Debanti Sengupta

Protein-engineered biomaterials: highly tunable tissue engineering scaffolds.

A common goal in tissue engineering is to attain the ability to tailor specific cell-scaffold interactions and thereby gain control over cell behavior. The tunable nature of protein-engineered biomaterials enables independent tailoring of a range of biomaterial properties, creating an attractive alternative to synthetic polymeric scaffolds or harvested natural scaffolds. Protein-engineered biomaterials are comprised of modular peptide domains with various functionalities that are encoded into a DNA plasmid, transfected into an organism of choice, and expressed and purified to yield a biopolymer with exact molecular-level sequence specification. Because of the modular design strategy of protein-engineered biomaterials, these scaffolds can be easily modified to enable optimization for specific tissue engineering applications. By including multiple peptide domains with different functionalities in a single, modular biomaterial, the scaffolds can be designed to mimic the diverse properties of the natural extracellular matrix, including cell adhesion, cell signaling, elasticity, and biodegradability. Recently, the field of protein-engineered biomaterials has expanded to include functional modules that are not normally present in the extracellular matrix, thus expanding the scope and functionality of these materials. For example, these modules can include noncanonical amino acids, inorganic-binding domains, and DNA-binding sequences. The modularity, tunability, and sequence specificity of protein-engineered biomaterials make them attractive candidates for use as substrates for a variety of tissue engineering applications.

Protein-engineered biomaterials: Nanoscale mimics of the Extracellular Matrix

BACKGROUND Traditional materials used as in vitro cell culture substrates are rigid and flat surfaces that lack the exquisite nano- and micro-scale features of the in vivo extracellular environment. While these surfaces can be coated with harvested extracellular matrix (ECM) proteins to partially recapitulate the bio-instructive nature of the ECM, these harvested proteins often exhibit large batch-to-batch variability and can be difficult to customize for specific biological studies. In contrast, recombinant protein technology can be utilized to synthesize families of 3 dimensional protein-engineered biomaterials that are cyto-compatible, reproducible, and fully customizable. SCOPE OF REVIEW Here we describe a modular design strategy to synthesize protein-engineered biomaterials that fuse together multiple repeats of nanoscale peptide design motifs into full-length engineered ECM mimics. MAJOR CONCLUSIONS Due to the molecular-level precision of recombinant protein synthesis, these biomaterials can be tailored to include a variety of bio-instructional ligands at specified densities, to exhibit mechanical properties that match those of native tissue, and to include proteolytic target sites that enable cell-triggered scaffold remodeling. Furthermore, these biomaterials can be processed into forms that are injectable for minimally-invasive delivery or spatially patterned to enable the release of multiple drugs with distinct release kinetics. GENERAL SIGNIFICANCE Given the reproducibility and flexibility of these protein-engineered biomaterials, they are ideal substrates for reductionist biological studies of cell-matrix interactions, for in vitro models of physiological processes, and for bio-instructive scaffolds in regenerative medicine therapies. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

Protein-engineered biomaterials to generate human skeletal muscle mimics.

Skeletal muscle function, which is essential in the human body, can be lost due to injury, trauma, or diseases such as muscular dystrophy, prompting the need for in vitro models of human skeletal muscle tissue. In vitro tissue models are indispensable in performing quantitative studies of injury and disease progression as well as in providing sources of tissue for potential regenerative medicine therapies. An ideal bioengineered model of skeletal muscle should promote myoblast (muscle cell precursor) alignment as well as subsequent myoblast fusion into mature, multinucleated muscle cells known as myotubes. Accordingly, we report the development of an engineered protein biomaterial that promotes the alignment and fusion of primary human myoblasts into organized and differentiated myotubes. Furthermore, these aligned human myotubes exhibit markers of functional maturation including organization of sarcomeres (the force-generating apparatus within muscle) and induced contractility upon electrical stimulation.

Imaging metabolic heterogeneity in cancer

As our knowledge of cancer metabolism has increased, it has become apparent that cancer metabolic processes are extremely heterogeneous. The reasons behind this heterogeneity include genetic diversity, the existence of multiple and redundant metabolic pathways, altered microenvironmental conditions, and so on. As a result, methods in the clinic and beyond have been developed in order to image and study tumor metabolism in the in vivo and in vitro regimes. Both regimes provide unique advantages and challenges, and may be used to provide a picture of tumor metabolic heterogeneity that is spatially and temporally comprehensive. Taken together, these methods may hold the key to appropriate cancer diagnoses and treatments in the future.

Bright Lu2O3:Eu Thin-Film Scintillators for High-Resolution Radioluminescence Microscopy

The performance of a new thin-film Lu2 O3 :Eu scintillator for single-cell radionuclide imaging is investigated. Imaging the metabolic properties of heterogeneous cell populations in real time is an important challenge with clinical implications. An innovative technique called radioluminescence microscopy has been developed to quantitatively and sensitively measure radionuclide uptake in single cells. The most important component of this technique is the scintillator, which converts the energy released during radioactive decay into luminescent signals. The sensitivity and spatial resolution of the imaging system depend critically on the characteristics of the scintillator, that is, the material used and its geometrical configuration. Scintillators fabricated using conventional methods are relatively thick and therefore do not provide optimal spatial resolution. A thin-film Lu2 O3 :Eu scintillator is compared to a conventional 500 μm thick CdWO4 scintillator for radioluminescence imaging. Despite its thinness, the unique scintillation properties of the Lu2 O3 :Eu scintillator allow us to capture single-positron decays with fourfold higher sensitivity, which is a significant achievement. The thin-film Lu2 O3 :Eu scintillators also yield radioluminescence images where individual cells appear smaller and better resolved on average than with the CdWO4 scintillators. Coupled with the thin-film scintillator technology, radioluminescence microscopy can yield valuable and clinically relevant data on the metabolism of single cells.

Single-Cell Characterization of 18F-FLT Uptake with Radioluminescence Microscopy

The radiotracer 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) is commonly used to measure cell proliferation in vivo. As a marker of cell proliferation, 18F-FLT is expected to be differentially taken up by arrested and actively dividing cells, but PET measures only aggregate uptake by tumor cells and therefore the single-cell distribution of 18F-FLT is unknown. We used a novel in vitro radioluminescence microscopy technique to measure the differential distribution of 18F-FLT radiotracer with single-cell precision. Methods: Using radioluminescence microscopy, we imaged the absolute uptake of 18F-FLT in live MDA-MB-231 cells grown under different serum conditions. We then compared 18F-FLT uptake with a standard measure of cell proliferation, using fluorescence microscopy of 5-ethynyl-2′-deoxyuridine incorporation in fixed cells. Results: According to 5-ethynyl-2′-deoxyuridine staining, few cells (1%) actively cycled under serum deprivation whereas most of them (71%) did under 20% serum. The distribution of 18F-FLT reflected this dynamic. At 0% serum, uptake of 18F-FLT was heterogeneous but relatively low. At 20% serum, a subpopulation of 18F-FLT–avid cells, representing 61% of the total population, emerged. Uptake of 18F-FLT in this population was 5-fold higher than in the remainder of the cells. Such a dichotomous distribution is not typically observed with other radiotracers, such as 18F-FDG. Conclusion: These results suggest that increased 18F-FLT uptake by proliferating cells is due to a greater fraction of 18F-FLT–avid cells rather than a change in 18F-FLT uptake by individual cells. This finding is consistent with the fact that 18F-FLT uptake is mediated by thymidine kinase 1 expression, which is higher in actively dividing cells. Overall, these findings suggest that, within the same patient, changes in 18F-FLT uptake reflect changes in the number of actively dividing cells, provided other parameters remain the same.

In silico optimization of radioluminescence microscopy

Radioluminescence microscopy (RLM) is a high-resolution method for imaging radionuclide uptake in live cells within a fluorescence microscopy environment. Although RLM currently provides sufficient spatial resolution and sensitivity for cell imaging, it has not been systematically optimized. This study seeks to optimize the parameters of the system by computational simulation using a combination of numerical models for the systems various components: Monte-Carlo simulation for radiation transport, 3D optical point-spread function for the microscope, and stochastic photosensor model for the electron multiplying charge coupled device (EMCCD) camera. The relationship between key parameters and performance metrics relevant to image quality is examined. Results show that Lu2 O3 :Eu yields the best performance among 5 different scintillator materials, and a thickness: 8 μm can best balance spatial resolution and sensitivity. For this configuration, a spatial resolution of ~20 μm and sensitivity of 40% can be achieved for all 3 magnifications investigated, provided that the user adjusts pixel binning and electron multiplying (EM) gain accordingly. Hence the primary consideration for selecting the magnification should be the desired field of view and magnification for concurrent optical microscopy studies. In conclusion, this study estimates the optimal imaging performance achievable with RLM and promotes further development for more robust imaging of cellular processes using radiotracers.

Performance Evaluation of 18F Radioluminescence Microscopy Using Computational Simulation

Purpose Radioluminescence microscopy can visualize the distribution of beta‐emitting radiotracers in live single cells with high resolution. Here, we perform a computational simulation of 18F positron imaging using this modality to better understand how radioluminescence signals are formed and to assist in optimizing the experimental setup and image processing. Methods First, the transport of charged particles through the cell and scintillator and the resulting scintillation is modeled using the GEANT4 Monte‐Carlo simulation. Then, the propagation of the scintillation light through the microscope is modeled by a convolution with a depth‐dependent point‐spread function, which models the microscope response. Finally, the physical measurement of the scintillation light using an electron‐multiplying charge‐coupled device (EMCCD) camera is modeled using a stochastic numerical photosensor model, which accounts for various sources of noise. The simulated output of the EMCCD camera is further processed using our ORBIT image reconstruction methodology to evaluate the endpoint images. Results The EMCCD camera model was validated against experimentally acquired images and the simulated noise, as measured by the standard deviation of a blank image, was found to be accurate within 2% of the actual detection. Furthermore, point source simulations found that a reconstructed spatial resolution of 18.5 μm can be achieved near the scintillator. As the source is moved away from the scintillator, spatial resolution degrades at a rate of 3.5 μm per μm distance. These results agree well with the experimentally measured spatial resolution of 30–40 μm (live cells). The simulation also shows that the system sensitivity is 26.5%, which is also consistent with our previous experiments. Finally, an image of a simulated sparse set of single cells is visually similar to the measured cell image. Conclusions Our simulation methodology agrees with experimental measurements taken with radioluminescence microscopy. This in silico approach can be used to guide further instrumentation developments and to provide a framework for improving image reconstruction.

Development and characterization of a scintillating cell imaging dish for radioluminescence microscopy

Radioluminescence microscopy is an emerging modality that can be used to image radionuclide probes with micron-scale resolution. This technique is particularly useful as a way to probe the metabolic behavior of single cells and to screen and characterize radiopharmaceuticals, but the quality of the images is critically dependent on the scintillator material used to image the cells. In this paper, we detail the development of a microscopy dish made of a thin-film scintillating material, Lu2O3:Eu, that could be used as the blueprint for a future consumable product. After developing a simple quality control method based on long-lived alpha and beta sources, we characterize the radioluminescence properties of various thin-film scintillator samples. We find consistent performance for most samples, but also identify a few samples that do not meet the specifications, thus stressing the need for routine quality control prior to biological experiments. In addition, we test and quantify the transparency of the material, and demonstrate that transparency correlates with thickness. Finally, we evaluate the biocompatibility of the material and show that the microscopy dish can produce radioluminescent images of live single cells.

WE‐H‐207A‐01: Computational Evaluation of High‐Resolution 18F Positron Imaging Using Radioluminescence Microscopy with Lu2O3: Eu Thin‐Film Scintillator

PURPOSE Radioluminescence microscopy, an emerging and powerful tool for high resolution beta imaging, has been applied to molecular imaging of cellular metabolism to understand tumor biology. A novel thin-film (10 µm thickness) scintillator made of Lu2 O3 : Eu has been developed to enhance the system performance. However the advances of radioluminescence imaging with Lu2 O3 scintillator compared with that using conventional scintillator have not been explored theoretically to date. To validate the advantages of the thin-film scintillator, this study uses a novel computational simulation framework to evaluate the performance of radioluminescence microscopy using both conventional and thin-film scintillators. METHODS Numerical models for different stages of positron imaging are established. Positron from 18 F passing through the scintillator and its neighbor structures are modeled by Monte-Carlo simulation using Geant4. The propagation and focus of photons by the microscope are modeled by convolution with a depth-varying point spread function generated by the Gibson-Lanni model. Photons focused on the detector plane are then captured and converted into electronic signals by an electron multiplication (EM) CCD camera, which is described by a photosensor model considering various noises and charge amplification. RESULTS The performance metrics of radioluminescence imaging with a thin-film Lu2 O3 and conventional CdWO4 scintillator are compared, including spatial resolution, sensitivity, positron track area and intensity. The spatial resolution of Lu2 O3 system can achieve 10 µm maximally, a 12 µm enhancement from that obtained from CdWO4 system. Meanwhile, the system with Lu2 O3 scintillator can provide a higher mean sensitivity: 40% compared with that (21.5%) obtained from CdWO4 system. Moreover, the simulation results are in good agreement with previous experimental measurements. CONCLUSION This study provides a new theoretical understanding of our imaging system and has the potential to promote the development of radioluminescence microscopy for more reliable and robust application on the functional imaging of delicate biological structures. The authors acknowledge funding from NIH grant R01CA186275 and SBIR grant 1R43GM110888-01.