Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems.

Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self-assembling bacterial microcompartment (BMC) shell proteins and liquid-liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid-liquid interfaces between either 1) the dextran-rich droplets and PEG-rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two-phase system, or 2) the polypeptide-rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically-driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three-phase system wherein coacervate droplets are contained within dextran-rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three-phase system by changing the polyelectrolyte charge ratio. The tens-of-micron scale BMC shell protein-coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

Highly modular hepatitis B virus-like nanocarriers for therapeutic protein encapsulation and targeted delivery to triple negative breast cancer cells.

Protein therapeutics offer enormous clinical impact in treating a variety of diseases by offering high selectivity with limited off-target effects. However, delivery challenges severely hinder functional proteins from reaching their target cells and necessitate frequent administration. To address these problems, nanocarrier encapsulation can provide protease protection and enhanced targeted transportation of functional proteins to their intended disease site. Inspired by their viral analogues, virus-like particles (VLPs) are non-infectious viral capsids that have potential for drug delivery applications because of their shared structural characteristics, such as high loading capacity, particle stability, and structural uniformity. Here, we describe a modular hepatitis B virus (HBV) VLP delivery platform offering tunable modifications of both the exterior and interior viral capsid surfaces via SpyCatcher-SpyTag bioconjugation and a multi-expression system, respectively. This new platform facilitates modification with epidermal growth factor receptor (EGFR)-targeting proteins and encapsulation with both model green fluorescent protein (GFP) and prodrug-converting yeast cytosine deaminase (yCD) enzyme. The resultant targeted VLPs demonstrated enhanced uptake and toxicity in EGFR-overexpressing triple negative breast cancer (TNBC) cells in contrast to non-malignant breast epithelial cells.

VEGF-Encoding, Gene-Activated Collagen-Based Matrices Promote Blood Vessel Formation and Improved Wound Repair.

Disruption in vascularization during wound repair can severely impair healing. Proangiogenic growth factor therapies have shown great healing potential; however, controlling growth factor activity and cellular behavior over desired healing time scales remains challenging. In this study, we evaluated collagen-mimetic peptide (CMP) tethers for their capacity to control growth factor gene transfer and growth factor activity using our recently developed gene-activated hyaluronic acid-collagen matrix (GAHCM). GAHCM was comprised of DNA/polyethyleneimine (PEI) polyplexes that were retained on hyaluronic acid (HA)-collagen hydrogels using CMPs. We hypothesized that using CMP-collagen tethers to control vascular endothelial growth factor-A (VEGF-A) gene delivery in fibroblasts would provide a powerful strategy to modulate the proangiogenic behaviors of endothelial cells (ECs) for blood vessel formation, resulting in enhanced wound repair. In co-culture experiments, we observed that CMP-modified GAHCM induced tunable gene delivery in fibroblasts as predicted, and correspondingly, VEGF-A produced by the fibroblasts led to increased growth and persistent migration of ECs for at least 7 days, as compared to non-CMP-modified GAHCM. Moreover, when ECs were exposed to fibroblast-containing VEGF-GAHCM with higher levels of CMP modification (50% CMP-PEI, or 50 CP), high CD31 expression was stimulated, resulting in the formation of an interconnected EC network with a significantly higher network volume and a larger diameter network structure than controls. Application of VEGF-GAHCM with 50 CP in murine splinted excisional wounds facilitated prolonged prohealing and proangiogenic responses resulting in increased blood vessel formation, improved granulation tissue formation, faster re-epithelialization, and overall enhanced repair. These findings suggest the benefits of CMP-collagen tethers as useful tools to control gene transfer and growth factor activity for improved treatment of wounds.

Peptide-Based Vectors: A Biomolecular Engineering Strategy for Gene Delivery.

From the first clinical trial by Dr. W.F. Anderson to the most recent US Food and Drug Administration-approved Luxturna (Spark Therapeutics, 2017) and Zolgensma (Novartis, 2019), gene therapy has revamped thinking and practice around cancer treatment and improved survival rates for adult and pediatric patients with genetic diseases. A major challenge to advancing gene therapies for a broader array of applications lies in safely delivering nucleic acids to their intended sites of action. Peptides offer unique potential to improve nucleic acid delivery based on their versatile and tunable interactions with biomolecules and cells. Cell-penetrating peptides and intracellular targeting peptides have received particular focus due to their promise for improving the delivery of gene therapies into cells. We highlight key examples of peptide-assisted, targeted gene delivery to cancer-specific signatures involved in tumor growth and subcellular organelle-targeting peptides, as well as emerging strategies to enhance peptide stability and bioavailability that will support long-term implementation.

Modified hyaluronic acid-collagen matrices trigger efficient gene transfer and prohealing behavior in fibroblasts for improved wound repair.

Growth factor therapy has demonstrated great promise for chronic wound repair, but controlling growth factor activity and cell phenotype over desired time frames remains a critical challenge. In this study, we developed a gene-activated hyaluronic acid-collagen matrix (GAHCM) comprising DNA/polyethylenimine (PEI) polyplexes retained on hyaluronic acid (HA)-collagen hydrogels using collagen mimetic peptides (CMPs). We hypothesized that manipulating both the number of CMP-collagen tethers and the ECM composition would provide a powerful strategy to control growth factor gene transfer kinetics while regulating cell behavior, resulting in enhanced growth factor activity for wound repair. We observed that polyplexes with 50% CMP-modified PEI (50 CP) showed enhanced retention of polyplexes in HCM hydrogels by 2.7-fold as compared to non-CMP modified polyplexes. Moreover, the incorporation of HA in the hydrogel promoted a significant increase in gene transfection efficiency based upon analysis of Gaussia luciferase (GLuc) reporter gene expression, and gene expression could be attenuated by blocking HA-CD44 signaling. Furthermore, when fibroblasts were exposed to vascular endothelial growth factor-A (VEGF-A)-GAHCM, the 50 CP matrix facilitated sustained VEGF-A production for up to 7 days, with maximal expression at day 5. Application of these VEGF-A-50 CP samples stimulated prolonged pro-healing responses, including the TGF-ß1-induced myofibroblast-like phenotypes and enhanced closure of murine splinted wounds. Overall, these findings demonstrate the use of ECM-based materials to stimulate efficient gene transfer and regulate cellular phenotype, resulting in improved control of growth factor activity for wound repair. GAHCM has significant potential to overcome key challenges in growth factor therapy for regenerative medicine. STATEMENT OF SIGNIFICANCE: Despite great promise for growth factor therapies in wound treatment, controlling growth factor activity and providing a microenvironment for cells that maximizes growth factor signaling have continued to limit the success of existing formulations. Our GAHCM strategy, combining CMP gene delivery and a hyaluronic acid-collagen matrix, enabled enhanced wound healing efficacy via the combination of controlled and localized growth factor expression and matrix-mediated regulation of cell behavior. Incorporation of CMPs and HA in the same matrix synergistically enhanced VEGF activity as compared with simpler matrices. Accordingly, GAHCM will advance our ability to leverage growth factor signaling for wound healing, resulting in new long-term treatments for recalcitrant wounds.

Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures.

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either fluorescence detection approaches, such as immunofluorescent staining or transfection with fluorescent proteins or edge detection techniques, which are often error-prone due to noise and other non-idealities in the image background. We designed a new algorithm that could accurately count and distinguish macrophages and fibroblasts, cells of different phenotypes that often colocalize during tissue regeneration. MATLAB was used to implement the algorithm, which differentiated distinct cell types based on differences in height from the background. A primary algorithm was developed using an area-based method to account for variations in cell size/structure and high-density seeding conditions. Non-idealities in cell structures were accounted for with a secondary, iterative algorithm utilizing internal parameters such as cell coverage computed using experimental data for a given cell type. Finally, an analysis of coculture environments was carried out using an isolation algorithm in which various cell types were selectively excluded based on the evaluation of relative height differences within the image. This approach was found to accurately count cells within a 5% error margin for monocultured cells and within a 10% error margin for cocultured cells.

EGFR Ligand Clustering on E2 Bionanoparticles for Targeted Delivery of Chemotherapeutics to Breast Cancer Cells.

Naturally occurring protein nanocages are promising drug carriers because of their uniform size and biocompatibility. Engineering efforts have enhanced the delivery properties of nanocages, but cell specificity and high drug loading remain major challenges. Herein, we fused the SpyTag peptide to the surface of engineered E2 nanocages to enable tunable nanocage decoration and effective E2 cell targeting using a variety of SpyCatcher (SC) fusion proteins. Additionally, the core of the E2 nanocage incorporated four phenylalanine mutations previously shown to allow hydrophobic loading of doxorubicin and pH-responsive release in acidic environments. We functionalized the surface of the nanocage with a highly cell-specific epidermal growth factor receptor (EGFR)-targeting protein conjugate, 4GE11-mCherry-SC, developed previously in our laboratories by employing unnatural amino acid (UAA) protein engineering chemistries. Herein, we demonstrated the benefits of this engineered protein nanocage construct for efficient drug loading, with a straightforward method for removal of the unloaded drug through elastin-like polypeptide-mediated inverse transition cycling. Additionally, we demonstrated approximately 3-fold higher doxorubicin internalization in inflammatory breast cancer cells compared to healthy breast epithelial cells, leading to targeted cell death at concentrations below the IC50 of free doxorubicin. Collectively, these results demonstrated the versatility of our UAA-based EGFR-targeting protein construct to deliver a variety of cargoes efficiently, including engineered E2 nanocages capable of site-specific functionalization and doxorubicin loading.

Incorporation of Endosomolytic Peptides with Varying Disruption Mechanisms into EGFR-Targeted Protein Conjugates: The Effect on Intracellular Protein Delivery and EGFR Specificity in Breast Cancer Cells.

Intracellular delivery of protein therapeutics remains a significant challenge limiting the majority of clinically available protein drugs to extracellular targets. Strategies to deliver proteins to subcellular compartments have traditionally relied on cell-penetrating peptides, which can drive enhanced internalization but exhibit unreliable activity and are rarely able to target specific cells, leading to off-target effects. Moreover, few design rules exist regarding the relative efficacy of various endosomal escape strategies in proteins. Accordingly, we developed a simple fusion modification approach to incorporate endosomolytic peptides onto epidermal growth factor receptor (EGFR)-targeted protein conjugates and performed a systematic comparison of the endosomal escape efficacy, mechanism of action, and capacity to maintain EGFR-targeting specificity of conjugates modified with four different endosomolytic sequences of varying modes of action (Aurein 1.2, GALA, HA2, and L17E). Use of the recently developed Gal8-YFP assay indicated that the fusion of each endosomolytic peptide led to enhanced endosomal disruption. Additionally, the incorporation of each endosomolytic peptide increased the half-life of the internalized protein and lowered lysosomal colocalization, further supporting the membrane-disruptive capacity. Despite this, only EGFR-targeted conjugates modified with Aurein 1.2 or GALA maintained EGFR specificity. These results thus demonstrated that the choice of endosomal escape moiety can substantially affect targeting capability, cytotoxicity, and bioactivity and provided important new insights into endosomolytic peptide selection for the design of targeted protein delivery systems.

Therapeutic nanocarriers comprising extracellular matrix-inspired peptides and polysaccharides.

INTRODUCTION: The extracellular matrix (ECM) is vital for cell and tissue development. Given its importance, extensive work has been conducted to develop biomaterials and drug delivery vehicles that capture features of ECM structure and function. AREAS COVERED: This review highlights recent developments of ECM-inspired nanocarriers and their exploration for drug and gene delivery applications. Nanocarriers that are inspired by or created from primary components of the ECM (e.g. elastin, collagen, hyaluronic acid (HA), or combinations of these) are explicitly covered. An update on current clinical trials employing elastin-like proteins is also included. EXPERT OPINION: Novel ECM-inspired nanoscale structures and conjugates continue to be of great interest in the materials science and bioengineering communities. Hyaluronic acid nanocarrier systems in particular are widely employed due to the functional activity of HA in mediating a large number of disease states. In contrast, collagen-like peptide nanocarriers are an emerging drug delivery platform with potential relevance to a myriad of ECM-related diseases, making their continued study most pertinent. Elastin-like peptide nanocarriers have a well-established tolerability and efficacy track record in preclinical analyses that has motivated their recent advancement into the clinical arena.

Engineering bionanoparticles for improved biosensing and bioimaging.

The importance of bioimaging and biosensing has been clear with the onset of the COVID-19 pandemic. In addition to viral detection, detection of tumors, glucose levels, and microbes is necessary for improved disease treatment and prevention. Bionanoparticles, such as extracellular vesicles and protein nanoparticles, are ideal platforms for biosensing and bioimaging applications because of their propensity for high density surface functionalization and large loading capacity. Scaffolding large numbers of sensing modules and detection modules onto bionanoparticles allows for enhanced analyte affinity and specificity as well as signal amplification for highly sensitive detection even at low analyte concentrations. Here we demonstrate the potential of bionanoparticles for bioimaging and biosensing by highlighting recent examples in literature that utilize protein nanoparticles and extracellular vesicles to generate highly sensitive detection devices with impressive signal amplification.

Modular Hepatitis B Virus-like Particle Platform for Biosensing and Drug Delivery.

The hepatitis B virus-like particle (HBV VLP) is an attractive protein nanoparticle platform due to the availability of 240 modification sites for engineering purposes. Although direct protein insertion into the surface loop has been demonstrated, this decoration strategy is restricted by the size of the inserted protein moieties. Meanwhile, larger proteins can be decorated using chemical conjugations; yet these approaches perturb the integrity of more delicate proteins and can unfavorably orient the proteins, impairing active surface display. Herein, we aim to create a robust and highly modular method to produce smart HBV-based nanodevices by using the SpyCatcher/SpyTag system, which allows a wide range of peptides and proteins to be conjugated directly and simply onto the modified HBV capsids in a controlled and biocompatible manner. Our technology allows the modular surface modification of HBV VLPs with multiple components, which provides signal amplification, increased targeting avidity, and high therapeutic payload incorporation. We have achieved a yield of over 200 mg/L for these engineered HBV VLPs and demonstrated the flexibility of this platform in both biosensing and drug delivery applications. The ability to decorate over 200 nanoluciferases per VLP improved detection signal by over 1500-fold, such that low nanomolar levels of thrombin could be detected by the naked eye. Meanwhile, a dimeric prodrug-activating enzyme was loaded without cross-linking particles by coexpressing orthogonally labeled monomers. This along with a epidermal growth factor receptor-binding peptide enabled tunable uptake of HBV VLPs into inflammatory breast cancer cells, leading to efficient suicide enzyme delivery and cell killing.

Enhanced wound healing via collagen-turnover-driven transfer of PDGF-BB gene in a murine wound model.

Wound healing is a complex biological process that requires coordinated cell proliferation, migration, and extracellular matrix production/remodeling, all of which are inhibited/delayed in chronic wounds. In this study, a formulation was developed that marries a fibrin-based, provisional-like matrix with collagen mimetic peptide (CMP)/PDGF gene-modified collagens, leading to the formation of robust gels that supported temporally controlled PDGF expression and facile application within the wound bed. Analysis employing in vitro co-gel scaffolds confirmed sustained and temporally controlled gene release based on matrix metalloproteinase (MMP) activity, with ~30% higher PDGF expression in MMP producing fibroblasts as-compared with non-MMP-expressing cells. The integration of fibrin with the gene-modified collagens resulted in co-gels that strongly supported both fibroblast cell recruitment/invasion as well as multiple aspects of the longer-term healing process. The excisional wound healing studies in mice established faster wound closure using CMP-modified PDGF polyplex-loaded co-gels, which exhibited up to 24% more wound closure (achieved with ~2 orders of magnitude lower growth factor dosing) after 9 days as compared to PDGF-loaded co-gels, and 19% more wound closure after 9 days as compared to CMP-free polyplex loaded co-gels. Moreover, minimal scar formation as well as improved collagen production, myofibroblast activity, and collagen orientation was observed following CMP-modified PDGF polyplex-loaded co-gel application on wounds. Taken together, the combined properties of the co-gels, including their stability and capacity to control both cell recruitment and cell phenotype within the murine wound bed, strongly supports the potential of the co-gel scaffolds for improved treatment of chronic non-healing wounds.

Manipulation of the dually thermoresponsive behavior of peptide-based vesicles through modification of collagen-like peptide domains.

Materials that respond to temporally defined exogenous cues continue to be an active pursuit of research toward on-demand nanoparticle drug delivery applications, and using one or more exogenous temperature stimuli could significantly expand the application of nanoparticle-based drug delivery formulations under both hyperthermal and hypothermal conditions. Previously we have reported the development of a biocompatible and thermoresponsive elastin-b-collagen-like polypeptide (ELP-CLP) conjugate that is capable of self-assembling into vesicles and encapsulating small molecule therapeutics that can be delivered at different rates via a single temperature stimulus. Herein we report the evaluation of multiple ELP-CLP conjugates, demonstrating that the inverse transition temperature (T t) of the ELP-CLPs can be manipulated by modifying the melting temperature (T m) of the CLP domain, and that the overall hydrophilicity of the ELP-CLP conjugate also may alter the T t. Based on these design parameters, we demonstrate that the ELP-CLP sequence (VPGFG)6-(GPO)7GG can self-assemble into stable vesicles at 25°C and dissociate at elevated temperatures by means of the unfolding of the CLP domain above its T m. We also demonstrate here for the first time the ability of this ELP-CLP vesicle to dissociate via a hypothermic temperature stimulus by means of exploiting the inverse transition temperature (T t) phenomena found in ELPs. The development of design rules for manipulating the thermal properties of these bioconjugates will enable future modifications to either the ELP or CLP sequences to more finely tune the transitions of the conjugates for specific biomedical applications.

Encapsulation of collagen mimetic peptide-tethered vancomycin liposomes in collagen-based scaffolds for infection control in wounds.

Wound infections are a significant clinical problem affecting millions of people worldwide. Topically applied antibacterial formulations with longer residence time and controlled antimicrobial release would offer significant benefits for improved prevention and treatment of infected wounds. In this study, we developed collagen mimetic peptide (CMP) tethered vancomycin (Van)-containing liposomes (Lipo) (CMP-Van-Lipo) hybridized to collagen-based hydrogels ('co-gels,' e.g., collagen/fibrin combination hydrogels) for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections in vitro and in vivo. Tethering CMP-Van-Lipo nanostructures to co-gels enabled sustained Van release and enhanced in vitro antibacterial effects against MRSA as compared to Van loaded co-gels or Van-Lipo loaded co-gels following multiple fresh bacterial inoculations over a period of 48 h. These results were successfully translated in vivo wherein MRSA infected wounds were effectively treated with CMP-Van-Lipo loaded co-gels for up to 9 days, whereas the activity of Van loaded co-gels and Van-Lipo loaded co-gels were limited to <2 days. Moreover, CMP-Van-Lipo retained in vivo antibacterial activity even after re-inoculation with bacteria; however, Van loaded co-gels and Van-Lipo loaded co-gels allowed significant bacterial growth demonstrating their limited efficacy. Altogether, these results provide proof-of-concept that CMP-Van-Lipo loaded co-gels can be effective topical formulations for preventive treatment of MRSA wound infections. STATEMENT OF SIGNIFICANCE: Current topical antimicrobial formulations (e.g., creams, gels, and ointments) do not control release, leaving antimicrobial concentrations either too high or too low at different time points, and provoking the development of antibacterial resistance and recurrence of wound infections. Here, collagen mimetic peptides (CMPs) were used to stably hybridize vancomycin-containing liposomal nanocarriers (CMP-Van-Lipo) within collagen-fibrin co-gels via triple-helical integration with collagen, enabling control over Van release for prolonged time periods and minimizing the adverse effects of the Lipo formulations on fibroblast cell viability in the wound bed. The CMP-Van-Lipo loaded co-gel's higher antibacterial effects in vitro were successfully translated in vivo for treatment of MRSA-infected mouse wounds, and thus the co-gels can be a potentially translatable treatment for improved clinical wound management.

Controlled Epidermal Growth Factor Receptor Ligand Display on Cancer Suicide Enzymes via Unnatural Amino Acid Engineering for Enhanced Intracellular Delivery in Breast Cancer Cells.

Proteins are ideal candidates for disease treatment because of their high specificity and potency. Despite this potential, delivery of proteins remains a significant challenge due to the intrinsic size, charge, and stability of proteins. Attempts to overcome these challenges have most commonly relied on direct conjugation of polymers and peptides to proteins via reactive groups on naturally occurring residues. While such approaches have shown some success, they allow limited control of the spacing and number of moieties coupled to proteins, which can hinder bioactivity and delivery capabilities of the therapeutic. Here, we describe a strategy to site-specifically conjugate delivery moieties to therapeutic proteins through unnatural amino acid (UAA) incorporation, in order to explore the effect of epidermal growth factor receptor (EGFR)-targeted ligand valency and spacing on internalization of proteins in EGFR-overexpressing inflammatory breast cancer (IBC) cells. Our results demonstrate the ability to enhance targeted protein delivery by tuning a small number of EGFR ligands per protein and clustering these ligands to promote multivalent ligand-receptor interactions. Furthermore, the tailorability of this simple approach was demonstrated through IBC-targeted cell death via the delivery of yeast cytosine deaminase (yCD), a prodrug converting enzyme.

Histone-Mimetic Gold Nanoparticles as Versatile Scaffolds for Gene Transfer and Chromatin Analysis.

Histone-inspired polymer assemblies (polyplexes) can regulate gene expression and subcellular transport in plasmids by harnessing the cellular machinery normally used for histone proteins. When grafted to polyplexes, histone tails promote nuclear accumulation, trigger plasmid DNA (pDNA) release, and enhance transcription. Herein, we developed multifunctional gold nanoparticles (AuNPs) decorated by histone motifs as histone-inspired scaffolds with improved pDNA binding, easy bioimaging, and increased potential for gene delivery and chromatin analysis applications. We hypothesized that polycationic AuNPs coupled to histone motifs would mimic the native presentation of these sequences on the histone octamer and thereby create structures with the capacity to both engage native histone effectors and condense pDNA into nucleosome-inspired nanostructures. AuNPs bearing ∼2 nm cores were prepared based on the well-established Brust-Schiffrin two-phase method involving tetrachloroaurate reduction in the presence of 1-pentanethiol. Solid phase peptide synthesis was employed to generate thiolated polycationic ligands and histone tail motifs, and the AuNPs and peptide ligands were combined in a two-step Murray place exchange reaction at various ratios to produce a collection of polycationic AuNPs modified with varying amounts of histone tails. Electron microscopy and thermal analyses demonstrated that these modified AuNPs exhibited tunable biochemical and biophysical properties that closely mimicked the properties of native histones. The histone-mimetic nanoscaffolds efficiently and sequence-specifically engaged histone effectors responsible for activating transcription. In addition, the nanoscaffolds condensed pDNA into complexes with high stability in the presence of physiological concentrations of heparin, a common extracellular polyanion. These combined properties of histone engagement and high stability led to a ∼6-fold enhancement in transfection efficiency as compared with typical polymeric transfection reagents, with the increased transfection efficiency correlated to the presence and amount of histone tails displayed on the surface of the nanoscaffolds. These findings demonstrate the utility of employing a biomimetic materials design approach to develop more effective and stable delivery vehicles for gene transfer and chromatin analysis applications.

Histone-targeted gene transfer of bone morphogenetic protein-2 enhances mesenchymal stem cell chondrogenic differentiation.

Skeletal tissue regeneration following traumatic injury involves a complex cascade of growth factor signals that direct the differentiation of mesenchymal stem cells (MSCs) within the fracture. The necessity for controlled and localized expression of these factors has highlighted the role gene therapy may play as a promising treatment option for bone repair. However, the design of nanocarrier systems that negotiate efficient intracellular trafficking and nuclear delivery represents a significant challenge. Recent investigations have highlighted the roles histone tail sequences play in directing nuclear delivery and activating DNA transcription. We previously established the ability to recapitulate these natural histone tail activities within non-viral nanocarriers, improving gene transfer and expression by enabling effective navigation to the nucleus via retrograde vesicular trafficking. Herein, we demonstrate that histone-targeting leads to ∼4-fold enhancements in osteogenic bone morphogenetic protein-2 (BMP-2) expression by MSCs over 6 days, as compared with standard polymeric transfection reagents. This improved expression augmented chondrogenesis, an essential first step in fracture healing. Importantly, significant enhancements of cartilage-specific protein expression were triggered by histone-targeted gene transfer, as compared with the response to treatment with equivalent amounts of recombinant BMP-2 protein. In fact, an ∼100-fold increase in recombinant BMP-2 was required to achieve similar levels of chondrogenic gene and protein expression. The enhancements in differentiation achieved using histone-targeting were in part enabled by an increase in transcription factor expression, which functioned to drive MSC chondrogenesis. These novel findings demonstrate the utility of histone-targeted gene transfer strategies to enable substantial reductions in BMP-2 dosing for bone regenerative applications. STATEMENT OF SIGNIFICANCE: This contribution addresses significant limitations in non-viral gene transfer for bone regenerative applications by exploiting a novel histone-targeting approach for cell-triggered delivery that induces osteogenic BMP-2 expression coincident with the initiation of bone repair. During repair, proliferating MSCs respond to a complex series of growth factor signals that direct their differentiation along cellular lineages essential to mature bone formation. Although these MSCs are ideal targets for enhanced transfection during cellular mitosis, few non-viral delivery approaches exist to enable maximization of this effect. Accordingly, this contribution seeks to utilize our histone-targeted nanocarrier design strategy to stimulate BMP-2 gene transfer in dividing MSCs. This gene-based approach leads to significantly augmented MSC chondrogenesis, an essential first step in bone tissue repair.

ECM turnover-stimulated gene delivery through collagen-mimetic peptide-plasmid integration in collagen.

Gene therapies have great potential in regenerative medicine; however, clinical translation has been inhibited by low stability and limited transfection efficiencies. Herein, we incorporate collagen-mimetic peptide (CMP)-linked polyplexes in collagen scaffolds to increase DNA stability by up to 400% and enable tailorable in vivo transgene expression at 100-fold higher levels and 10-fold longer time periods. These improvements were directly linked to a sustained interaction between collagen and polyplexes that persisted during cellular remodeling, polyplex uptake, and intracellular trafficking. Specifically, incorporation of CMPs into polyethylenimine (PEI) polyplexes preserved serum-exposed polyplex-collagen activity over a period of 14days, with 4 orders-of-magnitude more intact DNA present in CMP-modified polyplex-collagen relative to unmodified polyplex-collagen after a 10day incubation under cell culture conditions. CMP-modification also altered endocytic uptake, as indicated by gene silencing studies showing a nearly 50% decrease in transgene expression in response to caveolin-1 silencing in modified samples versus only 30% in unmodified samples. Furthermore, cellular internalization studies demonstrated that polyplex-collagen association persisted within cells in CMP polyplexes, but not in unmodified polyplexes, suggesting that CMP linkage to collagen regulates intracellular transport. Moreover, experiments in an in vivo repair model showed that CMP modification enabled tailoring of transgene expression from 4 to 25days over a range of concentrations. Overall, these findings demonstrate that CMP decoration provides substantial improvements in gene retention, altered release kinetics, improved serum-stability, and improved gene activity in vivo. This versatile technique has great potential for multiple applications in regenerative medicine. STATEMENT OF SIGNIFICANCE: In this work, we demonstrate a novel approach for stably integrating DNA into collagen scaffolds to exploit the natural process of collagen remodelling for high efficiency non-viral gene delivery. The incorporation of CMPs into DNA polyplexes, coupled with the innate affinity between CMPs and collagen, not only permitted improved control over polyplex retention and release, but also provided a series of substantial and highly unique benefits via the stable and persistent linkage between CMP-polyplexes and collagen fragments. Specifically, CMP-modification of polyplexes was demonstrated to (i) control release for nearly a month, (ii) improve vector stability under physiological-like conditions, and (iii) provide ligands able to efficiently transfer genes via endocytic collagen pathways. These unique properties overcome key barriers inhibiting non-viral gene therapy.

Anionic Polymer and Quantum Dot Excipients to Facilitate siRNA Release and Self-Reporting of Disassembly in Stimuli-Responsive Nanocarrier Formulations.

The incorporation of anionic excipients into polyplexes is a promising strategy for modulating siRNA binding versus release and integrating diagnostic capabilities; however, specific design criteria and structure-function relationships are needed to facilitate the development of nanocarrier-based theranostics. Herein, we incorporated poly(acrylic acid) (PAA) and quantum dot (QD) excipients into photolabile siRNA polyplexes to increase gene silencing efficiencies by up to 100% and enable self-reporting of nanocarrier disassembly. Our systematic approach identified the functional relationships between gene silencing and key parameters such as excipient loading fractions and molecular weights that facilitated the establishment of design rules for optimization of nanocarrier efficacy. For example, we found that PAA molecular weights ∼10-20× greater than that of the coencapsulated siRNA exhibited the most efficient release and silencing. Furthermore, siRNA release assays and RNAi modeling allowed us to generate a PAA "heat map" that predicted gene silencing a priori as a function of PAA molecular weight and loading fraction. QDs further promoted selective siRNA release and provided visual as well as Förster resonance energy transfer (FRET)-based monitoring of the dynamic changes in nanostructure in situ. Moreover, even with the addition of anionic components, our formulations exhibited substantially improved stability and shelf life relative to typical formulations, with complete stability after a week of storage and full activity in the presence of serum. Taken together, this study enabled synergistic improvements in siRNA release and diagnostic capabilities, along with the development of mechanistic insights that are critical for advancing the translation of nucleic acid theranostics into the clinic.

Efficient tuning of siRNA dose response by combining mixed polymer nanocarriers with simple kinetic modeling.

Two of the most prominent challenges that limit the clinical success of siRNA therapies are a lack of control over cargo release from the delivery vehicle and an incomplete understanding of the link between gene silencing dynamics and siRNA dosing. Herein, we address these challenges through the formulation of siRNA polyplexes containing light-responsive polymer mixtures, whose varied compositions and triggered release behavior provide enhanced gene silencing and controlled dose responses that can be predicted by simple kinetic models. Through the straightforward mixing of two block copolymers, the level of gene knockdown was easily optimized to achieve the maximum level of GAPDH protein silencing in NIH/3T3 cells (~70%) using a single siRNA dose. The kinetic model was used to describe the dynamic changes in mRNA and protein concentrations in response to siRNA treatment. These predictions enabled the application of a second dose of siRNA to maximally suppress gene expression over multiple days, leading to a further 50% reduction in protein levels relative to those measured following a single dose. Furthermore, polyplexes remained dormant in cells until exposed to the photo-stimulus, demonstrating the complete control over siRNA activity as well as the stability of the nanocarriers. Thus, this work demonstrates that pairing advances in biomaterials design with simple kinetic modeling provides new insight into gene silencing dynamics and presents a powerful strategy to control gene expression through siRNA delivery. STATEMENT OF SIGNIFICANCE: Our manuscript describes two noteworthy impacts: (1) we designed mixed polymer formulations to enhance gene silencing, and (2) we simultaneously developed a simple kinetic model for determining optimal siRNA dose responses to maintain silencing over several days. These advances address critical challenges in siRNA delivery and provide new opportunities in therapeutics development. The structure-function relationships prevalent in these formulations were established to enable tuning and forecasting of nanocarrier efficiency a priori, leading to siRNA dosing regimens able to maximally suppress gene expression. Our advances are significant because the mixed polymer formulations provide a straightforward and scalable approach to tailor siRNA delivery regimens. Moreover, the implementation of accurate dosing frameworks addresses a major knowledge gap that has hindered clinical implementation of siRNA.