Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
  • A61L24/0047—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
  • A61L24/0073—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix
  • A61L24/0089—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix containing inorganic fillers not covered by groups A61L24/0078 or A61L24/0084
• • A—HUMAN NECESSITIES
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  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
  • A61L24/001—Use of materials characterised by their function or physical properties
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
  • A61L24/001—Use of materials characterised by their function or physical properties
  • A61L24/0015—Medicaments; Biocides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
  • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
  • A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods
  • A61B17/12—Surgical instruments, devices or methods for ligaturing or otherwise compressing tubular parts of the body, e.g. blood vessels or umbilical cord
  • A61B17/12022—Occluding by internal devices, e.g. balloons or releasable wires
  • A61B17/12099—Occluding by internal devices, e.g. balloons or releasable wires characterised by the location of the occluder
  • A61B17/12109—Occluding by internal devices, e.g. balloons or releasable wires characterised by the location of the occluder in a blood vessel
  • A61B17/12113—Occluding by internal devices, e.g. balloons or releasable wires characterised by the location of the occluder in a blood vessel within an aneurysm
• • A—HUMAN NECESSITIES
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  • A61B17/00—Surgical instruments, devices or methods
  • A61B17/12—Surgical instruments, devices or methods for ligaturing or otherwise compressing tubular parts of the body, e.g. blood vessels or umbilical cord
  • A61B17/12022—Occluding by internal devices, e.g. balloons or releasable wires
  • A61B17/12131—Occluding by internal devices, e.g. balloons or releasable wires characterised by the type of occluding device
  • A61B17/12181—Occluding by internal devices, e.g. balloons or releasable wires characterised by the type of occluding device formed by fluidized, gelatinous or cellular remodelable materials, e.g. embolic liquids, foams or extracellular matrices
  • A61B17/1219—Occluding by internal devices, e.g. balloons or releasable wires characterised by the type of occluding device formed by fluidized, gelatinous or cellular remodelable materials, e.g. embolic liquids, foams or extracellular matrices expandable in contact with liquids
• • A—HUMAN NECESSITIES
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  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
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  • A61B2017/00526—Methods of manufacturing
• • A—HUMAN NECESSITIES
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  • A61B2017/00681—Aspects not otherwise provided for
  • A61B2017/00707—Dummies, phantoms; Devices simulating patient or parts of patient
• • A—HUMAN NECESSITIES
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  • A61B17/00—Surgical instruments, devices or methods
  • A61B2017/00681—Aspects not otherwise provided for
  • A61B2017/00725—Calibration or performance testing
• • A—HUMAN NECESSITIES
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  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods
  • A61B2017/00831—Material properties
  • A61B2017/00893—Material properties pharmaceutically effective
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2400/00—Materials characterised by their function or physical properties
  • A61L2400/06—Flowable or injectable implant compositions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2400/00—Materials characterised by their function or physical properties
  • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/36—Materials or treatment for tissue regeneration for embolization or occlusion, e.g. vaso-occlusive compositions or devices
• • G—PHYSICS
  • G09—EDUCATION; CRYPTOGRAPHY; DISPLAY; ADVERTISING; SEALS
  • G09B—EDUCATIONAL OR DEMONSTRATION APPLIANCES; APPLIANCES FOR TEACHING, OR COMMUNICATING WITH, THE BLIND, DEAF OR MUTE; MODELS; PLANETARIA; GLOBES; MAPS; DIAGRAMS
  • G09B23/00—Models for scientific, medical, or mathematical purposes, e.g. full-sized devices for demonstration purposes
  • G09B23/28—Models for scientific, medical, or mathematical purposes, e.g. full-sized devices for demonstration purposes for medicine

Definitions

• the present invention relates to shear thinning biomaterials and methods for making and using them.
• Shear-thinning hydrogels are non-Newtonian materials that behave as viscous fluids under shear stress and then recover solid-like properties upon elimination of the stress. Due to these properties, injectable shear-thinning biomaterials (STB) are attracting attention as a group of self-healing materials that allow for fluent infusion and local equilibrium after approaching the final application site.
• STBs can be delivered into the body using a needle or a general/microcatheters by manual pressure. To optimize the clinical application, it is necessary to adjust the physical properties of STB according to the specific clinical situations.
• the physical properties of conventional STBs can be modulated by a combination of several carbon-based, polymeric, and inorganic nanomaterials.
• biomaterials such as gelatin, hyaluronic acid, chitosan, collagen, and alginate have been previously used along with inorganic constituents to form STBs.
• gelatin limits the adsorption of nonspecific proteins, enhanced hemolysis, and ultimately prolongs clotting time, demonstrating substantially improved hemocompatibility of STB in vitro.
• STBs are prepared by mixing gelatin with synthetic clay nanoparticles, Laponite ® , for hemostasis and endovascular embolization. These STBs exhibit strong shear-thinning behavior as well as biocompatible properties ranging from blood coagulation to minimized inflammatory response. Others have extended this work to implement STBs as embolic agents, functionalized scaffolds, 3D-bioinks and drug delivery systems. Unfortunately, however, synthetic clay nanoparticles such as Laponite are crystallized nanoparticles and the size, surface chemistry of such materials are not easily tuned.
• Shear-thinning hydrogels are highly desirable biomaterials for catheter-based minimally invasive therapies.
• the tradeoff between injectability and mechanical integrity has limited their applications, particularly at high external shear stress such as endovascular procedures.
• Embodiments of the invention include, for example, shear-thinning biocompatible compositions of matter comprising silicate nanoparticles or nanoplatelets, one or more cationic polymers such as Polydiallyldimethylammonium chloride, and gelatin. While Polydiallyldimethylammonium chloride is used as an exemplary cationic polymer in the illustrative working embodiments of the invention that are disclosed herein, embodiments of the invention can utilize other cationic polymers such as Poly(2-dimethylamino)ethyl methacrylate) methyl chloride quaternary salt, Poly[(2-ethyldimethylammonioethyl methacrylate ethyl sulfate)-co- (1-vinylpyrrolidone)], Poly(acrylamide-co-diallyldimethylammonium chloride) and the like.
• cationic polymers such as Polydiallyldimethylammonium chloride, and gelatin.
• cationic monomers can be utilized to make cationic polymers useful in embodiments of the invention such as Diallyldimethylammonium chloride monomers, [2- (Methacryloyloxy)ethyl]trimethylammonium chloride monomers, [2- (Acryloyloxy)ethyl]trimethylammonium chloride monomers, (3- Acrylamidopropyl)trimethylammonium chloride monomers, [3- (Methacryloylamino)propyl]trimethylammonium chloride monomers and the like.
• cationic polymers can be formed using a single type of cationic monomer or mixture of different cationic monomers.
• the constituents or relative amounts the constituents are selected to tune or modulate one or more properties of the composition.
• the silicate nanoparticles or nanoplatelets have a median diameter of from 5 nm to 150 nm and/or comprise a negative charge at physiological pH.
• the silicate nanoparticles or nanoplatelets comprise from 5% to 45% (w/v) of the composition; and/or the cationic polymer comprises from 1% to 10% (w/w); and/or the gelatin comprises from 1% to 30% of the composition.
• the silicate nanoparticles or nanoplatelets comprise not more than 9% (w/v) of the composition; and/or the cationic polymer comprises at least 1, 2, 3, 4 or 5% (w/w) of the composition; and/or the cationic polymer comprises less than 10, 9, 8, 7 or 6% (w/w) of the composition.
• the composition is disposed within a vessel (e.g., a catheter) selected for its ability to facilitate a user modulating one or more rheological properties of the composition.
• compositions of the invention include additional agents such as a pharmaceutical excipient selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent.
• additional agents such as a pharmaceutical excipient selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent.
• the compositions of the invention include one or more therapeutic agents such as an anti-inflammatory agent, an agent that modulates coagulation, an antibiotic agent, a chemotherapeutic agent or the like.
• Another embodiment of the invention is a method of making a shear-thinning biocompatible composition disclosed herein comprising combining together silicate nanoparticles or nanoplatelets, a cationic polymer and gelatin, and optionally a pharmaceutical excipient and/or a therapeutic agent so as to form a shear-thinning biocompatible composition.
• a surface property of the silicate nanoparticles or nanoplatelets, a median diameter of the silicate nanoparticles or nanoplatelets, a relative amount of silicate nanoparticles or nanoplatelets; and/or a relative amount of cationic polymer, gelatin or the like is selected to tune or modulate one or more rheological properties of the shear-thinning biocompatible composition.
• Yet another embodiment of the invention is a method of delivering a shear- thinning biocompatible composition disclosed herein to a preselected site (e.g. an in vivo location where an individual has experienced trauma or injury).
• a preselected site e.g. an in vivo location where an individual has experienced trauma or injury.
• methods comprise disposing the composition in a vessel having a first end comprising an opening and a second end (e.g. a catheter); applying a force to the second end of the vessel, wherein the force is sufficient to liquify the composition; and then delivering the composition out of the vessel through the opening and to the preselected site.
• the pGL biomaterial (GL containing 5% w/w of PDDA, shown in the purple shade) is selected for further characterization and comparison with the GL biomaterial (shown in the gray shade).
• Shear-thinning hydrogels are suitable biomaterials for catheter-based minimally invasive therapies; however, the tradeoff between injectability and mechanical integrity has limited their applications, particularly at high external shear stress such as endovascular procedures. Extensive molecular crosslinking often results in stiff, hard-to-inject hydrogels that may block catheters, whereas weak crosslinking renders hydrogels mechanically weak and susceptible to shear-induced fragmentation. Thus, controlling molecular interactions is necessary to improve the cohesion of catheter-deployable hydrogels.
• embodiments of the invention include biocompatible compositions of matter comprising silicate nanoparticles or nanoplatelets, a cationic polymer such as Polydiallyldimethylammonium chloride, and gelatin.
• compositions of the invention can include further constituents such as additional polymers, excipients, therapeutic agents and the like.
• compositions of the invention can include one or more Food and Drug Administration (FDA) approved or cytocompatible polymers.
• FDA Food and Drug Administration
• Such polymers include alginate, chitosan, collagen, hyaluronic acid (HA), chondroitin sulfate (ChS), dextrin, gelatin, fibrin, peptide, and silk.
• Synthetic polymers such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poloxamer (Pluronic®) (PEO-PPO-PEO), polyoxamine (Tetronic®) (PEO-PPO), poly(vinyl alcohol) (PVA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), polycaprolactone (PCL), poly(L-glutamic acid) (PLga), polyanhydrides, poly(N-isopropylacrylamide) (PNIPAAm), polyaniline and the like can also be included in compositions of the invention.
• the nanoparticles or nanoplatelets comprise Laponite®, a synthetic smectite clay that has a number of technological applications. In biomedical applications, particularly in nanomedicine, this material holds great potential.
• Laponite® is a 2-dimensional (2D) nanomaterial composed of disk-shaped nanoscale crystals that have a high aspect ratio.
• compositions of the invention include, for example nanoparticles or nanoplatelets combined with a pharmaceutical excipient such as one selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent.
• a pharmaceutical excipient such as one selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent.
• excipient is meant to include, but is not limited to, those ingredients described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) the contents of which are incorporated by reference herein.
• compositions of the invention include one or more therapeutic agents such as an anti-inflammatory agent, an agent that modulates coagulation, an antibiotic agent, a chemotherapeutic agent or the like.
• Compositions of the invention can be formulated for use as carriers or scaffolds of therapeutic agents such as drugs, cells, proteins, and bioactive molecules (e.g., enzyme).
• therapeutic agents such as drugs, cells, proteins, and bioactive molecules (e.g., enzyme).
• carriers such compositions can incorporate the agents and deliver them to a desired site in the body for the treatments of a variety of pathological conditions. These include, for example, infectious and inflammatory diseases (e.g. Parkinson’s disease, bacterial and antimicrobial infection, diabetes and the like) as well as cancers (e.g. colon, lung, breast, ovarian, lymphoma cancers and the like).
• infectious and inflammatory diseases e.g. Parkinson’s disease, bacterial and antimicrobial infection, diabetes and the like
• cancers e.g. colon, lung, breast, ovarian
• compositions of the invention can provide a flexible dwelling space for cells and other agents for use in tissue repair and the regeneration of desired tissues (e.g. for cartilage, bone, retina, brain, and, neural tissue repair, vascular regeneration, wound healing and the like).
• desired tissues e.g. for cartilage, bone, retina, brain, and, neural tissue repair, vascular regeneration, wound healing and the like.
• embodiments of the invention can include immunomodulatory agents useful for immunotherapy in order to, for example, enhance components of the immune system.
• Certain illustrative materials and methods that can be adapted for use in such embodiments of the invention are found, for example in Hydrogels: Design, Synthesis and Application in Drug Delivery and Regenerative Medicine 1st Edition, Singh, Laverty and Donnelly Eds; and Hydrogels in Biology and Medicine (Polymer Science and Technology) UK ed. Edition by J.
• embodiments of the invention include methods of making a shear-thinning biocompatible composition disclosed herein comprising combining together silicate nanoparticles or nanoplatelets, a cationic polymer and gelatin, and optionally a pharmaceutical excipient and/or a therapeutic agent so as to form a shear- thinning biocompatible composition.
• a surface property of the silicate nanoparticles or nanoplatelets, a median diameter of the silicate nanoparticles or nanoplatelets, a relative amount of silicate nanoparticles or nanoplatelets; and/or a relative amount of cationic polymer, gelatin or the like is selected to tune or modulate one or more rheological properties of the shear-thinning biocompatible composition.
• the method is selected to form a shear-thinning biocompatible composition exhibiting an injection force of less than 10 Newtons when extruded through a 5F catheter at an injection rate of 2 mL min -1 under physiological conditions.
• the method is selected to form a shear-thinning biocompatible composition exhibiting a ⁇ -potential from -30 millivolts to -40 millivolts under physiological conditions.
• the silicate nanoparticles or nanoplatelets are selected to have a median diameter of from 5 nm to 150 nm; and/or the silicate nanoparticles or nanoplatelets are selected to comprise from 5% to 45% (w/w) of the composition; and/or the cationic polymer concentration in gelatin is selected to comprise at least 1, 2, 3, 4 or 5% (w/w); and/or the cationic polymer concentration in gelatin solution is selected to comprise less than 10, 9, 8, 7 or 6% (w/w); and/or the gelatin is selected to comprise from 1% to 30% of the composition.
• the silicate nanoparticles or nanoplatelets comprise not more than 9% (w/v) of the composition; and the gelatin comprises not more than 18% (w/v) of the composition.
• the methods combine the silicate nanoparticles or nanoplatelets with a pharmaceutical excipient and/or a therapeutic agent.
• Another embodiment of the invention is a method of delivering a shear- thinning biocompatible composition disclosed herein to a preselected site (e.g. an in vivo location where an individual has experienced trauma or injury).
• a preselected site e.g. an in vivo location where an individual has experienced trauma or injury.
• such methods comprise disposing the composition in a vessel having a first end comprising an opening and a second end (e.g.
• hydrogels such as injectability and mechanical robustness are regulated by the molecular interactions among their building blocks, such as polymer chains and nanoparticles via noncovalent and/or covalent binding within a three-dimensional (3D) network 6-9 .
• covalent bonds impart mechanical resilience to elastic hydrogel biomaterials
• noncovalent interactions such as ionic binding
• energy dissipation to withstand cyclic deformations and minimize mechanical mismatch at soft tissue interfaces 10-11 .
• various injectable biomaterials have been developed that form gels immediately after injection 12-13 .
• This gel formation mechanism is favorable in minimally invasive procedures, such as endovascular embolization or aneurysm treatment; 14 however, delays in biomaterial crosslinking and lack of cohesion may cause material loss in body fluids, e.g., blood, and block blood vessels 15 . Moreover, fast crosslinking may block the injection tools such as catheters or needles during the operation, endangering patients’ life 3, 15 . In general, although time-dependent crosslinking mechanisms may enable hydrogels to readily pass through needles, they often face major challenges with injection through long surgical catheters 3 .
• Injectability and stability of these hydrogels rely on noncovalent interactions among their components, which regulate their flow under shear and gel formation upon shear elimination, e.g., after injection 17-18 .
• these shear-thinning biomaterials have suitable catheter-injectability, they face severe challenges in terms of their mechanical stability (robustness) under physiological fluid flow conditions. This was clearly observed when a gelatin-based shear-thinning material was used to occlude a berry type, neck-less aneurysm model under physiologically relevant fluid flows 19 .
• the biomaterial was previously proven to be successful for endovascular embolization 17 , direct fluid flow permanently disrupted the hydrogel network, causing disintegration and fragmentation.
• Liquid/gel embolic materials that solidify upon injection in aqueous media are also limited in efficacy as they increase the risk of spilling and catheter entrapment/cementing 25 .
• Onyx one of such embolic materials, has already been used for the occlusion of aneurysms 26 .
• organic solvents in embolic materials such as dimethyl sulfoxide (DMSO) may result in systemic cardiovascular toxicity and vasospasm 27-28 . Accordingly, designing a catheter-injectable shear-thinning biomaterial with controlled molecular/colloidal interactions, specifically engineered to maximize cohesion may open new opportunities for the treatment of potentially fatal conditions, such as aneurysms.
• DMSO dimethyl sulfoxide
• PDMS Polydimethylsiloxane
• Ellsworth Adhesives Irvine, CA, USA
• Commercially available food- grade red dye was purchased from local store to improve the contrast of the images.
• NIH/3T3 murine fibroblasts ATCC® CRL-1658TM
• human umbilical vein endothelial cells HAVEC, ATCC® CRL-1730TM
• Dulbecco’s Phosphate-buffered saline (DPBS, 1X) used in the flow experiments and cell culture study was obtained from Fisher Scientific (Hampton, NH, USA).
• PrestoBlueTM cell viability reagent and Live/Dead viability/cytotoxicity kit for mammalian cells were purchased from Invitrogen (NY, USA).
• Dulbecco’s modified Eagle’s medium DMEM, fetal bovine albumin (FBS), qualified, heat inactivated, Penicillin/Streptomycin (10,000 U/mL), trypsin-EDTA phenol red (0.25%, 1X) were bought from Gibco (NY,USA).
• Endothelial basal medium (EBM-2) and endothelial growth BulletKit were obtained from Lonza (Basel, Switzerland).
• aqueous gelatin solution (18% w/v) at 37 ⁇ C was homogeneously mixed with PDDA solution at varying concentrations (0, 1, 2, 3, 5, 7, and 10% w/w).
• a 9% w/v LAPONITE gel was prepared via the exfoliation of LAPONITE in cold milliQ water (4 °C) using vigorous vortexing for at least 10 min, followed by mixing with the gelatin/PDDA polymer solutions and vortexing with intermittent spatula-assisted shearing.
• mixing was continued using a speed mixture for at least 5 min at 3000 rpm.
• the total solid mass content of biomaterials was maintained at 6% w/v.
• gelatin-LAPONITE biomaterial was prepared using the same method without including PDDA.
• the GL containing the optimum concentration of PDDA (5 % w/w) had the lowest injection force and highest cohesion.
• This biomaterial is called pGL as it is made up of an optimum PDDA (p) concentration, gelatin (G), and LAPONITE (L).
• Injection force measurements The injectability of biomaterials was characterized by injection force measurements using an Instron Universal Testing System (Model 5943). Several parameters pertaining to biomaterial injectability, including syringe volume and catheter diameter, were investigated to evaluate the applicability of the biomaterials in minimally invasive, catheter-based procedures.
• Syringes (BD Biosciences) loaded with the biomaterials (pGL or GL) were attached to medical catheters (Cook medical) and mounted in the material testing system using a tension grip around the luer lock connecting port of catheter.
• a compressive plate depressed the syringe plunger at a constant rate of 2 mL min -1 , and the material testing system was used to measure the force on the plate over time using the Bluehill ® universal software (version 3). The injection force was recorded as the load (N) when the injection force reached a plateau.
• Oscillatory strain sweeps were conducted at a range of 0.01-100% and a constant angular frequency of ⁇ 10 rad s -1 , and oscillatory angular frequency sweeps were performed at 0.1–100 rad s -1 and a constant stain of 0.1% at 25 °C. Viscosity was measured based on steady shear rheology at shear rates ranging from 0.01 to 10 s -1 at 25 °C. ⁇ -potential measurement As the electrostatic interactions of charged LAPONITE nanosilicates with gelatin and many other charged polymers are well investigated 29-30 , here, we only measure the ⁇ -potential of LAPONITE-gelatin aggregates at varying concentrations of added PDDA.
• hydrogels were synthesized with varying PDDA concentrations, disintegrated in water, and the ⁇ -potential of aggregates was measured using a previously established protocol with further modifications 31 .
• hydrogels (10 mg) were dispersed in Milli-Q water (10 mL) by sonication and vortexing for 2 h, and the ⁇ -potential of the aggregates was measured at 25 °C in at least triplicates with 20 scans using Zetasizer Nano series (Malvern Instruments).
• top portion consisted of an aneurysm sac hemisphere with a diameter of 6 mm.
• the bottom portion consisted of a cylindrical blood vessel with a diameter of 6 mm and a length of 75 mm, mimicking small- or medium-sized saccular cerebral aneurysms 32 .
• the hemisphere on the top portion was attached to the bottom portion, making a closed system, mimicking that of an aneurysm-affected blood vessel.
• the negative molds consisting of the (i) hemispheres and (ii) blood vessels were fabricated by 3D printing of acrylonitrile butadiene styrene (ABS) filaments using a Lulzbot (info) instrument. Next, these pieces were glued into custom-built laser-cut acrylic boxes, followed by filling with PDMS and curing in a hot air oven at 80 °C for 2 h. The cured PDMS was removed from the acrylic molds and the ABS was dissolved using acetone. For the flow experiments, completed models consisting of a top portion (hemisphere) and bottom portion (blood vessels) were assembled between two acrylic plates, bolted together to hold and seal the two pieces of aneurysm model.
• ABS acrylonitrile butadiene styrene
• the bifurcation model which contained a cylindrical blood vessel with a diameter of 5 mm.
• the diameter of aneurysm sac was 9 mm.
• Biomaterial retention quantification in aneurysm models in vitro The retention (stability) of biomaterials was investigated by filling the aneurysm models with GL or pGL, and exposing them to constant DPBS flow for 24 h, followed by mass loss quantification. The top hemisphere of each aneurysm model was filled with the biomaterials using a 1mL syringe attached to the 5F catheter, which was then assembled to the bottom section prior to flow experiments.
• the biomaterial-loaded aneurysm sac (top section) was weighed to obtain the initial biomaterial mass.
• the assembled model was exposed to 15 or 20 mL s -1 constant flow of DPBS. After 24 h, the flow was stopped, and the biomaterial was removed and freeze-dried. To quantify the mass loss, the dry weight of samples after flow experiments were compared with the dry weight of initial biomaterial placed in the model before the flow experiments. Fabrication of a patient-derived aneurysm model and filling it under pulsatile flow (carotid flow) The performance of pGL and GL biomaterials was compared with each other inside saccular aneurysms, specifically a patient-derived intracranial aneurysm under carotid flow.
• the basilar tip aneurysm model was created from human aneurysm 3D images using a previously developed method 33 . Briefly, the positive mold made of ABS was fabricated using a 3D printer, and the surface of printed objects was smoothed by dipping in the ABS solvent (eSolve). The vascular mold was dried and coated with PDMS, followed by curing at 60 °C. The ABS mold was then removed by immersion in acetone 53 .
• ABS solvent eSolve
• biomaterials were injected in the patient-derived aneurysm model using a 4F catheter under the pulsatile (carotid) flow of DPBS at 4 mL s -1 resembling the average blood flow in the basilar artery 34-36 . Images were acquired to investigate the occlusion of artery and the integrity (retention) of biomaterials. Cytotoxicity assessments Biomaterial cytotoxicity was evaluated using two different cell lines: NIH/3T3 murine fibroblasts and HUVECs. Fibroblast cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin.
• HUVECs were cultured in EBM-2 and supplemented with endothelial growth BulletKit and 1% (v/v) penicillin/streptomycin.
• Cells were cultured at 37 °C in a 5% CO 2 incubator (Forma incubators, ThermoFisher Scientific, USA), and the media was changed three times a week until a confluency of ⁇ 90% was reached to used them for the experiments.
• the confluent cells were trypsinized using trypsin-EDTA (0.25%) and counted using a hemocytometer.
• NIH/3T3 (2000 cells/well) and HUVECs (5000 cells/well) were seeded in a separate 12-well plate and placed at 37 °C in a CO2 incubator.
• UV ultraviolet
• biomaterials 0.1 mL of ultraviolet (UV) sterilized (wavelength ⁇ 250 nm, duration ⁇ 1 h) biomaterials was injected at the side of each well using a needle-free 1 mL syringe and was incubated with the cells for 1 and 4 days. Thereafter, the PrestoBlueTM cell viability assay was conducted following the manufacturer’s instructions to evaluate the metabolic activity of cells in contact with the biomaterials. Briefly, 1 mL of PrestoBlueTM reagent (10% in the complete medium) was added to each well in the dark and incubated at 37 °C for 1.5 h.
• PrestoBlueTM reagent 10% in the complete medium
• the stained cells were imaged for live cells (Calcein-AM, green fluorescent excitation/emission 495 nm/515nm) and dead cells (ethidium homodimer-1, red fluorescent excitation/emission 495 nm/635 nm) using an inverted fluorescence microscope (Axio Observer 5, Zeiss, Germany). The cell viability was quantified from 5 randomly selected areas of each well using ImageJ software 37 (Version 1.52e, USA). Hemolysis assessment To assess the hemolytic effects of biomaterials, a hemolysis assay was conducted following the ASTM E2524-08 standard. 38 Heparinized whole human blood was purchased from Zenbio (NC, USA).
• the concentration of the hemoglobin in the blood was calculated from the human hemoglobin standard curve using Drabkin’s reagent.
• the blood was diluted with DPBS to rectify hemoglobin concentration to 10 ⁇ 2 mg/mL.
• the biomaterial samples (0.1 mL) in 900 ⁇ L of DPBS were used as a blood-free control to identify possible false- positive assay results. All the samples were incubated at 37 °C for 3 h ⁇ 15 min, followed by centrifugation for 15 min at 14000 rpm. Subsequently, 100 ⁇ L of supernatant was transferred to a 96-well plate and an equal volume of Drabkin’s reagent was added to each well and allowed to react on a shaker for 15 min in the dark. The absorbance against the reagent was measured at 540 nm using a microplate reader, and the hemoglobin concentration in each sample was calculated from the standard curve.
• RESULTS AND DISCUSSION Figure 1a schematically represents the preparation of cohesive shear-thinning biomaterial (i.e., pGL), comprising biocompatible and biodegradable LAPONITE nanoplatelets 29 , gelatin, and PDDA.
• LAPONITE nanoplatelets are decorated with negative and positive charges on the surface and edge (rim), respectively, which interact with gelatin and many other biopolymers used for diverse biomedical applications 17, 29, 40 .
• modes of intermolecular interactions between LAPONITE and gelatin are limited because of their predominant negative charges, 41 weakening the biomaterial cohesion.
• controlling the dipolar interactions in LAPONITE-based hydrogel networks is challenging as the nanoplatelets readily form aggregates through electrostatic attractions with positively charged moieties/polymers and often disrupt the homogeneity of the biomaterial, leading to phase separation 29 .
• the pGL biomaterials were engineered by regulating the dipolar interactions though solvation-induced charge dilution, a well-known mechanism in peptide based therapeutics 42 .
• We hypothesize that the incorporation of positively charged PDDA in gelatin solution, followed by mixing with LAPONITE controls the electrostatic attraction of polymers with the anionic surface of LAPONITE nanoplatelets, reducing the net anionic group density of composite hydrogel (gelatin at the intrinsic pH of LAPONITE dispersion, i.e., pH 9-10).
• the extended attraction between the LAPONITE and polymers may improve the cohesion of the shear-thinning hydrogel, while maintaining the homogeneity and injectability.
• PDDA with distinct positive charges on the ammonium groups, introduces strong electrostatic interactions to the biomaterial via PDDA-LAPONITE and PDDA-gelatin binding (Figure 1b-iii&iv), improving the integrity and cohesion.
• Physical appearance of the GL and pGL is shown in Figure S1 in the supporting information found in Baidya et al., which is incorporated by reference.
• the engineered pGL biomaterial benefits from enhanced molecular and colloidal attractions, it can also be readily injected using clinically relevant catheters, e.g., 5F, as shown in Figure 1c. This is possibly a result of reversible, noncovalent interactions of pGL building blocks, which provides shear-thinning behavior while maintaining cohesion.
• FIG. 1d-i schematically shows the composition of homogeneous GL biomaterial, with the inset showing the biomaterial at ambient conditions.
• Increasing the PDDA concentration increases the binding strength between the solid components, as schematically presented in Figure 1d-ii,iii with insets showing the physical appearance of biomaterials.
• the charge induced enhancement in interactions is well studied in supramolecular aggregate formation 46 . Electrostatic interactions with PDDA may partially replace hydrogen bonding of solids with water, increasing the cohesion.
• Figure 2a shows the injectability of GL biomaterial at varying PDDA concentrations using a 5F catheter.
• the injection force was initially decreased as PDDA concentration was increased from 0 to 5 % w/w, followed by an increase at higher PDDA concentrations, yielding a minimum injection force ( ⁇ 10 N) at a PDDA concentration of 5% w/w.
• This behavior may be explained by the PDDA- induced enhanced molecular interactions 43 and dipolar replacement of water molecules in the biomaterial, as explained in Figures 1b,d.
• charged PDDA might release hydrogen bonded water molecules 49 upon dipolar interactions with gelatin backbone and LAPONITE nanoplatelets, which remain locally free (unbound) and form layers between the solid constituents in the hydrogel matrix.
• Figure 2a presents the injection force of GL hydrogel containing PDDA across all concentrations of PDDA.
• Figure 2b presents the ⁇ -potential of gelatin-LAPONITE aggregates at varying PDDA concentrations.
• pGL biomaterial at optimum PDDA concentration i.e., 5% w/w
• the injectability of pGL biomaterial was quantified based on various injection parameters, including injection rate, syringe volume, and catheter diameter.
• catheters that are commonly used for endovascular procedures were selected for the experiments.
• Figure 2c presents the injection force as a function of syringe volume while catheter diameter (5F), length (100 cm), and the flow rate (2 mL min -1 ) maintained constant. As the syringe volume increased, a higher force was required to inject the pGL biomaterial because of the displacement of a higher biomaterial volume from the syringe to the catheter at any time. All the injection forces remained within a range that can be applied by hand 50 .
• Figure 2d shows the force required to inject the pGL biomaterial through catheters with varying diameters while syringe volume (3 mL), length (100 cm), and injection rate (2 mL min -1 ) were constant. The smaller the catheter diameter the higher the injection force.
• the injection force of the pGL biomaterial remained nearly unchanged after 15 days and 1 month of synthesis ( Figure S4, Supporting Information as found in Baidya et al., which is incorporated by reference), attesting to a decent shelf life. Within this period, coarse aggregate formation/phase separation was not observed, which would have otherwise resulted in the severe fluctuations of injection force plateau 29 .
• Figure 2e presents the storage modulus (G’) and loss modulus (G”) of pGL and GL biomaterials versus shear strain at a constant angular frequency of ⁇ 10 rad s -1 .
• the pGL biomaterial has an improved solid-like behavior at strain ⁇ 20% compared with the GL biomaterial (G’pGL > G’GL), and at strain > 20%, the pGL attains a more liquid-like behavior (Figure 2e).
• Cyclic strain recovery of pGL and GL biomaterials is presented in Figure 2f, which shows the recovery of storage modulus for pGL and GL biomaterials with time when high (100%) and low (1%) external oscillatory strains were applied alternatively.
• Figure 2i presents the shear stress versus shear rate, which further demonstrates the shear-thinning behavior of pGL and GL biomaterials.
• the injectability and cohesion of pGL biomaterial was tested in an in vitro cerebral aneurysm model, a type of aneurysm which is fatal in many cases 21, 33 .
• Wide- neck saccular aneurysm the most frequently observed cerebral aneurysm 32 , was selected to be occluded with the pGL biomaterial.
• Figure 3a schematically represents the experimental setup for the side-wall wide-neck aneurysm.
• a dismantlable model was fabricated to quantify the loss of material upon fragmentation during the fluid flow experiments.
• Figure 3b shows the images of laboratory setup, including the loaded pGL biomaterial inside the aneurysm sac.
• a food-grade red dye was used to enhance the contrast of biomaterial against the PDMS model.
• different flow rates pertinent to cerebral blood flow were used.
• DPBS constant fluid
• Figure 3c shows the material recovery percentage at different flow rates (15 or 20 mL s -1 ) after 24 h, which match the visual observations, attesting to the significant loss of GL biomaterial and the near-complete retention of pGL.
• the stability of pGL biomaterial was further assessed in a bifurcation model, where the fluid directly contacts the material, increasing the risk of the fragmentation.
• Figure 3d schematically shows the anatomy of bifurcation aneurysm model. Images of the experimental setup with or without pGL biomaterial are presented in Figure 3e.
• a carotid flow pattern 35 was selected.
• the pGL biomaterial was injected inside the basilar tip aneurysm site using a 4F catheter (Figure 4b-ii), which is often used to treat complications in the basilar artery 36, 53 .
• the pGL biomaterial was injected in the model under a carotid fluid flow with a rate of 4 mL s -1 ( Figure 4b-ii). The flow rate was ⁇ 2 fold higher than the blood flow rate in the basilar artery 34 .
• Figure 4c shows the images of aneurysm site before (i, iii) and during (ii, iv) the delivery of pGL or GL biomaterials through a 4F catheter under the fluid flow.
• the GL biomaterial was immediately entrained under the flow, whereas the pGL occluded the aneurysm site consistently without undergoing fragmentation.
• the inset of Figure 4c-iv shows the loss of fragmented GL biomaterial during the delivery into the in vitro model.
• fluid flow was continued for 3 h after removing the catheter ( Figure S5, Supporting Information as found in Baidya et al., which is incorporated by reference).
• FIG. 4d The success rate of pGL biomaterial delivery into the aneurysm site under the carotid fluid flow was evaluated 20 times using the patient-derived basilar tip aneurysm model, which is shown in Figure 4d.
• the GL biomaterial in more than 70% of cases, the material was immediately fragmented during the in-flow injection into the aneurysm site under carotid fluid flow; however, the pGL biomaterial was injected and retained in the aneurysm site with a success rate of more than 90% without undergoing noticeable fragmentation.
• Figure 5a shows live/dead assay images of HUVECs when exposed to biomaterials (GL or pGL) for 4 days compared with the biomaterial-free system (control).
• Figures 5b and 5c demonstrate the quantification of live/dead assay fluorescence images for NIH/3T3 fibroblast cells and HUVECs, respectively. Approximately 96% of both cell lines remained viable after 4 days of incubation with the pGL biomaterial, similar to the GL and control samples. This indicates that the incorporation of PDDA in the GL biomaterial does not significantly affect the viability of cells.
• the pGL biomaterial did not have any significant hemolytic effects. Accordingly, this novel engineered biomaterial may potentially be used for catheter-based procedures.
• This work addresses a long-lasting shortcoming of shear-thinning biomaterials, i.e., cohesion while maintaining injectability, which may set the stage for novel minimally invasive therapies.
• REFERENCES (1) Zhang, K.; Feng, Q.; Fang, Z.; Gu, L.; Bian, L. Structurally Dynamic Hydrogels for Biomedical Applications: Pursuing a Fine Balance between Macroscopic Stability and Microscopic Dynamics. Chemical Reviews 2021, 121 (18), 11149-11193, DOI: 10.1021/acs.chemrev.1c00071.

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