Mussel-inspired biomaterials: From chemistry to clinic.

After several billions of years, nature still makes decisions on its own to identify, develop, and direct the most effective material for phenomena/challenges faced. Likewise, and inspired by the nature, we learned how to take steps in developing new technologies and materials innovations. Wet and strong adhesion by Mytilidae mussels (among which Mytilus edulis-blue mussel and Mytilus californianus-California mussel are the most well-known species) has been an inspiration in developing advanced adhesives for the moist condition. The wet adhesion phenomenon is significant in designing tissue adhesives and surgical sealants. However, a deep understanding of engaged chemical moieties, microenvironmental conditions of secreted proteins, and other contributing mechanisms for outstanding wet adhesion mussels are essential for the optimal design of wet glues. In this review, all aspects of wet adhesion of Mytilidae mussels, as well as different strategies needed for designing and fabricating wet adhesives are discussed from a chemistry point of view. Developed muscle-inspired chemistry is a versatile technique when designing not only wet adhesive, but also, in several more applications, especially in the bioengineering area. The applications of muscle-inspired biomaterials in various medical applications are summarized for future developments in the field.

Polylysine for skin regeneration: A review of recent advances and future perspectives.

There have been several attempts to find promising biomaterials for skin regeneration, among which polylysine (a homopolypeptide) has shown benefits in the regeneration and treatment of skin disorders. This class of biomaterials has shown exceptional abilities due to their macromolecular structure. Polylysine-based biomaterials can be used as tissue engineering scaffolds for skin regeneration, and as drug carriers or even gene delivery vectors for the treatment of skin diseases. In addition, polylysine can play a preservative role in extending the lifetime of skin tissue by minimizing the appearance of photodamaged skin. Research on polylysine is growing today, opening new scenarios that expand the potential of these biomaterials from traditional treatments to a new era of tissue regeneration. This review aims to address the basic concepts, recent trends, and prospects of polylysine-based biomaterials for skin regeneration. Undoubtedly, this class of biomaterials needs further evaluations and explorations, and many critical questions have yet to be answered.

Advanced Delivery Systems Based on Lysine or Lysine Polymers.

Polylysine and materials that integrate lysine form promising drug delivery platforms. As a cationic macromolecule, a polylysine polymer electrostatically interacts with cells and is efficiently internalized, thereby enabling intracellular delivery. Although polylysine is intrinsically pH-responsive, the conjugation with different functional groups imparts smart, stimuli-responsive traits by adding pH-, temperature-, hypoxia-, redox-, and enzyme-responsive features for enhanced delivery of therapeutic agents. Because of such characteristics, polylysine has been used to deliver various cargos such as small-molecule drugs, genes, proteins, and imaging agents. Furthermore, modifying contrast agents with polylysine has been shown to improve performance, including increasing cellular uptake and stability. In this review, the use of lysine residues, peptides, and polymers in various drug delivery systems has been discussed comprehensively to provide insight into the design and robust manufacturing of lysine-based delivery platforms.

Biodegradable zwitterionic poly(carboxybetaine) microgel for sustained delivery of antibodies with extended stability and preserved function.

Many recent innovative treatments are based on monoclonal antibodies (mAbs) and other protein therapies. Nevertheless, sustained subcutaneous, oral or pulmonary delivery of such therapeutics is limited by the poor stability, short half-life, and non-specific interactions between the antibody (Ab) and delivery vehicle. Protein stabilizers (osmolytes) such as carboxybetaine can prevent non-specific interactions within proteins. In this work, a biodegradable zwitterionic poly(carboxybetaine), pCB, based microgel covalently crosslinked with tetra(ethylene glycol) diacrylate (TTEGDA) was synthesized for Ab encapsulation. The resulting microgels were characterized via FTIR, diffusion NMR, small-angle neutron scattering (SANS), and cell culture studies. The microgels were found to contain up to 97.5% water content and showed excellent degradability that can be tuned with crosslinking density. Cell compatibility of the microgel was studied by assessing the toxicity and immunogenicity in vitro. Cells exposed to microgel showed complete viability and no pro-inflammatory secretion of interleukin 6 (IL6) or tumor necrosis factor-alpha (TNFα). Microgel was loaded with Immunoglobulin G (as a model Ab), using a post-fabrication loading technique, and Ab sustained release from microgels of varying crosslinking densities was studied. The released Abs (especially from the high crosslinked microgels) proved to be completely active and able to bind with Ab receptors. This study opens a new horizon for scientists to use such a platform for local delivery of Abs to the desired target with minimized non-specific interactions.

Injectable Cell-Laden Hydrogels for Tissue Engineering: Recent Advances and Future Opportunities.

Tissue engineering intends to create functionalized tissues/organs for regenerating the injured parts of the body using cells and scaffolds. A scaffold as a supporting substrate affects the cells' fate and behavior, including growth, proliferation, migration, and differentiation. Hydrogel as a biomimetic scaffold plays an important role in cellular behaviors and tissue repair, providing a microenvironment close to the extracellular matrix with adjustable mechanical and chemical features that can provide sufficient nutrients and oxygen. To enhance the hydrogel performance and compatibility with native niche, the cell-laden hydrogel is an attractive choice to mimic the function of the targeted tissue. Injectable hydrogels, due to the injectability, are ideal options for in vivo minimally invasive treatment. Cell-laden injectable hydrogels can be utilized for tissue regeneration in a noninvasive way. This article reviews the recent advances and future opportunities of cell-laden injectable hydrogels and their functions in tissue engineering. It is expected that this strategy allows medical scientists to develop a minimally invasive method for tissue regeneration in clinical settings. Impact statement Cell-laden hydrogels have been vastly utilized in biomedical application, especially tissue engineering. It is expected that this upcoming review article will be a motivation for the community. Although this strategy is still in its early stages, this concept is so alluring that it has attracted all scientists in the community and specialists at academic health centers. Certainly, this approach requires more development, and a bunch of crucial challenges have yet to be solved. In this review, we discuss this various aspects of this approach, the questions that must be answered, the expectations associated with it, and rational restrictions to develop injectable cell-laden hydrogels.

Surface Modification of Adenovirus Vector to Improve Immunogenicity and Tropism.

Although adenovirus is a popular vector for delivering genes, there are several drawbacks that limit its effectiveness, including tropism and both the innate and adaptive immune responses. One approach that has been used to ameliorate these drawbacks is PEGylation of the virus with subsequent modification to add functional moieties for the purpose of cell targeting or enhancing infection. Here, we describe a general approach for PEGylating adenovirus and conjugating cell-penetrating peptides to the surface of the virus to impart the ability to transduce CAR-negative cells.

Reprogramming the rapid clearance of thrombolytics by nanoparticle encapsulation and anchoring to circulating red blood cells.

Rapid clearance of thrombolytics from blood following intravenous injection is a major clinical challenge in cardiovascular medicine. To overcome this barrier, nanoparticle (NP) based drug delivery systems have been reported. Although superior than conventional therapy, a large proportion of the injected NP is still cleared by the reticuloendothelial system. Previously, we and others showed that ex vivo attachment of bioscavengers, thrombolytics, and nanoparticles (NPs) to glycophorin A receptors on red blood cells (RBCs) improved the blood half-life. This is promising, but ex-vivo approaches are cumbersome and challenging to translate clinically. Here, we developed a novel Ter119-polymeric NP containing tissue plasminogen activator for on-demand targeting of GPA receptors in vivo. Upon intravenous injection, the Ter119-NPs achieved remarkable RBC labeling efficiencies (>95%), resulting in marked enhancement of blood residence time of tPA from minutes to several days without any morphological, hematological, and histological complications. Our approach of RBC labeling with the NPs also prevented reticuloendothelial detections and the activations of innate and adaptive immune system. Data suggest that real-time targeting of therapeutics to RBC with NPs can potentially improve outcomes and reduce complications against a variety chronic disease.

Fabricating an electroactive injectable hydrogel based on pluronic-chitosan/aniline-pentamer containing angiogenic factor for functional repair of the hippocampus ischemia rat model.

The hippocampus, a critical cerebral region involved in learning and memory formation, is especially vulnerable to ischemic defect. Here, we developed an injectable electroactive hydrogel based on pluronic-chitosan/aniline-pentamer with proper conductivity around 10-4 S/cm to achieve the functional repair of the hippocampus following the ischemic defect. FTIR, DSC, and TGA measurements were performed to assess the chemical structure and thermal stability of the synthesized hydrogel. Aniline pentamer decreased the swelling capacity, degradation, and drug release rate. Further, contact angle, melting point, and gelation time of hydrogels were enhanced by addition of aniline oligomer. Moreover, it endowed the on-demand electro-responsive drug release. Injectability of hydrogel was evaluated by rheometry, exhibiting proper gelling time at the body temperature. The ionic/electrical conductivity and desired in vitro biocompatibility with PC12 cells were also achieved. Injection of VEGF-loaded electroactive hydrogel in the hippocampal ischemic animal model resulted in decreased infarction volume, improved hippocampal dependent learning, and memory performance. Taken all together, the results confirmed that fabricated injectable hydrogel would be a suitable candidate for ischemic defect treatment and can lead to new horizons to treat neurological disorders.

Agarose-based biomaterials for advanced drug delivery.

Agarose is a prominent marine polysaccharide representing reversible thermogelling behavior, outstanding mechanical properties, high bioactivity, and switchable chemical reactivity for functionalization. As a result, agarose has received particular attention in the fabrication of advanced delivery systems as sophisticated carriers for therapeutic agents. The ever-growing use of agarose-based biomaterials for drug delivery systems resulted in rapid growth in the number of related publications, however still, a long way should be paved to achieve FDA approval for most of the proposed products. This review aims at a classification of agarose-based biomaterials and their derivatives applicable for controlled/targeted drug delivery purposes. Moreover, it attempts to deal with opportunities and challenges associated with the future developments ahead of agarose-based biomaterials in the realm of advanced drug delivery. Undoubtedly, this class of biomaterials needs further advancement, and a lot of critical questions have yet to be answered.

Zeolites for theranostic applications.

Theranostic platforms bring about a revolution in disease management. During recent years, theranostic nanoparticles have been utilized for imaging and therapy simultaneously. Zeolites, because of their porous structure and tunable properties, which can be modified with various materials, can be used as a delivery agent. The porous structure of a zeolite enables it to be loaded and unloaded with various molecules such as therapeutic agents, photosensitizers, biological macromolecules, MRI contrast agents, radiopharmaceuticals, near-infrared (NIR) fluorophores, and microbubbles. Furthermore, theranostic zeolite nanocarriers can be further modified with targeting ligands, which is highly interesting for targeted cancer therapies.

A rapid millifluidic synthesis of tunable polymer-protein nanoparticles.

Polymeric nanoparticles have drawn recent attention for their ability to enhance the efficacy of therapeutic proteins through reduced immunogenicity and extended circulation time. Though effective, most nanoparticle drug delivery systems are currently produced in batch processes that are limited in control parameters and scalability. To address these deficiencies, a millifluidic process was developed to encapsulate bovine serum albumin in poly(L-lysine)-grafted-poly(ethylene glycol) through an electrostatic self-assembly mechanism. The millifluidic process utilized ultrasonication to overcome the diffusional barriers to self-assembly in a laminar flow regime and produce a nanoparticle tunable by controlling the feed flow rate, tubing material, and ultrasonic power input. Nanoparticle diameters ranged from 13 to 300 nm with polydispersity index measurements ranging from 0.1 to 0.4. The copolymer fully encapsulated the protein in all system configurations and protected the encapsulated protein in the presence of proteases. Notably, the enzymatic activity of the millifluidic nanoparticles was both comparable to that of nanoparticles produced through the batch process and greater than that of the free protein, suggesting there is little difference in the self-assembly induced through the batch and millifluidic processes. This study presents the utility of millifluidics in the synthesis of polymer-protein nanoparticles and provides insight into the development of continuous processes for the production of nanoparticle drug delivery systems.

Interactions between Biomolecules and Zwitterionic Moieties: A Review.

Throughout the past decade, zwitterionic moieties have gained attention as constituents of biocompatible materials for exhibiting superhydrophilic properties that prevent nonspecific protein adsorption. Researchers have been working to synthesize zwitterionic materials for diverse biomedical applications such as drug delivery, protein stabilization, and surface modification of implantable materials. These zwitterionic materials have been used in assorted architectures, including protein conjugates, surface coatings, nanoparticles, hydrogels, and liposomes. Herein, we summarize recent advancements that further our understanding of interactions between biomolecules and zwitterionic moieties. We focus on the solution behavior of zwitterions and zwitterionic polymers and the molecular interactions between these molecules and biomolecules as determined by both experimental and theoretical studies. Further, we discuss the implications of using such interactions in vivo and how zwitterionic moieties may be incorporated to facilitate targeted delivery of proteins, genes, or small molecules. Finally, we discuss current knowledge gaps that need to be addressed to advance the field.

Poloxamer: A versatile tri-block copolymer for biomedical applications.

Poloxamers, also called Pluronic, belong to a unique class of synthetic tri-block copolymers containing central hydrophobic chains of poly(propylene oxide) sandwiched between two hydrophilic chains of poly(ethylene oxide). Some chemical characteristics of poloxamers such as temperature-dependent self-assembly and thermo-reversible behavior along with biocompatibility and physiochemical properties make poloxamer-based biomaterials promising candidates for biomedical application such as tissue engineering and drug delivery. The microstructure, bioactivity, and mechanical properties of poloxamers can be tailored to mimic the behavior of various types of tissues. Moreover, their amphiphilic nature and the potential to self-assemble into the micelles make them promising drug carriers with the ability to improve the drug availability to make cancer cells more vulnerable to drugs. Poloxamers are also used for the modification of hydrophobic tissue-engineered constructs. This article collects the recent advances in design and application of poloxamer-based biomaterials in tissue engineering, drug/gene delivery, theranostic devices, and bioinks for 3D printing. STATEMENT OF SIGNIFICANCE: Poloxamers, also called Pluronic, belong to a unique class of synthetic tri-block copolymers containing central hydrophobic chains of poly(propylene oxide) sandwiched between two hydrophilic chains of poly(ethylene oxide). The microstructure, bioactivity, and mechanical properties of poloxamers can be tailored to mimic the behavior of various types of tissues. Moreover, their amphiphilic nature and the potential to self-assemble into the micelles make them promising drug carriers with the ability to improve the drug availability to make cancer cells more vulnerable to drugs. However, no reports have systematically reviewed the critical role of poloxamer for biomedical applications. Research on poloxamers is growing today opening new scenarios that expand the potential of these biomaterials from "traditional" treatments to a new era of tissue engineering. To the best of our knowledge, this is the first review article in which such issue is systematically reviewed and critically discussed in the light of the existing literature.

Effect of redox-responsive DTSSP crosslinking on poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles for delivery of proteins.

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins.

From microporous to mesoporous mineral frameworks: An alliance between zeolite and chitosan.

Microporous and mesoporous minerals are key elements of advanced technological cycles nowadays. Nature-driven microporous materials are known for biocompatibility and renewability. Zeolite is known as an eminent microporous hydrated aluminosilicate mineral containing alkali metals. It is commercially available as adsorbent and catalyst. However, the large quantity of water uptake occupies active sites of zeolite making it less efficient. The widely-used chitosan polysaccharide has also been used in miscellaneous applications, particularly in medicine. However, inferior mechanical properties hampered its usage. Chitosan-modified zeolite composites exhibit superior properties compared to parent materials for innumerable requests. The alliance between a microporous and a biocompatible material with the accompaniment of negative and positive charges, micro/nanopores and proper mechanical properties proposes promising platforms for different uses. In this review, chitosan-modified zeolite composites and their applications have been overviewed.

Intracellular Delivery of Glucose Oxidase for Enhanced Cytotoxicity toward PSMA-Expressing Prostate Cancer Cells.

Reactive oxygen species (ROS) forming enzymes are of significant interest as anticancer agents due to their potent cytotoxicity. A key challenge in their clinical translation is attaining site-specific delivery and minimizing biodistribution to healthy tissues. Here, complexes composed of the ROS enzyme glucose oxidase (GOX), poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG), and anti-prostate specific membrane antigen (anti-PSMA) monoclonal antibody are synthesized for localized delivery and uptake in prostate cancer cells. Formation of anti-PSMA-PLL-g-PEG/GOX results in nanoscale complexes ≈30 nm in diameter with a ζ-potential of 6 mV. The anti-PSMA-PLL-g-PEG/GOX complexes show significant cytotoxicity (≈60% reduction in cell viability) against PSMA-expressing LNCaP cells compared to unmodified GOX. Importantly, cytotoxicity in LNCaP cells occurrs concurrently with anti-PSMA-PLL-g-PEG/GOX uptake and increases in intracellular generation of ROS. These results demonstrate that cytotoxicity of ROS inducing enzymes can be enhanced by intracellular delivery compared to equivalent concentrations of free enzyme, providing a novel means for cancer therapy.

Using CFD simulations and statistical analysis to correlate oxygen mass transfer coefficient to both geometrical parameters and operating conditions in a stirred-tank bioreactor.

Optimization of a bioreactor design can be an especially challenging process. For instance, testing different bioreactor vessel geometries and different impeller and sparger types, locations, and dimensions can lead to an exceedingly large number of configurations and necessary experiments. Computational fluid dynamics (CFD), therefore, has been widely used to model multiphase flow in stirred-tank bioreactors to minimize the number of optimization experiments. In this study, a multiphase CFD model with population balance equations are used to model gas-liquid mixing, as well as gas bubble distribution, in a 50 L single-use bioreactor vessel. The vessel is the larger chamber in an early prototype of a multichamber bioreactor for mammalian cell culture. The model results are validated with oxygen mass transfer coefficient (kL a) measurements within the prototype. The validated model is projected to predict the effect of using ring or pipe spargers of different sizes and the effect of varying the impeller diameter on kL a. The simulations show that ring spargers result in a superior kL a compared to pipe spargers, with an optimum sparger-to-impeller diameter ratio of 0.8. In addition, larger impellers are shown to improve kL a. A correlation of kL a is presented as a function of both the reactor geometry (i.e., sparger-to-impeller diameter ratio and impeller-to-vessel diameter ratio) and operating conditions (i.e., Reynolds number and gas flow rate). The resulting correlation can be used to predict kL a in a bioreactor and to optimize its design, geometry, and operating conditions.

Polyionic complexes of butyrylcholinesterase and poly-l-lysine-g-poly(ethylene glycol): Comparative kinetics of catalysis and inhibition and in vitro inactivation by proteases and heat.

We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly-l-lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26-35 nm). The three copolymer-complexed enzymes exhibited reduced kcat (30-33% reduction), but no significant changes in Km. Inhibitory potency (as reflected by the bimolecular rate constant, ki) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in ki (16-60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers.

Molecular and Biocompatibility Characterization of Red Blood Cell Membrane Targeted and Cell-Penetrating-Peptide-Modified Polymeric Nanoparticles.

Red blood cells (RBCs) express a variety of immunomodulatory markers that enable the body to recognize them as self. We have shown that RBC membrane glycophorin A (GPA) receptor can mediate membrane attachment of protein therapeutics. A critical knowledge gap is whether attaching drug-encapsulated nanoparticles (NPs) to GPA and modification with cell-penetrating peptide (CPP) will impact binding, oxygenation, and the induction of cellular stress. The objective of this study was to formulate copolymer-based NPs containing model fluorescent-tagged bovine serum albumin (BSA) with GPA-specific targeting ligands such as ERY1 (ENPs), single-chain variable antibody (scFv TER-119, SNPs), and low-molecular-weight protamine-based CPP (LNPs) and to determine their biocompatibility using a variety of complementary high-throughput in vitro assays. Experiments were conducted by coincubating NPs with RBCs at body temperature, and biocompatibility was evaluated by Raman spectroscopy, hemolysis, complement lysis, and oxidative stress assays. Data suggested that LNPs effectively targeted RBCs, conferring 2-fold greater uptake in RBCs compared to ENPs and SNPs. Raman spectroscopy results indicated no adverse effect of NP attachment or internalization on the oxygenation status of RBCs. Cellular stress markers such as glutathione, malondialdehyde, and catalase were within normal limits, and complement-mediated lysis due to NPs was negligible in RBCs. Under the conditions tested, our data demonstrates that molecular targeting of the RBC membrane is a feasible translational strategy for improving drug pharmacokinetics and that the proposed high-throughput assays can prescreen diverse NPs for preclinical and clinical biocompatibility.

Identification of Excipients for Stabilizing Fiberless Adenovirus as Biopharmaceuticals.

Reducing the promiscuous tropism of native adenovirus by using fiberless adenovirus is advantageous toward its use as a gene therapy vector or vaccine component. The removal of the fiber protein on native adenovirus abrogates several undesirable interactions; however, this approach decreases the particle's physical stability. To create stable fiberless adenovirus for pharmaceutical use, the effects of temperature and pH on the particle's stability profile must be addressed. Our results indicate that the stability of fiberless adenovirus is increased when it is stored in mildly acidic conditions around pH 6. The stability of fiberless adenovirus can be further enhanced by using excipients. Excipient screening results indicate that the nonionic surfactant Pluronic F-68 and the amino acid glycine are potential stabilizers because of their ability to increase the thermal transition temperature of the virus particle and promote retention of biological activity after exposure to prolonged thermal stress. Our data indicate that the instability of fiberless adenovirus can be ameliorated by storing the virus in the appropriate environment, and it should be possible to further optimize the virus so that it can be used as a biopharmaceutical.