ABSTRACT
Nonspecific interactions between cells and implantable elastomers often leads to failure modes for devices such as catheters, cosmetic and reconstructive implants, and sensors. To reduce these interactions, device surfaces can be coated with hydrophilic polymers, where greater polymer density enhances antifouling properties. Although graft-from coating techniques result in higher density polymer films and lower fouling in controlled settings, simpler graft-to methods show similar results on complex implanted devices, despite limited density. To address the need for improved graft-to methods, we developed Graft then shrink (GtS) where elastomeric materials are temporarily swollen during polymer grafting. Herein, we demonstrate a graft-to based method for poly(oligo(ethylene glycol) methyl ether methacrylate) (pOEGMA) on swollen silicone, GtS, that enhances grafted polymer content and fouling resistance. Total grafted polymer content of pOEGMA on toluene swollen silicone increased over ~ 13 × compared to non-swollen controls, dependent on the degree of silicone swelling. Increases in total grafted polymer within the top 200 µm of the material led to bacterial and mammalian cell adhesion reductions of 75% and 91% respectively, compared to Shrink then Graft (StG) antifouling polymer coated controls. GtS allows for the simple 3D coating of swellable elastomers (e.g., silicone medical devices) with improved antifouling pOEGMA coatings.
Subject(s)
Biofouling , Polymers , Animals , Biofouling/prevention & control , Coated Materials, Biocompatible , Elastomers , Silicones , MammalsABSTRACT
Chemical immunotherapeutic strategies including Antibody Recruiting Molecules (ARMs - bivalent small molecules containing an antibody-binding domain (ABD) and a target-binding domain (TBD)) direct immune-mediated clearance of diseased cells. Anti-cancer ARM function relies on high tumor antigen valency, limiting function against lower antigen expressing tumors. To address this limitation, we report a tunable multivalent immune recruitment (MIR) platform to amplify/stabilize antibody recruitment to cells with lower antigen valencies. An initial set of polymeric ARMs (pARMs) were synthesized and screened to evaluate ABD/TBD copy number, ratio, and steric occlusion on specific immune induction. Most pARMs demonstrated simultaneous high avidity binding to anti-dinitrophenyl antibodies and prostate-specific membrane antigens on prostate cancer. Optimized pARMs mediated enhanced anti-cancer immune function against lower antigen expressing target cells compared to an analogous ARM.
Subject(s)
Antigens , Prostatic Neoplasms , Male , Humans , Antibodies/chemistry , PhagocytosisABSTRACT
Antifouling polymer coatings that are simple to manufacture are crucial for the performance of medical devices such as biosensors. "Grafting-to", a simple technique where presynthesized polymers are immobilized onto surfaces, is commonly employed but suffers from nonideal polymer packing leading to increased biofouling. Herein, we present a material prepared via the grafting-to method with improved antifouling surface properties and intrinsic localized surface plasmon resonance (LSPR) sensor capabilities. A new substrate shrinking fabrication method, Graft-then-Shrink, improved the antifouling properties of polymer-coated Au surfaces by altering graft-to polymer packing while simultaneously generating wrinkled Au structures for LSPR biosensing. Thiol-terminated, antifouling, hydrophilic polymers were grafted to Au-coated prestressed polystyrene (PS) followed by shrinking upon heating above the PS glass transition temperature. Interestingly, the polymer molecular weight and hydration influenced Au wrinkling patterns. Compared to Shrink-then-Graft controls, where polymers are immobilized post shrinking, Graft-then-Shrink increased the polymer content by 76% in defined footprints and improved the antifouling properties as demonstrated by 84 and 72% reduction in macrophage adhesion and protein adsorption, respectively. Wrinkled Au LSPR sensors had sensitivities of â¼200-1000 Δλ/ΔRIU, comparing favorably to commercial LSPR sensors, and detected biotin-avidin and desthiobiotin-avidin complexation in a concentration-dependent manner using a standard plate reader and a 96-well format.
ABSTRACT
Sustained release is being explored to increase plasma and tissue residence times of polymer-protein therapeutics for improved efficacy. Recently, poly(oligo(ethylene glycol) methyl ether methacrylate) (PEGMA) polymers have been established as potential PEG alternatives to further decrease immunogenicity and introduce responsive or sieving properties. We developed a drug delivery system that locally depresses the lower critical solution temperature (LCST) of PEGMA-protein conjugates within zwitterionic hydrogels for controlled release. Inside the hydrogel the conjugates partially aggregate through PEGMA-PEGMA chain interactions to limit their release rates, whereas conjugates outside of the hydrogel are completely solubilized. Release can therefore be tuned by altering hydrogel components and the PEGMA's temperature sensitivity without the need for traditional controlled release mechanisms such as particle encapsulation or affinity interactions. Combining local LCST depression technology and degradable zwitterionic hydrogels, complete release of the conjugate was achieved over 13 days.
ABSTRACT
Uncontrolled protein adsorption and cell binding to biomaterial surfaces may lead to degradation, implant failure, infection, and deleterious inflammatory and immune responses. The accurate characterization of biofouling is therefore crucial for the optimization of biomaterials and devices that interface with complex biological environments composed of macromolecules, fluids, and cells. Currently, a diverse array of experimental conditions and characterization techniques are utilized, making it difficult to compare reported fouling values between similar or different biomaterials. This review aims to help scientists and engineers appreciate current limitations and conduct fouling experiments to facilitate the comparison of reported values and expedite the development of low-fouling materials. Recent advancements in the understanding of protein-interface interactions and fouling variability due to experiment conditions will be highlighted to discuss protein adsorption and cell adhesion and activation on biomaterial surfaces.
ABSTRACT
Alpha synuclein (αS) oligomers are a key component of Lewy bodies implicated in Parkinson's disease (PD). Although primarily intracellular, extracellular αS exocytosed from neurons also contributes to PD pathogenesis through a prion-like transmission mechanism. Here, we show at progressive degrees of resolution that the most abundantly expressed extracellular protein, human serum albumin (HSA), inhibits αS oligomer (αSn) toxicity through a three-pronged mechanism. First, endogenous HSA targets αSn with sub-µM affinity via solvent-exposed hydrophobic sites, breaking the catalytic cycle that promotes αS self-association. Second, HSA remodels αS oligomers and high-MW fibrils into chimeric intermediates with reduced toxicity. Third, HSA unexpectedly suppresses membrane interactions with the N-terminal and central αS regions. Overall, our findings suggest that the extracellular proteostasis network may regulate αS cell-to-cell transmission not only by reducing the populations of membrane-binding competent αS oligomers but possibly also by shielding the membrane interface from residual toxic species.
Subject(s)
Molecular Chaperones/metabolism , Serum Albumin, Human/metabolism , alpha-Synuclein/metabolism , Cell Line, Tumor , Cell Survival , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Chaperones/chemistry , Serum Albumin, Human/chemistry , alpha-Synuclein/chemistryABSTRACT
Low-fouling and high-loading surfaces are increasingly important for biosensing and blood purification technologies. Selective and efficient target binding from complex media can be achieved with poly(carboxybetaine) (pCB) surfaces that consist of a dense brush layer to resist non-specific protein adsorption and a sparse "mushroom" upper layer for high-density capture agent immobilization (i.e. high-loading). We developed pH-controlled surface-reversible addition-fragmentation chain-transfer (S-RAFT) polymerization to simplify fabrication of multi-modal, low-fouling and high-loading pCB surfaces without the need for quenching or re-initiation steps, toxic transition metals or light irradiation. Multi-modal polymer layers were produced through partial polymer termination by temporarily raising the pH to aminolyse a fraction of dormant chain transfer agents (CTAs); remaining polymer chains with intact CTAs continued uninterrupted extension to create the "mushroom" upper layer. The multi-modal pCB surfaces were low-fouling towards proteins (<6.7 ng cm-2), and macrophages. Compared to mono-modal brush surfaces, multi-modal pCB surfaces were high-loading with 5-fold greater capture agent immobilization (e.g. antibody) and 4-fold greater target binding (e.g. biotin-fluorescein).
ABSTRACT
Soluble amyloid beta assemblies (Aß n ) are neurotoxic and play a central role in the early phases of the pathogenesis cascade leading to Alzheimer's disease. However, the current knowledge about the molecular determinants of Aß n toxicity is at best scant. Here, we comparatively analyze Aß n prepared in the absence or presence of a catechin library that modulates cellular toxicity. By combining solution NMR with dynamic light scattering, fluorescence spectroscopy, electron microscopy, wide-angle X-ray diffraction and cell viability assays, we identify a cluster of unique molecular signatures that distinguish toxic vs. nontoxic Aß assemblies. These include the exposure of a hydrophobic surface spanning residues 17-28 and the concurrent shielding of the highly charged N-terminus. We show that the combination of these two dichotomous structural transitions promotes the colocalization and insertion of ß-sheet rich Aß n into the membrane, compromising membrane integrity. These previously elusive toxic surfaces mapped here provide an unprecedented foundation to establish structure-toxicity relationships of Aß assemblies.
ABSTRACT
Low-fouling hydrogels with tunable degradation rates and biochemical environments have the potential to improve adoptive cell therapies for cancer immunotherapy and regenerative medicine. To this end, we developed in situ gelling hydrogels from low-fouling poly(carboxybetaine-co-maleimide) (pCBM) random copolymers and thiolated hyaluronic acid (HA-SH). pCBM-HA hydrogel enzymatic degradation rates were tuned 5 fold by altering pCBM composition (4, 11, and 16 maleimide mol%) and 2.3 fold by HA-SH concentration (1-2 wt%). pCBM-HA gels were low-fouling towards bovine serum albumin (BSA; adsorbed â¼20 µg cm-2) and resisted fibroblast adhesion. To control pCBM-HA bioactivity, the cell adhesive peptide CGRGDS was immobilized on pCBM to promote fibroblast adhesion (39% decrease in circularity), which increased metabolic activity by â¼50%. pCBM-HA modified with CGRGDS enhanced the metabolic activity of encapsulated T cells by â¼21% compared to gels without HA, indicating their potential for immunotherapies. Low-fouling pCBM-HA hydrogels provide a vehicle with tunable degradation rates and biochemical environments for encapsulation applications in cell adoptive therapies.
Subject(s)
Betaine/chemistry , Hyaluronic Acid/chemistry , Hydrogels/chemistry , Regenerative Medicine/methods , Tissue Engineering/methods , Acrylamides/chemistry , Adsorption , Animals , Cell Adhesion , Cell Encapsulation , Cell Survival , Cells, Cultured , Fibroblasts/metabolism , Immunotherapy , Leukocytes, Mononuclear/cytology , Materials Testing , Mice , NIH 3T3 Cells , Peptides/chemistry , Polymers , Serum Albumin, Bovine/chemistry , T-Lymphocytes/cytology , Tissue Engineering/instrumentationABSTRACT
Poly(carboxybetaine) (pCB) hydrogels do not elicit a foreign body response due to their low-fouling properties, making them ideal implantable materials for in vivo drug and cell delivery. Current reported pCB hydrogels are cross-linked using cytotoxic UV-initiated radical polymerization limiting clinical and in vivo translation. For clinical translation, we require in situ and biorthogonal cross-linking of pCB hydrogels that are both low-fouling and low-swelling to limit nonspecific interactions and minimize tissue damage, respectively. To this end, we synthesized carboxybetaine (CB) random copolymers (molecular weight (MW): â¼7-33 kDa; D: 1.1-1.36) containing azide (pCB-azide) or strained alkyne (Dibenzocyclooctyne (DBCO); pCB-DBCO) that rapidly cross-link upon mixing. Unlike CB homopolymers and other CB copolymers studied, high DBCO content pCB-DBCO30 (30% DBCO mole fraction) is thermoresponsive with a upper critical solution temperature (UCST; cloud point of â¼20 °C at 50 g/L) in water due to electrostatic associations. Due to the antipolyelectrolyte effect, pCB-DBCO30 is salt-responsive and is soluble even at low temperatures in 5 M NaCl, which prevents zwitterion electrostatic associations. pCB-azide and pCB-DBCO with 0.05 to 0.16 cross-linker mole fractions rapidly formed 10 wt % hydrogels upon mixing that were low-swelling (increase of â¼10% in wet weight) while remaining low-fouling to proteins (â¼10-20 µg cm-2) and cells, making them suitable for in vivo applications. pCB-X31 hydrogels composed of pCB-azide32 and pCB-DBCO30 formed opaque gels in water and physiological conditions that shrunk to â¼70% of their original wet weight due to pCB-DBCO30's greater hydrophobicity and interchain electrostatic interactions, which promotes nonspecific protein adsorption (â¼35 µg cm-2) and cell binding. Once formed, the electrostatic interactions in pCB-X31 hydrogels are not fully reversible with heat or salt. Although, pCB-X31 hydrogels are transparent when initially prepared in 5 M NaCl. This is the first demonstration of a thermo- and salt-responsive CB copolymer that can tune hydrogel protein and cell fouling properties.
Subject(s)
Betaine/analogs & derivatives , Hydrogels/chemistry , Polymers/chemistry , Animals , Betaine/metabolism , Betaine/pharmacology , Biofouling/prevention & control , Cattle , Cell Adhesion/drug effects , Elastic Modulus , Hydrogels/metabolism , Hydrogels/pharmacology , Hydrophobic and Hydrophilic Interactions , Mice , NIH 3T3 Cells , Polymers/metabolism , Polymers/pharmacology , Protein Binding , Serum Albumin, Bovine/metabolism , Static ElectricityABSTRACT
Antibodies are a growing class of cancer immunotherapeutics that facilitate immune-cell-mediated killing of tumors. However, the efficacy and safety of immunotherapeutics are limited by transport barriers and poor tumor uptake, which lead to high systemic concentrations and potentially fatal side effects. To increase tumor antibody immunotherapeutic concentrations while decreasing systemic concentrations, local delivery vehicles for sustained antibody release are being developed. The focus of this review is to define the material properties required for implantable controlled antibody delivery and highlight the controlled-release strategies that are applicable to antibody immunotherapeutics.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Hydrogels/chemistry , Neoplasms/therapy , Animals , Antibodies, Monoclonal/administration & dosage , Delayed-Action Preparations , Immunotherapy/methodsABSTRACT
Degradable low-fouling hydrogels are ideal vehicles for drug and cell delivery. For each application, hydrogel degradation rate must be re-optimized for maximum therapeutic benefit. We developed a method to rapidly and predictably tune degradation rates of low-fouling poly(oligo(ethylene glycol)methyl ether methacrylate) (P(EG) x MA) hydrogels by modifying two interdependent variables: (1) base-catalysed crosslink degradation kinetics, dependent on crosslinker electronics (electron withdrawing groups (EWGs)); and, (2) polymer hydration, dependent on the molecular weight (M W) of poly(ethylene glycol) (PEG) pendant groups. By controlling PEG M W and EWG strength, P(EG) x MA hydrogels were tuned to degrade over 6 to 52 d. A 6-member P(EG) x MA copolymer library yielded slow and fast degrading low-fouling hydrogels suitable for short- and long-term delivery applications. The degradation mechanism was also applied to RGD-functionalized poly(carboxybetaine methacrylamide) (PCBMAA) hydrogels to achieve slow (â¼50 d) and fast (â¼13 d) degrading low-fouling, bioactive hydrogels.
ABSTRACT
Cytokines play an important role in dysregulated immune responses to infection, pancreatitis, ischemia/reperfusion injury, burns, hemorrhage, cardiopulmonary bypass, trauma, and many other diseases. Moreover, the imbalance between inflammatory and antiinflammatory cytokines can have deleterious effects. Here, we demonstrated highly selective blood-filtering devices - antibody-modified conduits (AMCs) - that selectively eliminate multiple specific deleterious cytokines in vitro. AMCs functionalized with antibodies against human vascular endothelial growth factor A or tumor necrosis factor α (TNF-α) selectively eliminated the target cytokines from human blood in vitro and maintained them in reduced states even in the face of ongoing infusion at supraphysiologic rates. We characterized the variables that determine AMC performance, using anti-human TNF-α AMCs to eliminate recombinant human TNF-α. Finally, we demonstrated selective cytokine elimination in vivo by filtering interleukin 1 ß from rats with lipopolysaccharide-induced hypercytokinemia.
Subject(s)
Antibodies/blood , Antibodies/immunology , Cytokines/blood , Cytokines/immunology , Animals , Humans , Interleukin-1beta , Lipopolysaccharides/pharmacology , Male , Rats , Rats, Sprague-Dawley , Recombinant Proteins , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/genetics , Vascular Endothelial Growth Factor A/blood , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Methods to reversibly control the chemical environment of hydrogels have application in three-dimensional cell culture to study cell proliferation, migration and differentiation in environments more representative of in vivo environments. Herein, we have developed a method to temporally control the chemical environment of agarose hydrogels through non-covalent attachment of peptide motifs. Streptavidin-GRGDS conjugates were immobilized in desthiobiotin-modified agarose hydrogels through the desthiobiotin-streptavidin interaction (KD 10-11 M). Streptavidin-GRGDS was then displaced from the gel by the addition of biotin, which has a higher affinity for streptavidin (KD 10-15 M). This process was repeated to sequentially and simultaneously immobilize different biomolecules and model compounds in hydrogels over the course of several hours to weeks. The influence of dynamic chemical environments on cellular activity was demonstrated by monitoring HUVEC tube formation for 30 h.
Subject(s)
Cell Culture Techniques , Culture Media/chemistry , Hydrogels/chemistry , Alginates/chemistry , Biotin/analogs & derivatives , Biotin/chemistry , Cell Differentiation , Cell Movement , Cell Proliferation , Fluorescent Dyes/chemistry , Human Umbilical Vein Endothelial Cells , Humans , Oligopeptides/chemistry , Peptides/chemistry , Sepharose/chemistry , Streptavidin/chemistryABSTRACT
With increased clinical use of antibodies, long-term delivery strategies are needed to decrease injection frequency and improve health outcomes. A three-component drug-delivery system was developed for competitive affinity release of a streptavidin-antibody conjugate from agarose-desthiobiotin hydrogels via controlled dissolution of sparingly soluble biotin derivatives. The antibody conjugate was localized in the hydrogel through streptavidin-desthiobiotin complexation. Dissolution of sparingly soluble biotin derivatives disrupts streptavidin-desthiobiotin complexation for controlled release of the antibody conjugate. Release was tuned by altering the total biotin derivative concentration without further hydrogel or antibody modification. First-order tunable release of bioactive Avastin, a therapeutic anti-VEGF antibody, was demonstrated from a non-cytotoxic system for over 100â days.
Subject(s)
Antibodies/pharmacology , Drug Carriers/chemistry , Hydrogels/chemistry , Biotin/analogs & derivatives , Biotin/chemistry , Delayed-Action Preparations/chemistry , Humans , Sepharose/chemistry , Streptavidin/chemistryABSTRACT
Preparation of soft materials with diverse, customized shapes has been a topic of intense research interest. To this end, we have recently demonstrated photolithographic directed assembly as a strategy for customizing polyelectrolyte complex (PEC) shape. This process uses in situ photopolymerization of an anionic monomer in the presence of a cationic polymer, which drives localized PEC formation at the irradiation sites. Here, we show how such photolithographically assembled PECs can serve as structure-directing templates for tailoring the shapes of other soft materials; namely, thermoreversible gels. These templated hydrogels are prepared by adding a thermogelling polymer (agarose) to the anionic monomer/cationic polymer/photoinitiator precursor solutions so that, upon irradiation, custom-shaped PECs form within agarose gel matrices. Once these PECs are formed, the surrounding agarose gels are melted (through heating) and washed away which, upon returning the samples to room temperature, produces interpenetrating PEC/agarose gel networks with photopatterned shapes and dimensions. Dissolution of these sacrificial PEC templates in concentrated NaCl solutions then generates photolithographically templated agarose gels, whose shapes and dimensions match those of their PEC templates. Besides tuning their shapes and sizes, the mechanical properties of these gels can be easily tailored by varying the initial agarose concentrations used. Moreover, this PEC-templated gel synthesis appears to not adversely affect hydrogel cytocompatibility, suggesting its potential suitability for biological and biomedical applications. Though the present study uses only agarose as the model gel system, this PEC-based strategy for customizing gel shape can likely also be applied to other thermoreversible gel networks (e.g., those based on methylcellulose, poloxamers or thermoresponsive chitosan derivatives) and could have many attractive applications, ranging from drug delivery and tissue engineering, to sensing and soft robotics.
ABSTRACT
Vascular endothelial growth factor 165 (VEGF165) is an important extracellular protein involved in pathological angiogenesis in diseases such as cancer, wet age-related macular degeneration (wet-AMD) and retinitis pigmentosa. VEGF165 exists in two different isoforms: the angiogenic VEGF165a, and the anti-angiogenic VEGF165b. In some angiogenic diseases the proportion of VEGF165b may be equal to or higher than that of VEGF165a. Therefore, developing therapeutics that inhibit VEGF165a and not VEGF165b may result in greater anti-angiogenic activity and therapeutic benefit. To this end, we report the selective binding properties of sulfated hyaluronic acid (s-HA). Selective biopolymers offer several advantages over antibodies or aptamers including cost effective and simple synthesis, and the ability to make nanoparticles or hydrogels for drug delivery applications or VEGF165a sequestration. Limiting sulfation to the C-6 hydroxyl (C-6 OH) in the N-acetyl-glucosamine repeat unit of hyaluronic acid (HA) resulted in a polymer with strong affinity for VEGF165a but not VEGF165b. Increased sulfation beyond the C-6 OH (i.e. greater than 1 sulfate group per HA repeat unit) resulted in s-HA polymers that bound both VEGF165a and VEGF165b. The C-6 OH sulfated HA (Mw 150 kDa) showed strong binding properties to VEGF165a with a fast association rate constant (Ka; 2.8 × 10(6) M(-1) s(-1)), slow dissociation rate constant (Kd; 2.8 × 10(-3) s(-1)) and strong equilibrium binding constant (KD; â¼1.0 nM)), which is comparable to the non-selective VEGF165 binding properties of the commercialized therapeutic anti-VEGF antibody (Avastin(®)). The C-6 OH sulfated HA also inhibited human umbilical vein endothelial cell (HUVEC) survival and proliferation and human dermal microvascular endothelial cell (HMVEC) tube formation. These results demonstrate that the semi-synthetic natural polymer, C-6 OH sulfated HA, may be a promising biomaterial for the treatment of angiogenesis-related disease.
Subject(s)
Angiogenesis Inhibitors/metabolism , Hyaluronic Acid/analogs & derivatives , Vascular Endothelial Growth Factor A/metabolism , Amino Acid Sequence , Angiogenesis Inhibitors/chemical synthesis , Angiogenesis Inhibitors/pharmacology , Bevacizumab/metabolism , Carbohydrate Conformation , Chondroitin Sulfates/metabolism , Drug Evaluation, Preclinical , Human Umbilical Vein Endothelial Cells , Humans , Hyaluronic Acid/chemical synthesis , Hyaluronic Acid/metabolism , Hyaluronic Acid/pharmacology , Kinetics , Molecular Sequence Data , Molecular Weight , Neovascularization, Physiologic/drug effects , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
High-efficiency upconverted light would be a desirable stimulus for triggered drug delivery. Here we present a general strategy to achieve photoreactions based on triplet-triplet annihilation upconversion (TTA-UC) and Förster resonance energy transfer (FRET). We designed PLA-PEG micellar nanoparticles containing in their cores hydrophobic photosensitizer and annihilator molecules which, when stimulated with green light, would undergo TTA-UC. The upconverted energy was then transferred by FRET to a hydrophobic photocleavable group (DEACM), also in the core. The DEACM was bonded to (and thus inactivated) the cell-binding peptide cyclo-(RGDfK), which was bound to the PLA-PEG chain. Cleavage of DEACM by FRET reactivated the PLA-PEG-bound peptide and allowed it to move from the particle core to the surface. TTA-UC followed by FRET allowed photocontrolled binding of cell adhesion with green light LED irradiation at low irradiance for short periods. These are attractive properties in phototriggered systems.