Universal Nanocarrier Ink Platform for Biomaterials Additive Manufacturing.

Ink engineering is a fundamental area of research within additive manufacturing (AM) that designs next-generation biomaterials tailored for additive processes. During the design of new inks, specific requirements must be considered, such as flowability, postfabrication stability, biointegration, and controlled release of therapeutic molecules. To date, many (bio)inks have been developed; however, few are sufficiently versatile to address a broad range of applications. In this work, a universal nanocarrier ink platform is presented that provides tailored rheology for extrusion-based AM and facilitates the formulation of biofunctional inks. The universal nanocarrier ink (UNI) leverages reversible polymer-nanoparticle interactions to form a transient physical network with shear-thinning and self-healing properties engineered for direct ink writing (DIW). The unique advantage of the material is that a range of functional secondary polymers can be combined with the UNI to enable stabilization of printed constructs via secondary cross-linking as well as customized biofunctionality for tissue engineering and drug delivery applications. Specific UNI formulations are used for bioprinting of living tissue constructs and DIW of controlled release devices. The robust and versatile nature of the UNI platform enables rapid formulation of a broad range of functional inks for AM of advanced biomaterials.

Designed modular protein hydrogels for biofabrication.

Designing proteins that fold and assemble over different length scales provides a way to tailor the mechanical properties and biological performance of hydrogels. In this study, we designed modular proteins that self-assemble into fibrillar networks and, as a result, form hydrogel materials with novel properties. We incorporated distinct functionalities by connecting separate self-assembling (A block) and cell-binding (B block) domains into single macromolecules. The number of self-assembling domains affects the rigidity of the fibers and the final storage modulus G' of the materials. The mechanical properties of the hydrogels could be tuned over a broad range (G' = 0.1 - 10 kPa), making them suitable for the cultivation and differentiation of multiple cell types, including cortical neurons and human mesenchymal stem cells. Moreover, we confirmed the bioavailability of cell attachment domains in the hydrogels that can be further tailored for specific cell types or other biological applications. Finally, we demonstrate the versatility of the designed proteins for application in biofabrication as 3D scaffolds that support cell growth and guide their function. STATEMENT OF SIGNIFICANCE: Designed proteins that enable the decoupling of biophysical and biochemical properties within the final material could enable modular biomaterial engineering. In this context, we present a designed modular protein platform that integrates self-assembling domains (A blocks) and cell-binding domains (B blocks) within a single biopolymer. The linking of assembly domains and cell-binding domains this way provided independent tuning of mechanical properties and inclusion of biofunctional domains. We demonstrate the use of this platform for biofabrication, including neural cell culture and 3D printing of scaffolds for mesenchymal stem cell culture and differentiation. Overall, this work highlights how informed design of biopolymer sequences can enable the modular design of protein-based hydrogels with independently tunable biophysical and biochemical properties.

Biopolymer Nano-Network for Antimicrobial Peptide Protection and Local Delivery.

Antimicrobial resistance (AMR) develops when bacteria no longer respond to conventional antimicrobial treatment. The limited treatment options for resistant infections result in a significantly increased medical burden. Antimicrobial peptides offer advantages for treatment of resistant infections, including broad-spectrum activity and lower risk of resistance development. However, sensitivity to proteolytic cleavage often limits their clinical application. Here, a moldable and biodegradable colloidal nano-network is presented that protects bioactive peptides from enzymatic degradation and delivers them locally. An antimicrobial peptide, PA-13, is encapsulated electrostatically into positively and negatively charged nanoparticles made of chitosan and dextran sulfate without requiring chemical modification. Mixing and concentration of oppositely charged particles form a nano-network with the rheological properties of a cream or injectable hydrogel. After exposure to proteolytic enzymes, the formed nano-network loaded with PA-13 eliminates Pseudomonas aeruginosa during in vitro culture and in an ex vivo porcine skin model while the unencapsulated PA-13 shows no antibacterial effect. This demonstrates the ability of the nano-network to protect the antimicrobial peptide in an enzyme-challenged environment, such as a wound bed. Overall, the nano-network presents a useful platform for antimicrobial peptide protection and delivery without impacting peptide bioactivity.

Supramolecular Reinforcement of Polymer-Nanoparticle Hydrogels for Modular Materials Design.

Moldable hydrogels are increasingly used as injectable or extrudable materials in biomedical and industrial applications owing to their ability to flow under applied stress (shear-thin) and reform a stable network (self-heal). Nanoscale components can be added to dynamic polymer networks to modify their mechanical properties and broaden the scope of applications. Viscoelastic polymer-nanoparticle (PNP) hydrogels comprise a versatile and tunable class of dynamic nanocomposite materials that form via reversible interactions between polymer chains and nanoparticles. However, PNP hydrogel formation is restricted to specific interactions between select polymers and nanoparticles, resulting in a limited range of mechanical properties and constraining their utility. Here, a facile strategy to reinforce PNP hydrogels through the simple addition of α-cyclodextrin (αCD) to the formulation is introduced. The formation of polypseudorotoxanes between αCD and the hydrogel components resulted in a drastic enhancement of the mechanical properties. Furthermore, supramolecular reinforcement of CD-PNP hydrogels enabled decoupling of the mechanical properties and material functionality. This allows for modular exchange of structural components from a library of functional polymers and nanoparticles. αCD supramolecular binding motifs are leveraged to form CD-PNP hydrogels with biopolymers for high-fidelity 3D (bio)printing and drug delivery as well as with inorganic NPs to engineer magnetic or conductive materials.

Surface Tension-Assisted Additive Manufacturing of Tubular, Multicomponent Biomaterials.

The fabrication of functional biomaterials for organ replacement and tissue repair remains a major goal of biomedical engineering. Advances in additive manufacturing (AM) technologies and computer-aided design (CAD) are advancing the tools available for the production of these devices. Ideally, these constructs should be matched to the geometry and mechanical properties of the tissue at the needed implant site. To generate geometrically defined and structurally supported multicomponent and cell-laden biomaterials, we have developed a method to integrate hydrogels with 3D-printed lattice scaffolds leveraging surface tension-assisted AM.

Hierarchical biomaterials via photopatterning-enhanced direct ink writing.

Recent advances in additive manufacturing (AM) technologies provide tools to fabricate biological structures with complex three-dimensional (3D) organization. Deposition-based approaches have been exploited to manufacture multimaterial constructs. Stimulus-triggered approaches have been used to fabricate scaffolds with high resolution. Both features are useful to produce biomaterials that mimic the hierarchical organization of human tissues. Recently, multitechnology biofabrication approaches have been introduced that integrate benefits from different AM techniques to enable more complex materials design. However, few methods allow for tunable properties at both micro- and macro-scale in materials that are conducive for cell growth. To improve the organization of biofabricated constructs, we integrated direct ink writing (DIW) with digital light processing (DLP) to form multimaterial constructs with improved spatial control over final scaffold mechanics. Polymer-nanoparticle hydrogels were combined with methacryloyl gelatin (GelMA) to engineer dual inks that were compatible with both DIW and DLP. The shear-thinning and self-healing properties of the dual inks enabled extrusion-based 3D printing. The inclusion of GelMA provided a handle for spatiotemporal control of cross-linking with DLP. Exploiting this technique, complex multimaterial constructs were printed with defined mechanical reinforcement. In addition, the multitechnology approach was used to print live cells for biofabrication applications. Overall, the combination of DIW and DLP is a simple and efficient strategy to fabricate hierarchical biomaterials with user-defined control over material properties at both micro- and macro-scale.

Additive manufacturing in drug delivery: Innovative drug product design and opportunities for industrial application.

Additive manufacturing (AM) or 3D printing is enabling new directions in product design. The adoption of AM in various industrial sectors has led to major transformations. Similarly, AM presents new opportunities in the field of drug delivery, opening new avenues for improved patient care. In this review, we discuss AM as an innovative tool for drug product design. We provide a brief overview of the different AM processes and their respective impact on the design of drug delivery systems. We highlight several enabling features of AM, including unconventional release, customization, and miniaturization, and discuss several applications of AM for the fabrication of drug products. This includes products that have been approved or are in development. As the field matures, there are also several new challenges to broad implementation in the pharmaceutical landscape. We discuss several of these from the regulatory and industrial perspectives and provide an outlook for how these issues may be addressed. The introduction of AM into the field of drug delivery is an enabling technology and many new drug products can be created through productive collaboration of engineers, materials scientists, pharmaceutical scientists, and industrial partners.

Additive Manufacturing of Precision Biomaterials.

Biomaterials play a critical role in modern medicine as surgical guides, implants for tissue repair, and as drug delivery systems. The emerging paradigm of precision medicine exploits individual patient information to tailor clinical therapy. While the main focus of precision medicine to date is the design of improved pharmaceutical treatments based on "-omics" data, the concept extends to all forms of customized medical care. This includes the design of precision biomaterials that are tailored to meet specific patient needs. Additive manufacturing (AM) enables free-form manufacturing and mass customization, and is a critical enabling technology for the clinical implementation of precision biomaterials. Materials scientists and engineers can contribute to the realization of precision biomaterials by developing new AM technologies, synthesizing advanced (bio)materials for AM, and improving medical-image-based digital design. As the field matures, AM is poised to provide patient-specific tissue and organ substitutes, reproducible microtissues for drug screening and disease modeling, personalized drug delivery systems, as well as customized medical devices.

Automated and Continuous Production of Polymeric Nanoparticles.

Polymeric nanoparticles (NPs) are increasingly used as therapeutics, diagnostics, and building blocks in (bio)materials science. Current barriers to translation are limited control over NP physicochemical properties and robust scale-up of their production. Flow-based devices have emerged for controlled production of polymeric NPs, both for rapid formulation screening (~µg min-1) and on-scale production (~mg min-1). While flow-based devices have improved NP production compared to traditional batch processes, automated processes are desired for robust NP production at scale. Therefore, we engineered an automated coaxial jet mixer (CJM), which controlled the mixing of an organic stream containing block copolymer and an aqueous stream, for the continuous nanoprecipitation of polymeric NPs. The CJM was operated stably under computer control for up to 24 h and automated control over the flow conditions tuned poly(ethylene glycol)-block-polylactide (PEG5K -b-PLA20K ) NP size between ≈56 nm and ≈79 nm. In addition, the automated CJM enabled production of NPs of similar size (D h ≈ 50 nm) from chemically diverse block copolymers, PEG5K -b-PLA20K , PEG-block-poly(lactide-co-glycolide) (PEG5K -b-PLGA20K ), and PEG-block-polycaprolactone (PEG5K -b-PCL20K ), by tuning the flow conditions for each block copolymer. Further, the automated CJM was used to produce model nanotherapeutics in a reproducible manner without user intervention. Finally, NPs produced with the automated CJM were used to scale the formation of injectable polymer-nanoparticle (PNP) hydrogels, without modifying the mechanical properties of the PNP gel. In conclusion, the automated CJM enabled stable, tunable, and continuous production of polymeric NPs, which are needed for the scale-up and translation of this important class of biomaterials.