Patent Application
20240181136
The present invention relates to a non-cross-linked biocompatible low molecular weight (LMW) hydrogel having a high storage modulus. In some embodiments, the non-cross-linked biocompatible LMW hydrogel comprises water and thioglycolipid. The invention also relates to a carrier material and an article of manufacture comprising said LMW hydrogel. In particular, the present invention relates to low molecular hydrogels comprising thioglycolipids.
Hydrogels have been utilized in many biomedical fields, including dermatology, drug delivery systems, stem cell delivery systems, bonding and coating systems, tissue engineering or repairing systems, wound healing, cell culture, etc. One current field of study is concerned with tissue and tissue manipulation using poly(ethylene glycol) (PEG) compounds. Another field of study is using biopolymer-based hydrogels as scaffolds or artificial extracellular matrices that provide growth spaces for cells from the viewpoint of tissue engineering. The scaffolds or artificial extracellular matrices allow cells to grow better or in desired directions for the purpose of tissue engineering.
Some of the biopolymers currently used for producing hydrogels include polysaccharides such as glycogen, chitosan, cellulose, and hyaluronic acid. These biopolymers are often converted into hydrogels by physical crosslinking or irreversible chemical crosslinking. The process of cross-linking biopolymers to produce hydrogels results in high molecular weight hydrogels and requires additional time and cost.
Unlike high molecular weight hydrogels, conventional low molecular weight (LMW) gels are composed of small molecules that aggregate via noncovalent interactions and generally form fibrous 3-D networks or porous structures that immobilize solvent. LMW gels do not need any cross-linking steps, and therefore, are less costly and time consuming to prepare. In addition, LMW gels are known to be thermoreversible, gel at lower concentrations, and have high tolerance towards salts. LMW hydrogels are some of the most unique gels, as they are mainly composed of water; this allows their potential use in diverse applications in soft materials and biomaterials including drug delivery, cell growth, enzyme immobilization, and tissue engineering. Therefore, much effort has focused on the design of small molecules that serve as low molecular weight hydrogelators as well as improving their physicochemical properties, such as mechanical properties and biocompatibility, for various applications. The gelation behavior and mechanical properties of hydrogels are difficult to predict given the complex processes of self-assembly and three-dimensional network formation, although approaches to tune their properties such as improving mechanical properties based on known hydrogels have been reported. Among all kinds of LMW gels, sugar-based gelators have gained special interest for use in novel soft materials due to their biodegradability and biocompatibility, as well as for their sourcing from renewable raw materials. Recently, glycolipid-based hydrogels have been used in neural cell cultures, scaffold for stem cells and enzymes, and as cell-responsive capsules, although this work is still in its infancy and optimization of systems and properties is required.
Sugars have long been of interest for use in functional low molecular weight hydrogels. However, common functional compositions utilizing sugars have either relied on complex gelator molecules to increase intermolecular interaction strength, with relatively complex chemical syntheses, or are based on materials harvested from natural sources requiring extensive effort. Furthermore, many conventional LMW hydrogels, including those made from certain sugar-based materials, are fibrous. More importantly, regardless of microstructure, most LMW hydrogels made from sugar-based materials have a relatively low mechanical strength, with minimal design guidelines emerging from past efforts on simple alkyl glycosides and related systems that could lead to stronger hydrogels.
Therefore, there is a need for a simple method of producing functional LMW hydrogels using readily available sources. In particular, there is a need for LMW hydrogels that have high mechanical strength and are not a fibrous 3-D network.
Surprisingly and unexpectedly, in contrast to other conventional LMW hydrogels based on glycolipids, many of which are fibrous, the present inventors have discovered that functional hydrogels (i.e., those possessing mechanical properties adequate for various commercializable uses) can be produced in a layered 3-D network by utilizing a specific set of disaccharide-based thioglycolipids. Moreover, LMW hydrogels of the invention have a surprisingly high mechanical strength compared to other conventional LMW hydrogels, including those based on glycolipids that have been reported previously. Accordingly, LMW hydrogels are applicable in a wide variety of applications including, but not limited to, biomedicine and environmental science. Particular examples of application that are suitable for LMW hydrogels of the invention include, but are not limited to, wound healing, tissue engineering and repair, drug delivery, cell culture, as markers for fluid flow or pollutant plume tracking, in heavy metal extraction, and oil spill remediation.
One particular aspect of the invention provides a biocompatible low molecular weight (“LMW”) hydrogel. Some embodiments of the invention provide LMW hydrogels having a high storage modulus. In particular, LMW hydrogels of the invention are biocompatible. As used herein, the term “biocompatible” refers to substances that are not toxic to cells. For example, biocompatible can mean not producing a toxic, injurious, or immunologic response in living tissue, or the ability of a material to perform with an appropriate host response in a specific situation. In some embodiments, a substance is considered to be “biocompatible” if its addition to cells in vitro results in about 20% or less, typically about 10% or less, and often about 5% or less cell death. In other embodiments, a substance is considered to be “biocompatible” if its addition to cells in vivo does not induce inflammation and/or other adverse effects in vivo.
The term “LMW” refers to a compound or hydrogel having a molecular weight of about 1,500 g/mol or less, typically about 1,000 g/mol or less, and often about 750 g/mol or less.
Yet in other embodiments, the LMW hydrogels of the invention have a high storage modulus. The term “high storage modulus” refers to having a modulus of at least about 10 kPa, typically at least about 20 kPa, often at least about 50 kPa, and most often at least about 75 kPa at 25° C. When referring to a numerical value, the terms “about” and “approximately” are used interchangeably herein and refer to being within an acceptable error range for the particular value as determined by one of ordinary skill in the art. Such a value determination will depend at least in part on how the value is measured or determined, e.g., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose. For example, the term “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, the term “about” when referring to a numerical value can mean ±20%, typically ±10%, often ±5% and more often ±1% of the numerical value. In general, however, where particular values are described in the application and claims, unless otherwise stated, the term “about” means within an acceptable error range for the particular value, typically within one standard deviation.
Still in other embodiments, the LMW hydrogels of the invention comprises water and thioglycolipid.
Other aspects of the invention provide a carrier material and an article of manufacture comprising the LMW hydrogel disclosed herein. Still other aspects of the invention provide methods for producing and using the same.
One particular aspect of the invention provides a biocompatible low molecular weight (“LMW”) hydrogel having a high storage modulus, wherein said LMW hydrogel comprises water and thioglycolipid that are non-cross linked. In some embodiments, said hydrogel is a non-fibrous 3-D network. Still in other embodiments, at least 90% of said LMW hydrogel is water. Yet in other embodiments, the amount of thioglycolipid in said LMW hydrogel ranges from about 0.5% to about 10% by weight. In further embodiments, the storage modulus of said LMW hydrogel is at least about 10 kPa at 25° C. In other embodiments, the storage modulus of said LMW hydrogel is at least about 100 kPa at 25° C.
In one particular embodiment, said thioglycolipid comprises a disaccharide that is linked to a lipid via a thiol linkage. In general any disaccharides with 1→4 linkages known to one skilled in the art can be used. Particular examples of disaccharides used in the invention include, but are not limited to, lactose, maltose, cellobiose, lactulose, chitobiose or a combination thereof. For current cost considerations, typical disaccharides used include lactose, cellobiose, or a combination thereof.
Still in another embodiment, said thioglycolipid is of the formula: A-B, where A is a disaccharide sugar moiety that is linked to B through a thioether linkage, and B is C8-C20 n-alkyl. Exemplary alkyls used in the invention include, but are not limited to, C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, and C20 alkyl.
Another aspect of the invention provides a carrier material comprising (a) a LMW hydrogel disclosed herein and (b) a cargo. In some embodiments, said cargo is a therapeutic agent or prophylactic agent. In some instances, said therapeutic agent or prophylactic agent is a nutrient, pharmaceutical, drug, prodrug, peptide, glycopeptide, enzyme, polynucleotide, lipid, phospholipid, co-surfactant, metal complexant, steroid or other anti-inflammatory agent, antibacterial agent, antifungal agent, disinfecting agent, or combinations thereof.
Yet in other embodiments, said cargo is an absorbing material, a substrate in tissue engineering or cell culture.
Another aspect of the invention provides an article of manufacture comprising a LMW hydrogel disclosed herein. In some embodiments, said article of manufacture comprises a wound healing material, tissue engineering or repair material, cell culture material, or controlled-release drug delivery material. Yet in other embodiments, said article of manufacture comprises microparticles or nanoparticles of the LMW hydrogel, and wherein the microparticles or nanoparticles of LMW hydrogel comprises a cargo. In some instances, said cargo is a therapeutic agent, a prophylactic agent, an indicator moiety, a labelling moiety, an environmental tracer, a complex-forming agent that forms a complex with a heavy metal or a rare earth element, or an agrochemical. In other instances, said cargo comprises a rhamnolipid, a mono- or disaccharide analogue, or a combination thereof. Still in other instances, said cargo is a therapeutic agent.
Yet another aspect of the invention provides an article of manufacture comprising a microparticle or a nanoparticle, wherein said microparticle or a nanoparticle comprises a core and a shell, and wherein said core comprises a magnetic material and said shell comprises the LMW hydrogel disclosed herein and a cargo. In some embodiments, said core comprises iron, nickel, cobalt, or a combination thereof.
Still another aspect of the invention provides a low molecular weight physical hydrogel comprising water and thioglycolipid having a storage modulus of at least about 10 kPa at 25° C., wherein said hydrogel comprises at least about 80% by weight of water. In some embodiments, said hydrogel comprises at least about 90% by weight of water. Still in other embodiments, said hydrogel comprises at least about 1% by weight of thioglycolipid. Yet in other embodiments, said hydrogel comprises at least about 2% by weight of thioglycolipid. In further embodiments, said hydrogel comprises at least about 5% by weight of thioglycolipid.
Still in further embodiments, said thioglycolipid is of the formula: A-B, where A is a disaccharide sugar moiety that is linked to B through a thioether linkage, and B is C8-C20 alkyl. In some instances, B comprises C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, or C20 alkyl.
Yet in other embodiments, a storage modulus (G′) of said low molecular weight physical hydrogel ranges from about 10 kPa to about 570 kPa.
Some aspects of the present invention are based on development of a series of low molecular weight hydrogels based on simple alkyl glycolipids by the present inventors. The alkyl glycolipids are readily produced in large quantities through a scalable, high-yield synthetic process that provides tailoring of LMW hydrogel properties. Despite being held together through non-covalent interactions, these hydrogels exhibit mechanical strengths with storage moduli up to 10's to 100's of kPa in contrast to the 1,000's of Pa typical of other non-cross-linked hydrogels. These features facilitate potential utility of these hydrogels in a range of applications. Some of the advantages of LMW hydrogels of the invention include, but are not limited to, (i) their production by a simple, scalable, high-efficiency process with short formation time, (ii) direct hydrogel formation in water only without the need for additives, organic solvents or salts, (iii) resistance to acidic and basic environments and added salts, (iv) stability at body temperature without the need for cross linking, and (v) biocompatible and biodegradable since these materials are based on sugars.
In some embodiments, the LMW hydrogels of the invention can be made from simple alkyl thioglycolipids that spontaneously self-assemble through weak, noncovalent intermolecular interactions. The LMW hydrogels of the invention are formed rapidly from the alkyl thioglycolipids disclosed herein, have surprising and unexpected mechanical strength, and are biocompatible and biodegradable. LMW hydrogels of the invention can be used in a wide range of applications including, but not limited to, biomedicine to environmental science.
The invention provides a series of LMW hydrogels from simple glycolipids that possess surprising mechanical strength and stability at human body temperatures despite being self-assembled through noncovalent interactions. Moreover, these LMW hydrogels are thixotropic (rapidly and fully self-healing back to original storage moduli values) after repeated exposure to shear stress. Because LMW hydrogels are biocompatible and biodegradable, they are especially suitable for applications ranging from biomedicine to environmental science. For example, in the biomedical arena, LMW hydrogels of the invention can be used in a range of ex vivo and in vivo applications such as, but not limited to, wound healing, tissue engineering and repair, drug delivery, and cell culture. In the environmental applications, LMW hydrogels of the invention (e.g., in microparticle or nanoparticle form) can be used, for example, as markers for fluid flow or pollutant plume tracking, in heavy metal extraction, and oil spill remediation.
One particular aspect of the invention provides a biocompatible low molecular weight (“LMW”) hydrogel having a high storage modulus, wherein said LMW hydrogel comprises water and thioglycolipid. In some embodiments, the molecular weight of the thioglycolipid is 750 g/mol or less. Still in other embodiments, the LMW hydrogel is non-cross-linked hydrogel. Yet in other embodiments, the storage modulus of LMW hydrogel is at least 10 kPa at 25° C.
In other embodiments, at least about 80%, sometimes at least about 85%, typically at least about 95%, and often at least about 98% of said LMW hydrogel is water. Yet in other embodiments, the amount of thioglycolipid in said LMW hydrogel ranges from about 0.25% to about 20% by weight, typically from about 0.5% to about 10% by weight, and often from about 0.5% to about 5% by weight. Still in other embodiments, the storage modulus of said LMW hydrogel at 25° C. is at least about 105 Pa, typically at least about 10 kPa, more often at least about 20 kPa, and more often at least about 50 kPa, and most often at least about 75 kPa.
Still in other embodiments, the thioglycolipid comprises a thiol derivative of disaccharide. As used herein, the term “thiol derivative” refers to having a sugar moiety that is attached to a lipid moiety via a thioether linkage. The thiol moiety can be derived from the functional group of the lipid moiety or the sugar moiety.
In some embodiments, the LMW hydrogel comprises a mixture of a thioglycolipid and a glycolipid. In these embodiments, the amount of thioglycolipid can range from about 1% to about 99%, typically from about 5% to about 95%, often from about 5% to about 75%, more often from about 5% to about 50%, still more often from about 5% to about 25%, and most often from about 5% to about 10%. In other embodiments, the amount of glycolipid can range from about 1% to about 99%, typically from about 5% to about 95%, often from about 5% to about 75%, more often from about 5% to about 50%, still more often from about 5% to about 25%, and most often from about 5% to about 10%.
In one particular embodiment, the disaccharide comprises lactose, maltose, cellobiose, lactulose, chitobiose, or a combination thereof.
Yet in some embodiments, the thioglycolipid and/or the glycolipid is of the formula: A-B, where A is a sugar moiety (typically a disaccharide) and B is a lipid moiety such as C8-C20 alkyl. It should be appreciated that in thioglycolipids A and B are linked through a sulfur atom (i.e., thioether) linkage, whereas in glycolipids A and B are linked through an oxygen atom (i.e., ether) linkage. In some embodiments, B comprises C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, or C20 alkyl.
Another aspect of the invention provides a carrier material comprising (a) a LMW hydrogel disclosed herein and (b) a cargo. In some embodiments, the cargo comprises a therapeutic agent, including a wound healing material, or prophylactic agent. In general, a therapeutic agent refers to any drug or a prodrug known to one skilled in the art that is used to treat a clinical condition, disease, or a disorder in a subject. Typically, the subject is mammal, and often human. Exemplary therapeutic agents include drugs and biologics that have been approved by the Food and Drug Administration (FDA) for human or animal use, any drugs or biologics that are currently undergoing a clinical trial, and other drugs and biologics that are or will be developed for treatment of human or animals.
In some embodiments, the therapeutic agent is independently selected from the group consisting of an anticancer agent, an antiviral agent, an antibacterial agent, antifungal agent, an immunosuppressant agent, a hemostasis agent, and an anti-inflammatory agent.
Cell based therapies are important options for the treatment of clinical indications including diseases, tissue damage, neurological disorders, blood disorders, cancers, developmental defects, wounds and orthopedic impediments. Many cell-based therapies are target specific, with cells being administered directly to a target site. When cells are not suitably retained at a target site after administration, there is both a loss of cells available for the intended treatment as well as an increased risk of cell differentiation at an alternative site. When cells are administered to a target site without sufficient protection, the cells may go through physiochemical changes such as hypertrophy, necrosis, apoptosis, or senescence. Treatment efficacy is attenuated when the administered cells are physiochemically altered or not retained at the desired target site. In one particular embodiment of the invention, LMW hydrogels of the invention can be used to deliver cells to a desired therapeutic target area. In another embodiment, the LMW hydrogels can be used to deliver nutrients to cells within a desired target area.
In certain embodiments, the carrier can be prepared by admixing the precursor of LMW hydrogel and the cargo in an aqueous solution. In some embodiments, gelling or the formation of LMW hydrogel comprising the cargo can be controlled by time, the pH of the aqueous buffer, and/or other factors such as temperature, concentration, etc. In one particular embodiment, the gelling time (i.e., formation of LMW hydrogel comprising a cargo) ranges from about 20 seconds to about 30 minutes, typically, from about 1 minute to about 20 minutes.
In some embodiments, the cargo is released from the LMW hydrogel through diffusion, osmosis, degradation of the LMW hydrogel, including enzymatic degradation, or any combination thereof. In certain embodiments, the cargo is initially released from the LMW hydrogel through diffusion and later released through degradation of the LMW hydrogel. In some embodiments, the cargo is substantially released from the LMW hydrogel within one day, typically within 24 hours, often within 12 hours, and most often within 8 hours. In some embodiments, the LMW hydrogel precursor or the LMW hydrogel and the cargo do not form a covalent bond or cross-link during formation of the carrier. Yet in other embodiments, the LMW hydrogel precursor (e.g., thioglycolipid) or LMW hydrogel and the cargo form a covalent bond or become cross-linked during formation of the carrier.
The carrier can be made by mixing (a) LMW hydrogel precursor disclosed herein with (b) a cargo in an aqueous solution under conditions sufficient to form a carrier comprising a LMW hydrogel and a cargo. Alternatively, the carrier can be made using a droplet microfluidic. Exemplary method for using a droplet microfluidic can be found, for example, in U.S. Pat. No. 7,485,671, issued to Qiu et al. on Feb. 3, 2009, which is incorporated herein by reference in its entirety.
In some embodiments, the therapeutic agent or prophylactic agent is a nutrient, pharmaceutical, drug, prodrug, peptide (including polypeptide and protein), glycopeptide, enzyme, polynucleotide, lipid, phospholipid, co-surfactant, metal complexant, steroid or other anti-inflammatory agent including NSAID, antibacterial agent, antifungal agent, disinfecting agent, or combinations thereof. The terms “polynucleotide” and “oligonucleotide” are used interchangeably herein and refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally-occurring nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
Still in other embodiments, the cargo is an absorbing material or a substrate in tissue engineering or cell culture.
Another aspect of the invention provides an article of manufacture comprising a LMW hydrogel disclosed herein. In some embodiments, the article of manufacture comprises a wound healing material, tissue engineering or repair material, cell culture material, or controlled-release drug delivery material. Yet in other embodiments, the article of manufacture comprises microparticles or nanoparticles of the LMW hydrogel, where the micro- or nanoparticles of LMW hydrogel contain a cargo. In some embodiments, the cargo is a therapeutic or prophylactic agent including those disclosed herein. Still in other embodiments, the cargo is an indicator moiety, a labelling moiety, an environmental tracer, or a complex-forming agent that forms a complex with a heavy metal or a rare earth element. In one particular embodiment, the cargo comprises a rhamnolipid or an analogue of a rhamnolipid based on a different sugar. Yet in other embodiments, the cargo is an agrochemical. Exemplary agrochemicals that can be used in the invention include, but are not limited to, a fertilizer or other agricultural nutrient, herbicide, pesticide, biocide, fungicide, sporocide, and combinations thereof. In one particular embodiment, the article of manufacture comprises a controlled-release agrochemical delivery material.
The article of manufacture can be used in a variety of applications depending on the nature of the cargo. In some embodiments, the article of manufacture is used as a therapeutic agent. In other embodiments, the article of manufacture is used as an environmental bioremediation or sequestering agent. Yet in other embodiments, the article of manufacture is used as a metal harvesting agent.
Yet another aspect of the invention provides an article of manufacture that comprises a microparticle or a nanoparticle having a core and a shell. The core comprises a magnetic material and the shell comprises a LMW hydrogel. In some embodiments, the LMW hydrogel comprises a cargo. The cargo can be any one or more of the cargo as described herein. In certain embodiments, the core comprises iron, nickel, cobalt, or a mixture thereof.
Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting. In the Examples, procedures that are constructively reduced to practice are described in the present tense, and procedures that have been carried out in the laboratory are set forth in the past tense.
Materials. The thioglycolipids were synthesized via methodology previously reported by one of the present inventors with a modified deacetylation procedure. Szabó et al., Carbohydr. Res. 2016, 422, 1-4. https://doi.org/10.1016/j.carres.2015.12.008
Acetylation of sugars. To an oven-dried round bottom flask equipped with a stir bar were added sugar (1 eq) and sodium acetate (0.37 eq). The round bottom flask was evacuated and refilled with dry nitrogen. Anhydrous toluene (20 mL/mmol of sugar) and acetic anhydride (1.7 eq/OH of sugar) were then added via syringe. The contents of the round bottom flask were stirred and refluxed at 120° C. for 24 hours until the reaction was complete as indicated by TLC analysis. The reaction mixture was then cooled to room temperature. Water (10 mL/mmol of sugar) was then added to the reaction mixture. The contents of the round bottom flask were stirred for 1 hour. Then, diethyl ether (30 mL/mmol of sugar) was added to the reaction. The stirring was continued until white solid precipitated. Suction filtration was used to collect the products. Recrystallization by ethanol was then performed to give pure products.
Glycosylation of sugars with primary alkyl thiols. To an oven-dried round bottom flask equipped with a stir bar were added sugar octaacetate (1 eq) and indium bromide (3 wt %). The round bottom flask was evacuated and refilled with dry nitrogen. Anhydrous chloroform (5 mL/mmol of sugar octaacetate) and alkylthiol (2 eq) were then added via syringe. The contents of the round bottom flask were stirred and refluxed at 60° C. for 24 hours until the reaction was complete as indicated by TLC analysis. The reaction mixture was then cooled to room temperature and extracted with chloroform. The combined organic layers were washed three times with copious amounts of water and dried over magnesium sulfate. The organic phase was condensed by evaporation, and the resulting residue was purified by column chromatography on silica gel to give the product.
De-acetylation of acetyl glycolipids. To an oven-dried round bottom flask equipped with a stir bar were added alkyl-β-thioperacetyl sugar (1 eq) and Amberlyst resin (35 wt % of alkyl-β-thioperacetyl sugar). Methanol was then added to the mixture. The contents of the round bottom flask were stirred for 24 hours until the reaction was complete as indicated by TLC analysis. Gravity filtration was then performed to separate the resin from the organic phase. The organic phase was then condensed by evaporation. The chemical structures of the final compounds were characterized by 1H NMR, 13C NMR and mass spectrometry.
Preparation of Thioglycolipid Solutions and Hydrogel Fabrication. Aqueous solutions of thioglycolipids were prepared by weighing 2-3 mg of thioglycolipids and adding an appropriate volume of water to make solution samples at desired concentrations in vials. The vials were capped, sealed with Parafilm, and heated to 75° C. (or 85° C. for LacSC12) until the thioglycolipids were completely dissolved, which typically required about 1 min. The samples were then allowed to cool to ambient temperature. Above some minimal concentration, the solution forms hydrogels upon cooling (
A similar protocol for hydrogelation as described above was used to make cargo-containing gels for analysis. In this case, water was replaced with a dilute aqueous solution of the cargo.
Differential Scanning calorimetry. A Mettler Toledo DSC 823e differential scanning calorimeter, equipped with cooling apparatus, was used for measurement of the phase transition temperature and enthalpy of the gels. Hydrogel samples were first prepared using the fabrication method described above. Then, they were heated to 75° C. (or 85° C. for LacSC12) until they were in the solution state and then pipetted into aluminum, 40 μL DSC pans and capped with lids. The amount of hydrogel in each aluminum pan was controlled to about 6-8 mg. The pans were maintained at 85° C. for 1 min to ensure the samples were in the solution state. Then, they were cooled to 5° C. and maintained at this temperature for 20 min to ensure the formation of hydrogels. On subsequent cooling, the samples were heated from 5° C. to 85° C. at a heating rate at 5° C. per min.
Rheometry. The viscoelastic mechanical properties of the hydrogels were characterized using small amplitude oscillatory shear rheology on a Discovery Hybrid Rheometer 2 (TA Instruments, USA) with a sandblasted 20 mm cone-and-plate geometry at a 1° angle and a sandblasted stage. Inertia, friction, and rotational mapping calibrations were performed prior to each experiment. A Peltier temperature-controlled stage was maintained at 4° C., 10° C., 25° C., or 37° C. for rheology testing. Hydrogels were heated in a water bath above their transition temperature for 5 min, then transferred to the stage before the geometry was lowered quickly (1-3 sec) to the testing gap height of 50 μm. Excess gel was trimmed from the edges of the assembly. To control evaporation, the assembly was encased in a solvent trap with a water seal, and a mineral oil barrier was placed around the edges of the trap. Hydrogels were allowed to form and relax for 1 hour prior to experimentation. Strain sweeps were performed from 0.01% to 1000% shear strain at a constant 10 rad/s angular frequency. G′ (storage modulus) and G″ (loss modulus) were determined by averaging data within the linear viscoelastic region of the strain sweep. Frequency sweeps were performed from 0.01 rad/s to 100 rad/s at a constant 0.01% shear strain. Thixotropy experiments were conducted by cycling the shear strain between 0.01% and 100%.
Electron Microscopy. Scanning electron microscopy (SEM) was performed on an FEI Inspect S SEM equipped with a tungsten filament and a secondary electron detector for standard imaging. The preparation of xerogel samples for SEM analysis is described as follows: hydrogel samples were first heated to the solution state and then pipetted onto SEM sample stubs. Sample stubs containing the thiolactosides were then cooled at room temperature for 15 min for hydrogelation. For hydrogels from thiocellobiosides, the sample stubs with solution were quenched in at −4° C. ice/water bath for 15 min to form hydrogels. Then, samples were flash-frozen in liquid nitrogen followed by lyophilization to yield xerogels. Samples were gold coated prior to SEM characterization to reduce sample charging.
A Hitachi HF-5000 Transmission Electron Microscope (TEM) was used for TEM analysis to probe nanostructure of the xerogels. The preparation of xerogel samples for TEM is described as follows: lyophilization was performed after hydrogelation in 1-dram vials. After lyophilization, hexane was added to the vial followed by sonication for several seconds. Then, 1 μL of the resulting solution was pipetted onto a TEM grid. The grid was allowed to dry overnight prior to TEM imaging.
Fluorescence Spectroscopy. Steady-state fluorescence spectroscopy was performed on a Photon Technologies, Inc. (PTI), Quanta Master 40 spectrofluorometer. Prodan fluorescence was excited at 350 nm with emission observed between 370 nm and 640 nm. LacSC8, LacSC10, LacSC12, CelSC8, CelSC10 and CelSC12 gels and solutions were studied. Fluorescence spectra were initially acquired at 5° C. Then, using a 10° C. increment, fluorescence spectra were acquired between 5° C. and 75° C. for all thioglycolipids except LacSC12, which was studied to 85° C. Given the optical opacity of the gels, fluorescence spectra were acquired in a front face illumination geometry. Hydrogel samples containing the fluorophore prodan were first heated to the solution state, pipetted into a 1.7 mL-triangular fluorescence cuvette (Wilmad Lab-Glass) and then capped with a stopper to prevent evaporation. The cuvette was then quenched either at room temperature or at −4° C. to form hydrogels, and the sample allowed to equilibrate for 30 min prior to spectral acquisition. For samples that remain as solutions under all conditions, a similar preparation method was employed for characterization using a right-angle geometry, but a 10 mm-path rectangular fluorescence cell (Starna cell) was used instead. Normal right-angle fluorescence sampling was only conducted on 1 wt % LacSC8 and 1 wt % CelSC10 samples that were visually clear.
Visual Assessment of Thioglycolipid Hydrogel Formation. A wide range of thioglycolipid solutions were evaluated to establish concentration conditions (wt %) under which hydrogelation occurs and to assess the formation of other phases. Visual assessments of these aqueous solutions for different concentration conditions at room temperature are summarized in Table 1. For LacSC8 and LacSC12 solutions at concentrations <0.5 wt %, the solutions were either clear (C) or contain large aggregates (O) (i.e., cloudy). These solutions formed partial hydrogels surrounded by clear solution between 0.5 but <1 wt %, and were completely gelled at concentrations of 1 wt % and greater. Solutions of LacSC10 at <2 wt % formed partial hydrogels surrounded by clear liquid but were completely gelled at or above 2 wt %. CelSC8 solutions remained as clear solutions at all concentrations studied. CelSC10 and CelSC12 solutions formed settled fibrous aggregates in clear solution after equilibrating at ambient temperature overnight. However, CelSC10 and CelSC12 solutions completely hydrogelled when quenched at temperatures <20° C., although these slowly transformed into settled fibrous aggregates over time at room temperature, suggesting that they are metastable. If retained at 5° C. , the CelSC10 and CelSC12 gels were stable for at least 6 months. Interestingly, the metastable fibrous hydrogel structure was maintained, even in inverted vials, but any vibrational perturbation caused these fibrous gels to readily expel their water. Powder x-ray diffraction confirmed that these fibrous aggregates were crystalline. Overall, the thioglycolipid concentration at which hydrogelation can be observed can be as low as 0.05 wt %, depending on thioglycolipid system, compared to the typical concentration for hydrogelation of at least 1 wt % commonly reported in the literature, although 0.25-2 wt % is the concentration range wherein these thioglycolipid solutions became fully hydrogelled. The existence of metastable hydrogels and the ability to tune the transformation of hydrogels into crystals may allow their potential use as smart materials for different applications.
Thermal Properties by DSC. Differential scanning calorimetry (DSC) was used to determine the phase transition temperature as well as to explore the possible existence of other lyotropic phases. DSC involves monitoring the enthalpy change during a phase transition, which can be used as a measure of hydrogel stability. The results (Table 2) indicated an endothermic transition between 30° C. and 80° C. for all thioglycolipid hydrogels studied, and these transitions matched those observed visually in bulk solutions. Overall, hydrogels with longer carbon chains had higher thermostability (i.e., higher phase transition temperature). In addition, the LacSCx (x=10,12) hydrogels had higher thermostability than the CelSCx (x=10,12) hydrogels, despite similar phase transition enthalpies. LacSC8, LacSC10 and CelSC10 hydrogels exhibited similar enthalpies for the gel-sol transition. However, the LacSC12 and CelSC12 hydrogels required twice the enthalpy compared to the other three. Collectively, these results suggest that thioglycolipid chain length is the determining contributor to hydrogel thermostability.
Mechanical Properties by Rheology. Viscoelasticity is an important property of hydrogels, as different mechanical properties dictate utility in different applications. Rheology was used to characterize the viscoelastic properties of these thioglycolipid hydrogels. The two parameters of interest in these studies were storage modulus (G′) and loss modulus (G″). Materials for which G′>G″ are considered more solid-like, the desired behavior for hydrogels; in contrast, systems for which G″>G′ are considered more liquid-like. The magnitude of G′ is also an important parameter, as different hydrogel applications require different storage moduli. For instance, hydrogel scaffolds used for the replacement of functional tissues such as cardiovascular or musculoskeletal connective tissues require hydrogels with G′>1 kPa; similarly, stiff peptide hydrogels with G′>10 kPa are candidates for tissue engineering applications.
Strain sweep experiments were conducted on thioglycolipid hydrogels from 0.01% to 1000% shear strain at a constant 10 rad/s angular frequency at different temperatures, and frequency sweep experiments were performed from 0.01 rad/s to 100 rad/s at a constant 0.01% shear strain. All samples tested were at 1 wt % concentration except LacSC10 which was studied at 2 wt % given its required higher concentration for full gelation. All hydrogels tested exhibited solid-like behavior under certain conditions (
The rheology crossover point was generally observed between 50% and 100% shear strain for these materials (
Among all hydrogels tested, LacSC10 and LacSC12 hydrogels retained their gel properties at 37° C., making these gels candidates for medical applications. Thixotropy experiments were conducted by cycling the shear strain between 0.01% and 100%, and the results validated the self-healing properties of these two hydrogels (
Stability of some of the representative hydrogels of the invention in various pH aqueous media was investigated.
Microstructure of Hydrogels and Xerogels. One of the most unpredictable and perplexing topics when studying LMW hydrogels is the self-assembly process of hydrogelators which is usually estimated from the microstructure of hydrogels. The microstructure of LMW hydrogels provides information about how the molecules interact with each other and form the network that maintains the hydrogel structure. SEM and TEM are the two most used techniques for investigating hydrogel microstructure through analysis of the corresponding xerogels after flash freezing in liquid nitrogen followed by lyophilization.
SEM was performed on xerogel samples to reveal structural details on a 100's of nm to μm scale. Representative images are shown in
Xerogels from the fibrous hydrogels formed by CelSC10 and CelSC12 were also analyzed. Representative images are shown in
When looking at these hydrogel samples and fibrous samples under SEM, the microstructure can be very diverse given that the gelation process is a competition between molecules aggregating into the hydrogel network and molecules precipitating into other morphologies. The fibrous samples observed clearly represented a situation wherein most of the molecules aggregated into crystals within a small portion of hydrogel network. Similarly, even in hydrogel samples from LacSCx (x=8,10,12), amorphous aggregates that do not seem to contribute much to the hydrogel network were observed as well. In some cases, for instance, such as for LacSC12 hydrogels, small parts of the surface resembled the fibrous samples from CelSCx (x=10,12).
Although the structures of the layered xerogels were revealed by SEM, greater detail on the scale of 10's of nm can be extracted from transmission electron spectroscopy (TEM). TEM analysis was undertaken on the LacSC10 hydrogel as a representative system to better understand nanostructure of these xerogels. Representative TEM images are shown in
Panel a in
Confirmation of the crystalline nature of the fibrils of CelSC10 and CelSC12 came from powder and single-crystal x-ray crystallography. Although the molecular packing pattern in single crystals does not necessarily represent the aggregation pattern of molecules in the hydrogel structure, they do provide some possible insight. These results indicate that the molecules are packed in an interdigitated fashion with the cellobiose sugar groups pointing away from the thioalkyl core, consistent with a layered structure.
Aggregation Properties by Fluorescence Spectroscopy. Insight into the
structure and microenvironment of these thioglycoside hydrogel systems at different temperatures and concentrations came from fluorescence spectroscopy experiments using the polarity-sensitive fluorophore, prodan. The peak fluorescence emission wavelength for prodan is indicative of the polarity, and hence organization, of its microenvironment, thereby providing insight into the nature of the aggregation occurring. It has been demonstrated previously using aggregation of octyl glucoside that the wavelength of maximum of prodan emission uniquely reports on lyotropic phase state. This emission maximum shifts to shorter wavelengths with increasing order due to a decrease in polarity of the sequestered prodan environment. Free prodan in aqueous solutions had an emission maximum of 525 nm; this emission maximum systematically decreased with increasing organizational order through micellar (M), hexagonal (H), lamellar (L), cubic (C), gel (Gel) and crystalline (Cry) lyotropic phases. For octyl glucoside, the peak emission wavelength for each phase has been reported as follows. These values are expected to shift slightly for different molecular systems, although the order for each phase should remain the same.
For these thioglycolipid hydrogels, the change in organization and phase with concentration and temperature are portrayed by the representative prodan fluorescence behavior shown in
Combining the results obtained from prodan fluorescence, differential scanning calorimetry, visual assessment, and rheology, approximate phase diagrams were constructed for each thioglycolipid system studied. These are shown in
Typical phase diagrams of amphiphilic molecules include mainly three regions: solid, monomers and micelles. The solid region usually lies in the low temperature and high concentration regime. This regime represents the concentration and temperature range wherein the amphiphile is not soluble in the solvent. The monomer region usually lies in the low concentration regime across all temperatures. In this range of concentration and temperature, the amphiphilic molecules are soluble in the solvent but not at a concentration high enough to drive aggregation and formation of micelles. The third regime, wherein the concentration is above the critical micelle concentration (CMC), is the regime wherein the concentration and temperature are sufficient to support aggregation and the formation of different lyotropic phases. The phase diagrams depicted in
Hydrogel Cargo Release Characteristics. Release profiles for these hydrogels for two major classes of molecular cargo, hydrophobic and hydrophilic, were characterized using fluorescence spectroscopy. Such release properties are of interest for therapeutic agent delivery or agrochemical delivery applications of the invention. Doxorubicin was chosen as a representative example of hydrophobic molecular cargo and 6-carboxyfluorescein (6-FAM) was chosen as a representative example of hydrophilic molecular cargo. In these studies, the hydrogels were loaded with the cargo species during hydrogelation and the resulting loaded gels were then submerged in aqueous solutions for varying amounts of time. The aqueous media above the hydrogel was periodically exchanged for fresh media and the medium removed was analyzed for the presence of molecular cargo species using fluorescence spectroscopy. Release profiles for these cargo compounds are shown in
The release profiles demonstrated the delivery of some level of both hydrophobic and hydrophilic cargo into solution around the hydrogel. In general, a hydrophilic cargo is released more completely and more rapidly than hydrophobic cargo.
In physiological milieu such as might be experienced in therapeutic agent delivery, or environmental soil or water milieu such as might be experienced in agrochemical delivery, there is the additional possibility of enzymatically-assisted or microbially-assisted release of molecular cargo through partial degradation of the hydrogel. This possibility was tested using β-glycosidase as an enzyme contained in the aqueous medium surrounding the hydrogel loaded with molecular cargo. The results shown in
LMW hydrogels have attracted considerable interest with many attempts to utilize sugar-based LMW hydrogelators for various applications. The present invention provides novel disaccharide-based thioglycolipids through green synthetic processes along with characterization of the hydrogel physical properties.
The present inventors have discovered a new analogue of sugar-based LMWGs that possess high G′ values with intriguing microstructures, and surprising and unexpected properties and behaviors. The linear β(1→4) linkage in the lactose and cellobioside headgroups impose unique structural effects to the assembly process. Not only do the distinguished properties of these materials attribute a new analogue of LMW hydrogels for potential applications, but also the design of molecular structure that leads to these unique properties provide deeper insight in self-assembly and molecular packing of glycosylated-amphiphiles as potential low molecular weight hydrogelators.
Hydrogel Microparticles by Droplet Microfluidics. Particles in the size range of 40-60 μm diameter were fabricated using droplet microfluidics. LacSC8 hydrogel particles were formed using a 1 wt % aqueous LacSC8 solution as the droplet phase delivered at 10 μL/min into the droplet forming junction of a droplet microfluidic device held at 45° C. with toluene, dibutyl phthalate (DBP) or a mixture of toluene+DBP as the carrier phase at 20.8 μL/min. Droplets were collected in a chilled vessel containing either water, toluene or DBP. Particles of CelSC10 were similarly formed in an identical microfluidic device at 45° C. using an aqueous 1 wt % droplet solution of CelSC10 at 10 μL/min with a carrier phase of DBP at 20.8 μL/min. Droplets were collected in a chilled vessel containing either water, toluene or DBP. After collection, these particles were stable in solution at room temperature for over 1 week (the maximum time studied).
Bright field microscopy and fluorescence microscopy with Texas Red staining were used to characterize the resulting hydrogel microparticles. See
The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. All references cited herein are incorporated by reference in their entirety.
This application claims the priority benefit of U.S. Provisional Application No. 63/166,534, filed Mar. 26, 2021, which is incorporated herein by reference in its entirety.
This invention was made with government support under grant numbers 1339597 and 1954467 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/021835 | 3/25/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63166534 | Mar 2021 | US |
