The Future of Generic Biologics: Should the United States “Follow-On” the European Pathway?

The United States is embarking on a biotechnology drug revolution. In the last few decades, biotech drugs have saved millions of lives, and the market for these miracle cures continues to grow at an astronomical rate. Unfortunately, as the market for biotech drugs is skyrocketing, drug prices are following suit. As Congress strives to make these new drugs more affordable, it must not ignore significant safety concerns unique to these revolutionary therapies. Congress should follow the lead of the European Union to create an accessible pathway for generic forms of biotech drugs that includes strict regulatory measures to ensure drug safety and efficacy

Self-assembly of mechanoplasmonic bacterial cellulose-metal nanoparticle composites

Nanocomposites of metal nanoparticles (NPs) and bacterial nanocellulose (BC) enable fabrication of soft and biocompatible materials for optical, catalytic, electronic, and biomedical applications. Current BC-NP nanocomposites are typically prepared by in situ synthesis of the NPs or electrostatic adsorption of surface functionalized NPs, which limits possibilities to control and tune NP size, shape, concentration, and surface chemistry and influences the properties and performance of the materials. Here a self-assembly strategy is described for fabrication of complex and well-defined BC-NP composites using colloidal gold and silver NPs of different sizes, shapes, and concentrations. The self-assembly process results in nanocomposites with distinct biophysical and optical properties. In addition to antibacterial materials and materials with excellent senor performance, materials with unique mechanoplasmonic properties are developed. The homogenous incorporation of plasmonic gold NPs in the BC enables extensive modulation of the optical properties by mechanical stimuli. Compression gives rise to near-field coupling between adsorbed NPs, resulting in tunable spectral variations and enhanced broadband absorption that amplify both nonlinear optical and thermoplasmonic effects and enables novel biosensing strategies

Polypeptide functionalized gold nanoparticles for bioanalytical applications

Detection strategies that allow for simple, rapid, cost efficient and sensitive monitoring of proteins and their interactions with biomolecules are of great importance in drug development and diagnostics. This thesis describes the development of bioanalytical applications based on the tunable self-assembly of gold nanoparticles functionalized with a de novo designed polypeptide. Strategies for protein affinity sensing and for detection of several fundamentally important biological processes have been investigated, including Zn2+-mediated coordination between polypeptides and low molecular weight chelants and protease and phosphatase activity. A Zn2+ responsive synthetic polypeptide designed to fold into a helix-loop-helix motif and dimerize into a four-helix bundle has been used to control the stability and self-assembly of gold nanoparticles. This polypeptide has a high negative net charge at neutral pH as a consequence of its many glutamic acid residues, efficiently preventing folding and dimerization due to charge repulsion. Zn2+ coordination provides a means to trigger folding and dimerization at neutral pH. The polypeptide can be readily attached to gold nanoparticles via a cysteine residue in the loop region, retaining its folding properties and responsiveness to Zn2+. The polypeptide functionalized gold nanoparticles display excellent colloidal stability but aggregate reversibly after addition of millimolar concentrations of Zn2+. Aggregates are dense with a defined interparticle distance corresponding to the size of the four-helix bundle, resulting in a distinct red shift of the localized surface plasmon resonance band. Three completely different strategies for colorimetric biosensing have been developed, all being based on the same responsive hybrid nanomaterial. In the first strategy a synthetic receptor was co-immobilized on the gold nanoparticles together with the Zn2+ responsive polypeptide. Protein analyte binding to the receptor could be detected as this interaction sterically prevented aggregation induced by Zn2+. In the second strategy the reduction in colloidal stability caused by specific proteolytic cleavage of the immobilized polypeptide was exploited to monitor the enzymatic activity. The third strategy utilized the sensitivity of the system to small variations in Zn2+ concentration. The presence of low molecular weight chelants was found to influence the mode of aggregation, both by sequestering Zn2+ and through the formation of ternary complexes involving the polypeptides, which prevented dimerization and thus aggregation. This approach was further developed into a generic concept for phosphatase detection exploiting the different affinity of enzyme substrates and reaction products for Zn2+. The flexibility of the different detection schemes enables detection of a large number of analytes by exploiting the tunable stability of the nanoparticles and the possibilities to effectively decouple the recognition event and the nanoparticle stability modulation

Generic Phosphatase Activity Detection using Zinc Mediated Aggregation Modulation of Polypeptide-Modified Gold Nanoparticles

A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme. Phosphatase activity generates inorganic phosphate that forms an insoluble complex with Zn2+. In a sample containing a preset concentration of Zn2+, phosphatase activity will markedly reduce the concentration of dissolved Zn2+ from the original value, which in turn affects the aggregation of gold nanoparticles functionalized with a designed Zn2+ responsive polypeptide. The change in nanoparticle stability thus provides a rapid and sensitive readout of the phosphatase activity. The assay is not limited to a particular enzyme or enzyme substrate, which is demonstrated using three completely different phosphatases and five different substrates, and thus constitutes a highly interesting system for drug screening and diagnostics

Zinc-Triggered Hierarchical Self-Assembly of Fibrous Helix-Loop-Helix Peptide Superstructures for Controlled Encapsulation and Release

We demonstrate a novel route for hierarchical self-assembly of sub-micrometer-sized peptide superstructures that respond to subtle changes in Zn2+ concentration. The self-assembly process is triggered by a specific folding-dependent coordination of Zn2+ by a de novo designed nonlinear helix-loop-helix peptide, resulting in a propagating fiber formation and formation of spherical superstructures. The superstructures further form larger assemblies that can be completely disassembled upon removal of Zn2+ or degradation of the nonlinear peptide. This flexible and reversible assembly strategy of the superstructures enables facile encapsulation of nanoparticles and drugs that can be released by means of different stimuli.Funding Agencies|Swedish Research Council [621-2011-4319]; Swedish Foundation for Strategic Research [ICA10-0002]; Linkoping University; Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linkoping University [2009 00971]</p

Coiled coil-based therapeutics and drug delivery systems

Coiled coils are characterized by an arrangement of two or more alpha-helices into a superhelix and one of few protein motifs where the sequence-to-structure relationship to a large extent have been decoded and understood. The abundance of both natural and de novo designed coil coils provides a rich molecular toolbox for self assembly of elaborate bespoke molecular architectures, nanostructures, and materials. Leveraging on the numerous possibilities to tune both affinities and preferences for polypeptide oligomerization, coiled coils offer unique possibilities to design modular and dynamic assemblies that can respond in a predictable manner to biomolecular interactions and subtle physicochemical cues. In this review, strategies to use coiled coils in design of novel therapeutics and advanced drug delivery systems are discussed. The applications of coiled coils for generating drug carriers and vaccines, and various aspects of using coiled coils for controlling and triggering drug release, and for improving drug targeting and drug uptake are described. The plethora of innovative coiled coil-based molecular systems provide new knowledge and techniques for improving efficacy of existing drugs and can facilitate development of novel therapeutic strategies. (c) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). 1. Introduction 27 2. The coiled coil motif 27 3. Coiled coils and coiled coil-hybrids for drug delivery and therapeutics 30 3.1. Coiled coils in liposome drug delivery systems 30 3.2. Lipidated coiled coils for assembly of virus-like particles 31 3.3. Coiled coil nanoparticles 31 3.4. Coiled coil nanocarriers 33 3.5. Coiled coil polymer-hybrids 33 3.6. Coiled coil-based hydrogels 36 3.7. Coiled coil inorganic nanoparticle hybrids 37 3.8. Coiled coils combined with cell penetrating peptides 37 3.9. Coiled coils for improved targeting 38Funding Agencies|Swedish Foundation for Strategic Research (SFF)Swedish Foundation for Strategic Research [FFL15-0026, RMX18-0039]; Swedish Government Strategic Research Area inMaterials Science on Functional Materials at Linkoping University [2009-00971]; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation [KAW 2016.0231]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2017-04475]; Cancer Foundation [17 0534]</p

Peptide Decorated Gold Nanoparticle/Carbon Nanotube Electrochemical Sensor for Ultrasensitive Detection of Matrix Metalloproteinase-7

Matrix Metalloproteinase-7 (MMP-7) is a proteolytic enzyme overexpressed in different pathological conditions, including cancer, infection, and cardiovascular diseases, and is a relevant diagnostic biomarker and potential drug target. Here we demonstrate rapid and selective detection of MMP-7 with a limit-of-detection of 6 pg/mL and a dynamic range from 1 × 10−2 to 1 × 103 ng/mL using a peptide decorated gold nanoparticle/carbon nanotube electrochemical sensor. The sensor could be operated in diluted human serum and synthetic urine, with high specificity towards MMP-7. Moreover, the integration of nanomaterials in the sensing electrode significantly increased the signal-to-background ratio and strongly improved the stability of the sensor when compared to a conventional gold electrode. The simple and cost-effective fabrication and the ease of use make this sensor a very promising protease detection device for diagnostics and drug development

Folding driven self-assembly of a stimuli-responsive peptide-hyaluronan hybrid hydrogel

Protein-metal ion interactions are ubiquitous in nature and can be utilized for controlling the self-assembly of complex supramolecular architectures and materials. Here, a tunable supramolecular hydrogel is described, obtained by self-assembly of a Zn2+-responsive peptide-hyaluronic acid hybrid synthesized using strain promoted click chemistry. Addition of Zn2+ triggers folding of the peptides into a helix-loop-helix motif and dimerization into four-helix bundles, resulting in hydrogelation. Removal of the Zn2+ by chelators results in rapid hydrogel disassembly. Degradation of the hydrogels can also be time-programed by encapsulation of a hydrolyzing enzyme within the gel, offering multiple possibilities for modulating materials properties and release of encapsulated species. The hydrogel further shows potential antioxidant properties when evaluated using an in vitro model for reactive oxygen species.Funding Agencies|Swedish Research Council (VR); Swedish Foundation for Strategic Research (SSF); Carl Trygger Foundation; Forum Scientium; Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linkoping University (Faculty Grant SFO Mat LiU) [2009 00971]</p