNA tags. Upon binding to nucleolin, the aptamer causes the tube to open, exposing the enzyme to the blood. This triggers in situ activation of platelets and the formation of blood clots in the tumor vasculature, inducing tumor infarction and ultimately tumor shrinkage. By creating an autonomous system programmed to respond specifically to the local biology of the tumor vascular tissue, Li et al.3 have managed to ‘druggify’ thrombin, a protein that has been excluded from cancer therapies Technologies for delivering cytotoxic drugs to tumors while sparing healthy tissues have lagged behind our rapidly growing understanding of cancer biology. Improved methods for drug delivery would undoubtedly translate into higher efficacy and lower toxicity for many cancer therapies1. A promising approach is the use of drug nanocarriers equipped with tumorspecific targeting ligands or environmentally responsive molecular switches. Nanoparticles built from DNA origami can integrate molecular information to autonomously perform a complex task, but so far therapeutic applications of DNA origami have remained elusive2. In this issue, Li et al.3 report the first in vivo application of a DNA origami nanoparticle for cancer therapy. Their 100-nm cylindrical device carries thrombin within it, shielded from the blood, and unfolds to expose the enzyme only after binding a molecular trigger in the tumor. The authors show that delivery of thrombin induces induces local formation of blood clots, occludes tumor blood vessels and leads to tumor necrosis. In principle, the approach could be effective against a wide array of solid tumors, as all solid-tumor–feeding vessels are essentially the same4. Scientists have been making shapes out of DNA for over three decades, but the field took off in the 2000s with the advent of a folding technique called DNA origami5. The creation of complex three-dimensional shapes from a single-stranded DNA molecule relies on selfassembly through Watson–Crick base pairing. Computational methods have been developed to predict how a long DNA strand (the scaffold) will fold in three-dimensional space in Smart cancer therapy with DNA origami

Electrospun Patch Functionalized with Nanoparticles Allows for Spatiotemporal Release of VEGF and PDGF-BB Promoting In Vivo Neovascularization

Abstract: The use of nanomaterials as carriers for the delivery of growth factors has been applied to a multitude of applications in tissue engineering. However, issues of toxicity, stability, and systemic e...

Inflammation and Cancer: In Medio Stat Nano

Abstract: Cancer treatment still remains a challenge due to the several limitations of currently used chemotherapeutics, such as their poor pharmacokinetics, unfavorable chemical properties, as well as inability to discriminate between healthy and diseased tissue. Nanotechnology offered potent tools to overcome these limitations. Drug encapsulation within a delivery system permitted i) to protect the payload from enzymatic degradation/ inactivation in the blood stream, ii) to improve the physicochemical properties of poorly water-soluble drugs, like paclitaxel, and iii) to selectively deliver chemotherapeutics to the cancer lesions, thus reducing the off-target toxicity, and promoting the intracellular internalization. To accomplish this purpose, several strategies have been developed, based on biological and physical changes happening locally and systemically as a consequence of tumorigenesis. Here, we will discuss the role of inflammation in the different steps of tumor development and the strategies based on the use of nanoparticles that exploit the inflammatory pathways in order to selectively target the tumor-associated microenvironment for therapeutic and diagnostic purposes.

Trends towards Biomimicry in Theranostics

Abstract: Over the years, imaging and therapeutic modalities have seen considerable progress as a result of advances in nanotechnology. Theranostics, or the marrying of diagnostics and therapy, has increasingly been employing nano-based approaches to treat cancer. While first-generation nanoparticles offered considerable promise in the imaging and treatment of cancer, toxicity and non-specific distribution hindered their true potential. More recently, multistage nanovectors have been strategically designed to shield and carry a payload to its intended site. However, detection by the immune system and sequestration by filtration organs (i.e., liver and spleen) remains a major obstacle. In an effort to circumvent these biological barriers, recent trends have taken inspiration from biology. These bioinspired approaches often involve the use of biologically-derived cellular components in the design and fabrication of biomimetic nanoparticles. In this review, we provide insight into early nanoparticles and how they have steadily evolved to include bioinspired approaches to increase their theranostic potential.

Microfluidic Assembly of Liposomes with Tunable Size and Coloading Capabilities.

Abstract: Liposomes used for the delivery of pharmaceuticals have difficulties scaling up and reaching clinical translation as they suffer from batch-to-batch variability. Here, we describe a microfluidic approach for creating reproducible, homogenous nanoparticles with tunable characteristics. These nanoparticles of sizes ranging from 30 to 500 nm are rapidly self-assembled by controlling the flow rates of ethanol and aqueous streams. This method of microfluidic assembly allows for the efficient encapsulation of both hydrophobic and hydrophilic drugs in the lipid bilayer and particle core, respectively, either separately or in combination.

Non-invasive assessment of the spatial and temporal distributions of interstitial fluid pressure, fluid velocity and fluid flow in cancers in vivo

Abstract: Interstitial fluid pressure (IFP), interstitial fluid velocity (IFV), interstitial permeability (IP) and vascular permeability (VP) are cancer mechanopathological parameters of great clinical significance. To date, there is a lack of non-invasive techniques that can be used to estimate these parameters in vivo. In this study, we designed and tested new ultrasound poroelastography methods capable of estimating the magnitude and spatial distribution of fluid pressure, fluid velocity and fluid flow inside tumors. We theoretically proved that fluid pressure, velocity and flow estimated using poroelastography from a tumor under creep compression are directly related to the underlying IFP, IFV and fluid flow, respectively, differing only in peak values. We also proved that, from the spatial distribution of the fluid pressure estimated using poroelastography, it is possible to derive: the parameter alpha, which quantifies the spatial distribution of the IFP; the ratio between VP and IP and the ratio between the peak IFP and effective vascular pressure in the tumor. Finally, we demonstrated that axial strain time constant (TC) elastograms are directly related to VP and IP in tumors. Our techniques were validated using finite element and ultrasound simulations, while experiments on a human breast cancer animal model were used to show the feasibility of these methods in vivo.

In vivo imaging of radiopaque resorbable inferior vena cava filter infused with gold nanoparticles

Abstract: Radiopaque resorbable inferior vena cava filter (IVCF) were developed to offer a less expensive alternative to assessing filter integrity in preventing pulmonary embolism for the recommended prophylactic period and then simply vanishes without intervention. In this study, we determined the efficacy of gold nanoparticle (AuNP)-infused poly-p-dioxanone (PPDO) as an IVCF in a swine model. Infusion into PPDO loaded 1.14±0.08 % AuNP by weight as determined by elemental analysis. The infusion did not alter PPDO’s mechanical strength nor crystallinity (Kruskal−Wallis one-way ANOVA, p As a proof-of-concept, two pigs were deployed with IVCF, one with AuNP-PPDO and the other without coating. Results show that the stent ring of AuNP-PPDO was highly visible even in the presence of iodine-based contrast agent and after clot introduction, but not of the uncoated IVCF. Autopsy at two weeks post-implantation showed AuNP-PPDO filter was endothelialized onto the IVC wall, and no sign of filter migration was observed. The induced clot was also still trapped within the AuNP-PPDO IVCF. As a conclusion, we successfully fabricated AuNP-infused PPDO IVCF that is radiopaque, has robust mechanical strength, biocompatible, and can be imaged effectively in vivo. This suggests the efficacy of this novel, radiopaque, absorbable IVCF for monitoring its position and integrity over time, thus increasing the safety and efficacy of deep vein thrombosis treatment.

A Model-Based Approach to Investigate the Effect of a Long Bone Fracture on Ultrasound Strain Elastography

Abstract: The mechanical behavior of long bones and fractures has been under investigation for many decades due to its complexity and clinical relevance. In this paper, we report a new subject-specific methodology to predict and analyze the mechanical behavior of the soft tissue at a bone interface with the intent of identifying the presence and location of bone abnormalities with high accuracy, spatial resolution, and contrast. The proposed methodology was tested on both intact and fractured rabbit femur samples with finite element-based 3-D simulations, created from actual femur computed tomography data, and ultrasound elastography experiments. The results included in this study demonstrate that elastographic strains at the bone/soft tissue interface can be used to differentiate fractured femurs from the intact ones on a distribution level. These results also demonstrate that coronal plane axial shear strain creates a unique contrast mechanism that can be used to reliably detect fractures (both complete and incomplete) in long bones. Kruskal–Wallis test further demonstrates that the contrast measure for the fracture group (simulation: 2.1286±0.2206; experiment: 2.7034 ± 1.0672) is significantly different from that for the intact group (simulation: 0 ± 0; experiment: 1.1540±0.6909) when using coronal plane axial shear strain elastography ( $p$ < 0.01). We conclude that: 1) elastography techniques can be used to accurately identify the presence and location of fractures in a long bone and 2) the proposed model-based approach can be used to predict and analyze strains at a bone fracture site and to better interpret experimental elastographic data.

Controlled Release of Small Molecules for Cardiac Differentiation of Pluripotent Stem Cells.

Abstract: Induced pluripotent stem cells (iPSCs) have been shown to differentiate to functional cardiomyocytes (CM) with high efficiency through temporally controlled inhibition of the GSK3/Wnt signaling pat...

Bone surface enhancement in ultrasound images using a new Doppler-based acquisition/processing method

Abstract: Ultrasound (US) imaging has long been considered as a potential aid in orthopedic surgeries. US technologies are safe, portable and do not use radiations. This would make them a desirable tool for real-time assessment of fractures and to monitor fracture healing. However, image quality of US imaging methods in bone applications is limited by speckle, attenuation, shadow, multiple reflections and other imaging artifacts. While bone surfaces typically appear in US images as somewhat 'brighter' than soft tissue, they are often not easily distinguishable from the surrounding tissue. Therefore, US imaging methods aimed at segmenting bone surfaces need enhancement in image contrast prior to segmentation to improve the quality of the detected bone surface. In this paper, we present a novel acquisition/processing technique for bone surface enhancement in US images. Inspired by elastography and Doppler imaging methods, this technique takes advantage of the difference between the mechanical and acoustic properties of bones and those of soft tissues to make the bone surface more easily distinguishable in US images. The objective of this technique is to facilitate US-based bone segmentation methods and improve the accuracy of their outcomes. The newly proposed technique is tested both in in vitro and in vivo experiments. The results of these preliminary experiments suggest that the use of the proposed technique has the potential to significantly enhance the detectability of bone surfaces in noisy ultrasound images.

Non-invasive imaging of Young's modulus and Poisson's ratio in cancers in vivo

Abstract: Objective: Alterations of Young's modulus (YM) and Poisson's ratio (PR) in biological tissues are often early indicators of the onset of pathological conditions Knowledge of these parameters has been proven to be of great clinical significance for the diagnosis, prognosis and treatment of cancers Currently, however, there are no non-invasive modalities that can be used to image and quantify these parameters in vivo without assuming incompressibility of the tissue, an assumption that is rarely justified in human tissues Methods: In this paper, we develop a new method to simultaneously reconstruct YM and PR of a tumor and of its surrounding tissues, irrespective of the boundary conditions and the shape of the tumor based on ellipsoidal approximation This new non-invasive method allows the generation of high spatial resolution YM and PR maps from axial and lateral strain data obtained via ultrasound elastography The method was validated using finite element (FE) simulations and controlled experiments performed on phantoms with known mechanical properties The clinical feasibility of the developed method was also demonstrated in an orthotopic mouse model of breast cancer Results: Our results from simulations and controlled experiments demonstrate that the proposed reconstruction technique is accurate and robust Conclusion: Availability of the proposed technique could address the clinical need of a non-invasive modality capable of imaging, quantifying and monitoring the mechanical properties of tumors with high spatial resolution and in real time Significance: This technique can have a significant impact on the clinical translation of elasticity imaging methods

Corrigendum: Heparan Sulfate: A Potential Candidate for the Development of Biomimetic Immunomodulatory Membranes.

Abstract: [This corrects the article on p. 54 in vol. 5, PMID: 28983481.].

Implantable electrospun patches for site-directed drug delivery

Abstract: Disclosed are biocompatible, biodegradable, implantable devices for the controlled release of bioactive molecules. In particular embodiments, nanotechnology-based tunable implants are disclosed for 1) localized delivery of analgesics to treat postoperative pain; 2) sustained delivery of growth factors to promote vascularization; and 3) directing tissue regeneration, including the self-direction of autologous stem cells for organ remodeling.