Endosomal escape pathways for delivery of biologicals

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR

Catalytic in vivo protein knockdown by small-molecule PROTACs

Injectable microporous scaffolds assembled from annealed microgel building blocks whose properties can be tailored by microfluidic fabrication facilitate rapid wound healing in vivo.

/pdf/accelerated-wound-healing-by-injectable-microporous-gel-112forfdcm.pdf

Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks

The extracellular matrix (ECM) is a dynamic environment that constantly provides physical and chemical cues to embedded cells. Much progress has been made in engineering hydrogels that can mimic the ECM, but hydrogel properties are, in general, static. To recapitulate the dynamic nature of the ECM, many reversible chemistries have been incorporated into hydrogels to regulate cell spreading, biochemical ligand presentation and matrix mechanics. For example, emerging trends include the use of molecular photoswitches or biomolecule hybridization to control polymer chain conformation, thereby enabling the modulation of the hydrogel between two states on demand. In addition, many non-covalent, dynamic chemical bonds have found increasing use as hydrogel crosslinkers or tethers for cell signalling molecules. These reversible chemistries will provide greater temporal control of adhered cell behaviour, and they allow for more advanced in vitro models and tissue-engineering scaffolds to direct cell fate.

/pdf/the-design-of-reversible-hydrogels-to-capture-extracellular-47corb5k1s.pdf

The design of reversible hydrogels to capture extracellular matrix dynamics

Adaptable hydrogels have recently emerged as a promising platform for three-dimensional (3D) cell encapsulation and culture. In conventional, covalently crosslinked hydrogels, degradation is typically required to allow complex cellular functions to occur, leading to bulk material degradation. In contrast, adaptable hydrogels are formed by reversible crosslinks. Through breaking and re-formation of the reversible linkages, adaptable hydrogels can be locally modified to permit complex cellular functions while maintaining their long-term integrity. In addition, these adaptable materials can have biomimetic viscoelastic properties that make them well suited for several biotechnology and medical applications. In this review, an overview of adaptable-hydrogel design considerations and linkage selections is presented, with a focus on various cell-compatible crosslinking mechanisms that can be exploited to form adaptable hydrogels for tissue engineering.

/pdf/adaptable-hydrogel-networks-with-reversible-linkages-for-39n3pvmzbl.pdf

Adaptable Hydrogel Networks with Reversible Linkages for Tissue Engineering

The ability to specifically silence genes using RNA interference (RNAi) has wide therapeutic applications for the treatment of disease or the augmentation of tissue formation. RNAi is the sequence-specific gene silencing mediated by a 21-25 nucleotide double-stranded small interfering RNA (siRNA) molecule. siRNAs are incorporated into the RNA-induced silencing complex (RISC), which mediates mRNA sequence-specific binding and cleavage. Although RNAi has the potential to be a powerful therapeutic drug, its delivery remains a major limitation. The generation of nanosized particles is being investigated to enhance the delivery of siRNA-based drugs. These nanoparticles are generally designed to overcome one or more of the barriers encountered by the siRNA when trafficked to the cytosol. In this review, we will discuss recent advances in the design of delivery strategies for siRNA, focusing our attention to those strategies that have had in vivo success or have introduced novel functionality that allowed enhanced intracellular trafficking and/or cellular targeting. First, we will discuss the different barriers that must be overcome for efficient siRNA delivery. Second, we will discuss the approaches for siRNA delivery by size including direct modification of siRNAs (less than 10 nm), self-assembled particles based on cationic polymers and cationic lipids (100-300 nm), neutral liposomes ( 100 microm). Finally, we will briefly discuss recent in vivo therapeutic success.

/pdf/sirna-applications-in-nanomedicine-1vrc4k44b9.pdf

siRNA applications in nanomedicine.

The treatment of impaired wounds requires the use of biomaterials that can provide mechanical and biological queues to the surrounding environment to promote angiogenesis, granulation tissue formation, and wound closure. Porous hydrogels show promotion of angiogenesis, even in the absence of proangiogenic factors. It is hypothesized that the added delivery of nonviral DNA encoding for proangiogenic growth factors can further enhance this effect. Here, 100 and 60 μm porous and nonporous (n-pore) hyaluronic acid-MMP hydrogels with encapsulated reporter (pGFPluc) or proangiogenic (pVEGF) plasmids are used to investigate scaffold-mediated gene delivery for local gene therapy in a diabetic wound healing mouse model. Porous hydrogels allow for significantly faster wound closure compared with n-pore hydrogels, which do not degrade and essentially provide a mechanical barrier to closure. Interestingly, the delivery of pDNA/PEI polyplexes positively promotes granulation tissue formation even when the DNA does not encode for an angiogenic protein. And although transfected cells are present throughout the granulation tissue surrounding, all hydrogels at 2 weeks, pVEGF delivery does not further enhance the angiogenic response. Despite this, the presence of transfected cells shows promise for the use of polyplex-loaded porous hydrogels for local gene delivery in the treatment of diabetic wounds.

/pdf/porous-hyaluronic-acid-hydrogels-for-localized-nonviral-dna-4zg9cetf51.pdf

Porous Hyaluronic Acid Hydrogels for Localized Nonviral DNA Delivery in a Diabetic Wound Healing Model

The effective delivery of DNA locally would increase the applicability of gene therapy in tissue regeneration, where diseased tissue is to be repaired in situ. One promising approach is to use hydrogel scaffolds to encapsulate and deliver plasmid DNA in the form of nanoparticles to the diseased tissue, so that cells infiltrating the scaffold are transfected to induce regeneration. This study focuses on the design of a DNA nanoparticle-loaded hydrogel scaffold. In particular, this study focuses on understanding how cell-matrix interactions affect gene transfer to adult stem cells cultured inside matrix metalloproteinase (MMP) degradable hyaluronic acid (HA) hydrogel scaffolds. HA was cross-linked to form a hydrogel material using a MMP degradable peptide and Michael addition chemistry. Gene transfer inside these hydrogel materials was assessed as a function of polyplex nitrogen to phosphate ratio (N/P = 5 to 12), matrix stiffness (100-1700 Pa), RGD (Arg-Gly-Asp) concentration (10-400 μM), and RGD presentation (0.2-4.7 RGDs per HA molecule). All variables were found to affect gene transfer to mouse mensenchymal stem cells culture inside the DNA loaded hydrogels. As expected, higher N/P ratios lead to higher gene transfer efficiency but also higher toxicity; softer hydrogels resulted in higher transgene expression than stiffer hydrogels, and an intermediate RGD concentration and RGD clustering resulted in higher transgene expression. We believe that the knowledge gained through this in vitro model can be utilized to design better scaffold-mediated gene delivery for local gene therapy.

/pdf/utilizing-cell-matrix-interactions-to-modulate-gene-transfer-sgh8opf1c9.pdf

Utilizing Cell–Matrix Interactions To Modulate Gene Transfer to Stem Cells Inside Hyaluronic Acid Hydrogels

Non-viral DNA delivery from porous hyaluronic acid hydrogels in mice.

Although the natural extracellular matrix (ECM) is primarily a self-assembled structure, a large emphasis has been placed in recent years on the development of covalently crosslinked hydrogel scaffolds for tissue regeneration and stem cell transplantation. These hydrogels allow for cell spreading and migration through cell-mediated protease degradation [1, 2] or hydrolytic hydrogel degradation [3, 4]. However, in vivo cells do not always migrate and spread following matrix degradation; cells are able to navigate through protein fibers [5, 6]. Further, the requirement of matrix degradation to achieve cell migration and spreading results in a change in the mechanical properties of these hydrogels with time, which makes it challenging to decouple chemical cues from mechanical cues. Hydrogel materials that can mimic non-protease mediated cell spreading and migration and do not require active matrix degradation to achieve these cellular processes can offer an alternative system to study stem cell differentiation in vitro and overcome some of the current limitations of purely covalently crosslinked hydrogels.

/pdf/physically-associated-synthetic-hydrogels-with-long-term-2y02dt0osk.pdf

Talar Tokatlian

Papers

siRNA applications in nanomedicine.

Porous Hyaluronic Acid Hydrogels for Localized Nonviral DNA Delivery in a Diabetic Wound Healing Model

Utilizing Cell–Matrix Interactions To Modulate Gene Transfer to Stem Cells Inside Hyaluronic Acid Hydrogels

Non-viral DNA delivery from porous hyaluronic acid hydrogels in mice.

Physically Associated Synthetic Hydrogels with Long-Term Covalent Stabilization for Cell Culture and Stem Cell Transplantation