A key end goal of gene delivery research is to develop clinically-relevant vectors that can be used to combat elusive diseases such as AIDS. Despite promising engineering strategies, efficiency and ultimately gene modulation efficacy of nonviral vectors have been hindered by numerous in vitro and in vivo barriers that have resulted in sub-viral performance. In this perspective, we concentrate on the gene delivery barriers associated with the two most common classes of nonviral vectors, cationic-based lipids and polymers. We present the existing delivery barriers and summarize current vector-specific strategies to overcome said barriers.

Given the rise of antibiotic resistance and other difficult-to-treat diseases, genetic vaccination is a promising preventative approach that can be tailored and scaled according to the vector chosen for gene delivery. However, most vectors currently utilized rely on ubiquitous delivery mechanisms that ineffectively target important immune effectors such as antigen presenting cells (APCs). As such, APC targeting allows the option for tuning the direction (humoral vs cell-mediated) and strength of the resulting immune responses. In this work, we present the development and assessment of a library of mannosylated poly(beta-amino esters) (PBAEs) that represent a new class of easily synthesized APC-targeting cationic polymers. Polymeric characterization and assessment methodologies were designed to provide a more realistic physiochemical profile prior to in vivo evaluation. Gene delivery assessment in vitro showed significant improvement upon PBAE mannosylation and suggested that mannose-mediated uptake and processing influence the magnitude of gene delivery. Furthermore, mannosylated PBAEs demonstrated a strong, efficient, and safe in vivo humoral immune response without use of adjuvants when compared to genetic and protein control antigens. In summary, the gene delivery effectiveness provided by mannosylated PBAE vectors offers specificity and potency in directing APC activation and subsequent immune responses.

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Genetic vaccines offer a treatment opportunity based upon successful gene delivery to specific immune cell modulators. Driving the process is the vector chosen for gene cargo packaging and subsequent delivery to antigen-presenting cells (APCs) capable of triggering an immune cascade. As such, the delivery process must successfully navigate a series of requirements and obstacles associated with the chosen vector and target cell. In this work, we present the development and assessment of a hybrid gene delivery vector containing biological and biomaterial components. Each component was chosen to design and engineer gene delivery separately in a complimentary and fundamentally distinct fashion. A bacterial (Escherichia coli) inner core and a biomaterial [poly (beta-amino ester)]-coated outer surface allowed the simultaneous application of molecular biology and polymer chemistry to address barriers associated with APC gene delivery, which include cellular uptake and internalization, phagosomal escape, and intracellular cargo concentration. The approach combined and synergized normally disparate vector properties and tools, resulting in increased in vitro gene delivery beyond individual vector components or commercially available transfection agents. Furthermore, the hybrid device demonstrated a strong, efficient, and safe in vivo humoral immune response compared with traditional forms of antigen delivery. In summary, the flexibility, diversity, and potential of the hybrid design were developed and featured in this work as a platform for multivariate engineering at the vector and cellular scales for new applications in gene delivery immunotherapy.

The salivary gland is a complex, secretory tissue that produces saliva and maintains oral homeostasis. Radiation induced salivary gland atrophy, manifested as “dry mouth” or xerostomia, poses a significant clinical challenge. Tissue engineering recently has emerged as an alternative, long-term treatment strategy for xerostomia. In this review, we summarize recent efforts towards the development of functional and implantable salivary glands utilizing designed polymeric substrates or synthetic matrices/scaffolds. Although the in vitro engineering of a complex implantable salivary gland is technically challenging, opportunities exist for multidisciplinary teams to harvest the regenerative potential of stem/progenitor cells found in the adult glands and combine them with biomimetic and cell-instructive materials to assemble implantable tissue modules.

In vitro engineering of salivary glands relies on the availability of synthetic matrices presenting essential cell-instructive signals to guide tissue growth. Here, we describe a biomimetic, hyaluronic acid (HA)-based hydrogel platform containing covalently immobilized bioactive peptides derived from perlecan domain IV (TWSKV), laminin-111 (YIGSR, IKVAV), and fibronectin (RGDSP). The HA network was established by the thiol/acrylate reaction, and bioactive peptides were conjugated to the network with high efficiency without significantly altering the mechanical property of the matrix. When encapsulated as single cells in peptide-modified HA hydrogels, human salivary gland stem/progenitor cells (hS/PCs) spontaneously organized into multicellular spheroids with close cell–cell contacts. Conjugation of RGDSP and TWSKV signals in HA gels significantly accelerated cell proliferation, with the largest spheroids observed in RGDSP-tagged gels. Peptide conjugation did not significantly alter the expression of acinar (AMY1), ductal (TFCP2L1), and progenitor (KRT14) markers at the mRNA level. Characterization of three-dimensional (3D) cultures by immunocytochemistry showed positive staining for keratin-5 (K5), keratin-14 (K14), integrin-β1, and α-amylase under all culture conditions, confirming the maintenance of the secretory progenitor cell population. Two-dimensional (2D) adhesion studies revealed that integrin-β1 played a key role in facilitating cell–matrix interaction in gels with RGDSP, IKVAV, and TWSKV signals. Overall, conjugation of the RGDSP peptide to HA gels improved cell viability, accelerated the formation of epithelial spheroids, and promoted the expansion of the progenitor cell population in 3D. This work represents an essential first step toward the development of an engineered salivary gland.

Epithelial-to-mesenchymal transition (EMT) is a well-studied biological process that takes place during embryogenesis, carcinogenesis and tissue fibrosis. During EMT, the polarized epithelial cells with a cuboidal architecture adopt an elongated fibroblast-like morphology. This process is accompanied by the expression of many EMT-specific molecular markers. While the molecular mechanism leading to EMT has been well established, the effects of matrix topography and microstructure have not been clearly elucidated. Synthetic scaffolds mimicking the mesh-like structure of the basement membrane with an average fiber diameter of 0.5 μm and 5 μm were produced via electrospinning of poly(ε-caprolactone) (PCL) and were used to test the significance of fiber diameter on EMT. Cell-adhesive peptide motifs were conjugated to the fiber surface to facilitate cell attachment. Madin-Darby Canine Kidney (MDCK) cells grown on these substrates showed distinct phenotypes. On 0.5 μm substrates, cells grew as compact colonies with an epithelial phenotype. On 5 μm scaffolds, cells were more individually dispersed and appeared more fibroblastic. Upon addition of hepatocyte growth factor (HGF), an EMT inducer, cells grown on the 0.5 μm scaffold underwent pronounced scattering, as evidenced by the alteration of cell morphology, localization of focal adhesion complex, weakening of cell-cell adhesion, and upregulation of mesenchymal markers. By contrast, HGF did not induce a pronounced scattering of MDCK cells cultured on the 5.0 μm scaffold. Collectively, our results show that the alteration of the fiber diameter of proteins found in the basement membrane may create enough disturbances in epithelial organization and scattering that might have important implications in disease progression.

Improvements to bacterial vectors have resulted in non-viral gene therapy vehicles that are easily prepared and can achieve high levels of transfection efficacy. However, these vectors are plagued by potential cytotoxicity and immunogenicity, prompting means of attenuation to reduce unwanted biological outcomes while maintaining transfection efficiency. In this study, listeriolysin O (LLO) producing Escherichia coli BL21(DE3) strains were pre-treated with polymyxin B (PLB), a pore-forming antibiotic, and tested as a delivery vector for gene transfer to a murine RAW264.7 macrophage cell line using a 96-well high-throughput assay. PLB treatment resulted in statistically significant higher levels of gene delivery and lower cytotoxicity. The results suggest a fine balance between bacterial cellular damage, heightened gene and protein release, and increased mammalian cell gene delivery. Overall, the approach presented provides a simple and effective way to enhance bacterial gene delivery while simultaneously reducing unwanted outcomes as a function of using a biological vector.