“Keep it simple stupid” -- KISS is a common idiom used in engineering (and elsewhere), which instructs the designer to design simple systems.  The main reason: complex systems are more prone to failure. Implanted devices in the brain, spinal cord, and peripheral nervous system have many exciting applications for treating chronic clinical conditions (e.g., chronic pain, Parkinson’s tremor, epilepsy, spinal cord injury, and others).  These devices electrically stimulate or record neural activity.  A significant amount of funding from public and private entities is being invested to discover and further develop devices for a wide range of clinical applications.  These applications have been classified as ‘neuromodulation’, ‘electroceuticals’, or ‘bioelectronics’.  While non-invasive solutions exist, due to their reliability and target engagement specificity, implanted neural interface devices remain the gold standard for engaging with the nervous system tissues.  However, the surgery to implant devices may limit the wide-spread adoption of neural interface technology by patients due to the real, or even just perceived, risks of the invasive surgery. In this talk, recent progress on a number of approaches to achieve a minimally invasive neural interface, coined the Injectrode® will be presented.  We will present several iterations of the device concept, design and prototypes which we have progressively simplified to remove unnecessary complexity from the system, specifically for the purpose of sacral nerve stimulation to restore bladder function to individuals with spinal cord injury.

Synthetic hydrogels allow us to mimic many properties of the extracellular matrix (ECM) and assess their impact on cell function. Our recent studies showed that upon embedding into 3D hydrogels, cells actively secrete and remodel the hydrogel-cell interface by depositing nascent ECM. While hydrogel mechanics direct the amount of nascent ECM deposition1, there is limited understanding of how other hydrogel properties, such as ligand exposure and wettability, guide the dynamic interplay between cells, their nascent ECM, and the hydrogel. To address this, we engineered hyaluronic acid (HA) hydrogels of matched mechanics but different degrees of modifications. We then leveraged metabolic labeling to investigate the spatiotemporal dynamics of nascent ECM in response to HA modifications. Chondrocytes were isolated from juvenile bovine joints and passaged once prior embedding into norbornene-modified HA with varying degrees of substitution (DS%): 9.5%, 23%, and 43%, quantified by 1H NMR2. Crosslinking with dithiols was adjusted to obtain moduli of 5 kPa. Embedded chondrocytes were cultured for 7 days in azidohomoalanine media, and nascent ECM was visualized with dibenzocyclooctyne-amine-488 or dibenzocyclooctyne-biotin mediated click chemistry for mass spectrometry. Total ECM protein production was quantified with Bradford Protein Assay and dsDNA quantification, and cell proliferation was assessed by EdU incorporation. Staining of nascent ECM after 7 days showed increased thickness as a function of DS%, ranging from 1.21±0.54 µm (DS9.5%) up to 2.72±1.23 um (DS43%). Quantitative bioassays confirmed increased ECM production for DS43% with 4.43 μg ECM protein/μg DNA compared to DS9.5% with 2.74 μg/μg ECM protein/μg DNA. Additionally, at day 7, EdU incorporation showed a 6-fold increase in dividing cells in DS43% (76.3%±7.5%) compared to DS9.5% hydrogels (12.6%±4.6%). These results suggest that higher HA modifications enhance cell proliferation and nascent ECM production. This study established a functional link between HA modifications and ECM production and cell proliferation independent of mechanics. Ongoing studies are using pulse labeling and proteomic analysis towards gaining mechanistic insight into the contributions of hydrogel modification to nascent ECM production in 3D hydrogels.

Biomaterials derived from polyproline (PPII) peptide sequences offer significant promise due to their diverse applications, including as antifouling agents for biomaterials and as anchors that encourage high density of other peptide sequences. However, the precise mechanisms underlying their antifouling behavior is not fully understood. This study comprehensively investigates antifouling properties of PPII peptides using bovine serum albumin (BSA) as a model foulant while varying guest amino acids within the PPII sequence. By varying the sequence and controlling their adsoprtion, aim to investigate multiple properties, including secondary structure, hydrophobicity, rate constant of rearrangement, and surface coverage, to elucidate their impact on these on antifouling mechanisms. Our findings indicate that surface coverage and incorporation of proline in the sequence has a significant positive impact on antifouling properties. Moreover, this research marks the first exploration of human mesenchymal cells (hMSCs) as foulants on PPII-functionalized surfaces, revealing successful minimization of cell adhesion that correlates with the model foulant results. In conclusion, our research underscores the potential of PPII design and application as an antifouling biomaterial, paving the way for future advancements in this field including use of these sequences as way to control hMSC spreading and attachment.

Cell and tissue reprogramming have the potential to enable the development of highly promising cell therapies for a wide variety of conditions. Current approaches to cell reprogramming, however, face major practical and translational hurdles, including heavy reliance on viral vectors, and a highly stochastic nature, which often leads to inefficient and/or unpredictable reprogramming outcomes. We developed a novel nanotechnology-based approach that overcomes these barriers by enabling deterministic transfection of reprogramming factors into tissues with single-cell resolution and without the need for viral vectors. Cell and tissue nano-transfection (TNT) promote remarkably fast and efficient direct cellular reprogramming in vivo. Such platform technology could be applicable to virtually any reprogramming model, and its minimally disruptive and non-viral nature make it an ideal candidate for use in highly complex disease systems, including neurodegenerative conditions and metabolic disorders, among others. Cell and tissue nano-transfection chips were manufactured from silicon, as described previously. These chips were then used to nanotransfect fibroblasts with different cocktails of reprogramming factors, which were then evaluated (for their therapeutic potential) in different murine models of health and disease. Our results indicate that Si nanochip-based cell and tissue nanotransfection could potentially be used to effectively drive different reprogramming processes, in vivo, to recover damage or diseased tissue structure and function. Additional studies are also being conducted to evaluate the extent to which cell and tissue-nanotransfection-based approaches can be used to mitigate disease burden in different preclinical models of cancer.

It is estimated that there will be 66,440 new pancreatic ductal adenocarcinoma (PDAC) cases in 2024 in the US with nearly 80% presenting with non-resectable disease, resulting in a dismal five-year survival rate of 10% [1,2]. To increase treatment options, early PDAC detection tools are needed. Nanobubbles (NB) are a submicron ultrasound (US) contrast agent which extravasate through permeable vasculature, increasing tumor accumulation [3]. Addition of a PDAC targeting moiety increases contrast agent retention in tumors. Of many biomarkers, extra domain B-fibronectin (EDB-FN) is overexpressed in PDAC [4]. We propose the addition of ZD2, an EDB-FN targeting molecule, to increase PDAC lesion signal intensity, thus improving diagnosis. Nanobubbles (NB) were prepared as previously reported [3]. ZD2-NBs were prepared by adding ZD2 solution to nanobubble precursor lipid solution. The stability of ZD2-NBs was confirmed with US in an agarose phantom (18MHz fc, 4% power, 1fps). NB diameter was measured using dynamic light scattering. ZD2-NBs’ cell uptake was determined by co-incubating rhodamine tagged ZD2-NBs with human PDAC cell lines (Capan-1, BxPC3) and measuring fluorescence intensity. NBs targetability to PDAC was examined using a flank tumor model in immunocompromised mice inoculated with Capan-1 cells. NBs were administered via I.V. injection, and tumor and kidney US signal was monitored in B and NLC mode for 20 minutes post injection. Signal intensity was analyzed using MATLAB. Tissues were preserved after the final time point for histological analysis. We successfully incorporated an EDB-FN targeting peptide, ZD2, into our ultrasound contrast agents. The addition of ZD2 did not significantly alter NB size, charge, or US stability. ZD2-NBs had comparable US contrast signal to untargeted NBs and peptide addition did not negatively affect signal intensity or stability for 300s. ZD2-NBs had approximately 2-fold higher fluorescence signal compared to untargeted NBs in BxPC3 cells. ZD2-NBs had 2.86-fold higher tumor NLC signal 16 minutes after injection compared to untargeted NBs. Experiments to assess biodistribution and in vivo targeting are ongoing.

The ability to rapidly and efficiently identify novel safe and effective non-viral delivery vehicles for use in gene therapy and drug delivery remains a challenge. To overcome this challenge, we have created a design-build-test-learn (DBTL) polymer nanoparticle (PNP) discovery platform capable of rapidly producing and efficiently identifying PNP designs with clear utility for a variety of applications. During each DBTL cycle by this platform, hundreds to thousands of diverse PNP designs are systematically and efficiently screened in vitro and in vivo within months using standardized, reproducible methodology. To date, we have synthesized, characterized, and assessed more than 6,000 novel polymers produced via reversible addition–fragmentation chain transfer (RAFT) polymerization. Each polymer underwent high-throughput purification and characterization, including measurements of molecular weight, monomer conversion, composition, size, polydispersity, zeta potential, loading efficiency, cytotoxicity, and transfection efficiency in vitro. In addition, PNP biodistribution performance was evaluated in C57BL/6 wild type mice. Early proof-of-concept work has yielded multiple examples where this DBTL approach has proven successful in as few as 2 or 3 iterative DBTL cycles. Indeed, we have identified lead PNP delivery candidates that distribute to multiple desired tissue types following intravenous injection in mice. Moreover, we have identified lead PNP candidates capable of distributing specifically to murine sciatic nerve and/or brain tissue following intrathecal injection. Finally, we have identified multiple lead PNP candidates that leveraged specific design parameters, such as controlled molecular weights and monomer composition percentages, to maintain certain physicochemical characteristics (e.g., size, zeta potential, etc.) upon loading with nucleic acid cargo. Altogether, the empirical and modeling insights provided by data collected during each DBTL cycle has enabled us to identify clear patterns in PNP design parameters with significant utility for specific gene and drug delivery applications. The results from these studies have demonstrated the capability of our DBTL platform to rapidly and efficiently identify lead PNP delivery candidates that warrant further investigation. Therefore, this iterative high-throughput synthesis, characterization, and assessment approach to non-viral delivery vehicle discovery is well-positioned to soon offer potentially paradigm-shifting capabilities to the gene therapy and drug delivery fields.